WO2010001600A1 - Positive affect marker gene and use thereof - Google Patents

Positive affect marker gene and use thereof Download PDF

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Publication number
WO2010001600A1
WO2010001600A1 PCT/JP2009/003040 JP2009003040W WO2010001600A1 WO 2010001600 A1 WO2010001600 A1 WO 2010001600A1 JP 2009003040 W JP2009003040 W JP 2009003040W WO 2010001600 A1 WO2010001600 A1 WO 2010001600A1
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gene
group
genes
stimulus
rat
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PCT/JP2009/003040
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French (fr)
Japanese (ja)
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村上和雄
林隆志
堀美代
坂本成子
浦山修
中川嘉
山中陽子
原康弘
Original Assignee
トヨタ自動車株式会社
国立大学法人筑波大学
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Publication of WO2010001600A1 publication Critical patent/WO2010001600A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/30—Psychoses; Psychiatry
    • G01N2800/304—Mood disorders, e.g. bipolar, depression

Definitions

  • the present invention relates to a genetic marker that is an objective index of positive emotion and a method of using the same.
  • a genetic marker that is an objective indicator of positive emotions can be found, based on such indicators, it can be assessed whether a given stimulus is one that leads the subject to positive emotions or health promotion, It can be used in the field of product development that pursues comfort.
  • the present invention provides a genetic marker that can easily and reliably evaluate the positive emotion or health level of a subject, and provides a positive emotion or health evaluation system based on the genetic marker. For the purpose.
  • rat positive emotion marker gene a gene that a gene exists.
  • the vicinity of 50 KHz refers to a sound range of 45 to 65 KHz, preferably 45 to 55 KHz.
  • the present inventors conducted a comprehensive analysis of gene expression levels in humans under positive emotions that induced laughter, and found that genes whose expression levels were significantly increased in humans with such positive emotions (hereinafter, It was found that there is also a “human positive emotion marker gene”.
  • the present invention has been completed based on the above findings, and includes the following features.
  • the present invention is a first step of loading a rat or human with a stimulus to be evaluated, and a first step of measuring the expression level of at least one rat positive emotion marker gene or human positive emotion marker gene in a biological sample.
  • the given stimulus is a positive stimulus for a rat or a human, including the second step and a third step of evaluating whether the target stimulus is a positive stimulus based on the measured expression level of the gene. It relates to a method for evaluating whether or not.
  • the expression level of the gene in the second step, can be measured using a support on which a nucleic acid containing at least a part of the base sequence of the gene to be measured is immobilized.
  • the present invention also provides at least one nucleic acid selected from the group consisting of nucleic acids comprising at least part of the base sequence of a rat positive emotion marker gene or a human positive emotion marker gene, or a rat positive emotion marker gene or a human positive emotion marker gene
  • the present invention relates to a test kit for evaluating a positive emotion of a rat or a human or a human health level, comprising at least one molecule selected from the group consisting of molecules that specifically bind to a protein encoded by
  • the molecule may be an antibody, a ligand protein, or a receptor protein.
  • the nucleic acid or molecule may be fixed to a support.
  • a rat or human positive emotion or health evaluation system using a positive emotion marker gene as an index, a test kit for performing positive emotion evaluation in a rat or human, and a human health evaluation A test kit for performing is provided.
  • FIG. 1 shows a time chart of the Tickling stimulus load employed in the example.
  • FIG. 2 is a frequency component analysis diagram of Tickling (+) 4-week-old rat-generated speech on the stimulation start date.
  • FIG. 3 is a frequency component analysis diagram of Tickling ( ⁇ ) 4-week-old rat-generated speech on the stimulation start date.
  • FIG. 4 is a frequency component analysis diagram of Light Touch (+) 4-week-old rat-generated speech on the stimulation start date.
  • FIG. 5 is a frequency component analysis diagram of Light Touch ( ⁇ ) 4-week-old rat-generated speech on the stimulation start date.
  • FIG. 6 is a frequency component analysis diagram of control (+) 4-week-old rat-generated speech on the stimulation start date.
  • FIG. 1 shows a time chart of the Tickling stimulus load employed in the example.
  • FIG. 2 is a frequency component analysis diagram of Tickling (+) 4-week-old rat-generated speech on the stimulation start date.
  • FIG. 3 is a frequency component analysis diagram of Tickling
  • FIG. 7 is a frequency component analysis diagram of control ( ⁇ ) 4-week-old rat-generated speech on the stimulation start date.
  • FIG. 8 is a frequency component analysis diagram of Tickling (+) 6-week-old rat-generated speech on the stimulation start date.
  • FIG. 9 is a frequency component analysis diagram of Tickling ( ⁇ ) 6-week-old rat-generated speech on the stimulation start date.
  • FIG. 10 is a frequency component analysis diagram of Tickling (+) 8-week-old rat-generated speech on the stimulation start date.
  • FIG. 11 is a frequency component analysis diagram of Tickling ( ⁇ ) 8-week-old rat-generated speech on the stimulation start date.
  • FIG. 12 is a frequency component analysis diagram of Tickling (+) 4-week-old rat-generated speech on the day following the start of stimulation.
  • FIG. 13 is a frequency component analysis diagram of Tickling ( ⁇ ) 4-week-old rat-generated speech the day after the start of stimulation.
  • FIG. 14 is a frequency component analysis diagram of Light Touch (+) 4-week-old rat-generated speech on the day after the start of stimulation.
  • FIG. 15 is a frequency component analysis diagram of Light Touch ( ⁇ ) 4-week-old rat-generated speech the day after the start of stimulation.
  • FIG. 16 is a frequency component analysis diagram of control (+) 4-week-old rat-generated speech on the day following the start of stimulation.
  • FIG. 17 is a frequency component analysis diagram of control ( ⁇ ) 4-week-old rat-generated speech on the day following the start of stimulation.
  • FIG. 18 is a frequency component analysis diagram of Tickling (+) 6-week-old rat-generated speech on the day following the start of stimulation.
  • FIG. 19 is a frequency component analysis diagram of Tickling ( ⁇ ) 6-week-old rat-generated speech the day after the start of stimulation.
  • FIG. 20 is a frequency component analysis diagram of Tickling (+) 8-week-old rat-generated speech the day after the start of stimulation.
  • FIG. 21 is a frequency component analysis diagram of Tickling ( ⁇ ) 8-week-old rat-generated speech on the day following the start of stimulation.
  • FIG. 22 is a frequency component analysis diagram of Tickling (+) 4-week-old rat-generated speech in the second week after the start of stimulation.
  • FIG. 23 is a frequency component analysis diagram of Tickling ( ⁇ ) 4-week-old rat-generated speech in the second week after the start of stimulation.
  • FIG. 24 is a frequency component analysis diagram of Light Touch (+) 4-week-old rat-generated speech in the second week after the start of stimulation.
  • FIG. 25 is a frequency component analysis diagram of Light Touch ( ⁇ ) 4-week-old rat-generated speech in the second week after the start of stimulation.
  • FIG. 26 is a frequency component analysis diagram of control (+) 4-week-old rat-generated speech at the second week after the start of stimulation.
  • FIG. 27 is a frequency component analysis diagram of control ( ⁇ ) 4-week-old rat-generated speech at the second week after the start of stimulation.
  • FIG. 24 is a frequency component analysis diagram of Light Touch (+) 4-week-old rat-generated speech in the second week after the start of stimulation.
  • FIG. 25 is a frequency component analysis diagram of Light Touch ( ⁇ ) 4-week-old rat-generated speech in the second week after the start of stimulation.
  • FIG. 28 is a frequency component analysis diagram of Tickling (+) 6-week-old rat-generated speech in the second week after the start of stimulation.
  • FIG. 29 is a frequency component analysis diagram of Tickling ( ⁇ ) 6-week-old rat-generated speech in the second week after the start of stimulation.
  • FIG. 30 is a frequency component analysis diagram of Tickling (+) 8-week-old rat-generated speech in the second week after the start of stimulation.
  • FIG. 31 is a frequency component analysis diagram of Tickling ( ⁇ ) 8-week-old rat-generated speech in the second week after the start of stimulation.
  • FIG. 32 is a frequency component analysis diagram of Tickling (+) 4-week-old rat-generated speech at the fourth week after the start of stimulation.
  • FIG. 33 is a frequency component analysis diagram of Tickling ( ⁇ ) 4-week-old rat-generated speech at the fourth week after the start of stimulation.
  • FIG. 34 is a frequency component analysis diagram of Light Touch (+) 4-week-old rat-generated speech in the fourth week after the start of stimulation.
  • FIG. 35 is a frequency component analysis diagram of Light Touch ( ⁇ ) 4-week-old rat-generated speech at the fourth week after the start of stimulation.
  • FIG. 36 is a frequency component analysis diagram of control (+) 4-week-old rat-generated speech at 4 weeks after the start of stimulation.
  • FIG. 37 is a frequency component analysis diagram of control ( ⁇ ) 4-week-old rat-generated speech at 4 weeks after the start of stimulation.
  • FIG. 38 is a frequency component analysis diagram of Tickling (+) 6-week-old rat-generated speech at the fourth week after the start of stimulation.
  • FIG. 39 is a frequency component analysis diagram of Tickling ( ⁇ ) 6-week-old rat-generated speech at 4 weeks after the start of stimulation.
  • FIG. 40 is a frequency component analysis diagram of Tickling (+) 8-week-old rat-generated speech at 4 weeks after the start of stimulation.
  • FIG. 41 is a frequency component analysis diagram of Tickling ( ⁇ ) 8-week-old rat-generated speech at the fourth week after the start of stimulation.
  • FIG. 42 shows the waiting time for approach immediately after each treatment on a 4-week-old rat immediately after the start of stimulation.
  • FIG. 43 shows the waiting time for approach immediately after each treatment on a 4-week-old rat on the day following the start of stimulation.
  • FIG. 44 shows the approach waiting time immediately after each treatment on a 4-week-old rat in the second week after the start of stimulation.
  • FIG. 45 shows the approach waiting time immediately after each treatment on a 4-week-old rat at 4 weeks after the start of stimulation.
  • FIG. 46 shows the signal intensity distribution of each probe on the DNA chip when the difference in gene expression between Tickling / control and Tickling / Light Touch was analyzed by the DNA chip method.
  • FIG. 47 is an image obtained by clustering probes having signal intensities of Cy3 and Cy5 of 50 or more in any two sets of analysis.
  • FIG. 48 is a clustering image of probes in which an expression difference of more than 2 times or less than 0.5 times was observed in at least two sets of analyses.
  • FIG. 49 is a clustered image of 593 probes in which an expression difference of more than 1.5 times was observed in the salivary gland tissue of the Tickling group compared to the control group.
  • FIG. 50 is a clustered image of 171 probes in which an expression difference of more than 1.5 times was observed in the salivary gland tissue of the Tickling group compared to the Light Touch group.
  • FIG. 51 is a Venn diagram showing the number of genes (including transcripts) in which an expression difference was observed in the Tickling group compared to the control group or the Light Touch group.
  • FIG. 52 is a clustering image of 525 probes in which an expression difference of less than 1 / 1.5 times was observed in the salivary gland tissue of the Tickling group compared to the control group.
  • FIG. 53 is a clustered image of 166 probes in which an expression difference of less than 1 / 1.5 times was observed in the salivary gland tissue of the Tickling group compared to the Light Touch group.
  • FIG. 54 is a Venn diagram showing the number of salivary gland tissue genes in which a difference in expression was found in the Tickling group compared to the control group or the Light Touch group.
  • FIG. 55 is a clustered image of 157 probes in which an expression difference of more than 1.5 times was observed in the hypothalamic tissue of the Tickling group compared to the control group.
  • FIG. 56 is a clustered image of 136 probes in which an expression difference of more than 1.5 times was observed in the hypothalamic tissue of the Tickling group compared to the Light Touch group.
  • FIG. 57 is a Venn diagram showing the number of hypothalamic genes (including transcripts) in which expression differences were observed in the Tickling group compared to the control group or the Light Touch group.
  • FIG. 58 is a clustered image of 57 probes in which an expression difference of less than 1 / 1.5 times was observed in the hypothalamus of the Tickling group compared to the control group.
  • FIG. 59 is a clustered image of 185 probes in which an expression difference of less than 1 / 1.5-fold was observed in the hypothalamic tissue of the Tickling group compared to the Light Touch group.
  • FIG. 60 is a Venn diagram showing the number of hypothalamic genes in which a difference in expression of less than 1 / 1.5-fold was observed in the Tickling group compared to the control group or the Light Touch group.
  • FIG. 61 shows the color shading and P value of the GO hierarchy diagram.
  • FIG. 62 is a GO hierarchy diagram of brain and blood genes whose expression was increased in the Tickling group compared to the control group.
  • FIG. 63 is a GO hierarchy diagram of brain and blood genes whose expression is increased in the Tickling group compared to the Light Touch group.
  • FIG. 64 is a GO hierarchy diagram of brain and blood genes whose expression is decreased in the Tickling group compared to the Light Touch group.
  • FIG. 65 is a GO hierarchy diagram of salivary gland genes whose expression was increased in the Tickling group compared to the control group.
  • FIG. 66 is a GO hierarchy diagram of salivary gland genes whose expression was decreased in the Tickling group compared to the control group.
  • FIG. 67 is a GO hierarchy diagram of salivary gland genes whose expression is increased in the Tickling group compared to the Light Touch group.
  • FIG. 68 is a GO hierarchy diagram of salivary gland genes whose expression is decreased in the Tickling group compared to the Light Touch group.
  • FIG. 64 is a GO hierarchy diagram of brain and blood genes whose expression is decreased in the Tickling group compared to the Light Touch group.
  • FIG. 65 is a GO hierarchy diagram of salivary gland genes whose expression was increased in the Tickling group compared to the control group.
  • FIG. 69 is a GO hierarchy diagram of hypothalamic genes whose expression was increased in the Tickling group compared to the control group.
  • FIG. 70 is a GO hierarchy diagram of hypothalamic genes whose expression was decreased in the Tickling group compared to the control group.
  • FIG. 71 is a GO hierarchy diagram of hypothalamic genes whose expression is increased in the Tickling group compared to the Light Touch group.
  • FIG. 72 is a GO hierarchy diagram of hypothalamic genes whose expression is decreased in the Tickling group compared to the Light Touch group.
  • FIG. 73 is a frequency component analysis diagram of rat-generated speech on the second day of Tickling load.
  • FIG. 74 is a frequency component analysis diagram of rat-generated speech at the start of Light Touch load.
  • FIG. 73 is a frequency component analysis diagram of rat-generated speech on the second day of Tickling load.
  • FIG. 75 shows the signal intensity distribution of each probe on the DNA chip when the difference in gene expression between Tickling / Light Touch was analyzed by the DNA chip method.
  • FIG. 76 is an image obtained by clustering probes in which the signal intensity of Cy3 and Cy5 is 50 or more in any analysis.
  • FIG. 77 is a clustered image of 868 probes in which an expression difference of 1.5 times or more or 1 / 1.5 times or less was recognized in any analysis.
  • FIG. 78 is a clustering image of 88 probes in which an expression difference of 2 times or more or 0.5 times or less was recognized.
  • FIG. 79 is a clustering image of 4,397 probes that recognized an expression difference of 1.5 times or more or 1 / 1.5 times or less in any analysis (probe alignment is based on long-term stimulation).
  • FIG. 80 is a clustering image of 4,397 probes that recognized an expression difference of 1.5 times or more or 1 / 1.5 times or less in any analysis (probe alignment is based on short-term stimulation).
  • FIG. 81 is a clustered image of 865 probes in which an expression difference of 2 times or more or 0.5 times or less was recognized in any analysis (probe alignment is based on long-term stimulation).
  • FIG. 82 is a clustered image of 865 probes in which an expression difference of 2 times or more or 0.5 times or less was recognized in any analysis (probe alignment is based on short-term stimulation).
  • FIG. 83 shows the color shading and P value of the GO hierarchy diagram.
  • FIG. 84 is a GO hierarchy diagram of genes whose expression is commonly increased in the brain and scratched cells.
  • FIG. 85 is a GO hierarchy diagram of genes whose expression is increased in the hypothalamus.
  • FIG. 86 is a GO hierarchy diagram of genes with decreased expression in the hypothalamus.
  • FIG. 87 is a GO hierarchy diagram of genes whose expression is increased in the striatum.
  • FIG. 88 is a GO hierarchy diagram of genes with decreased expression in the striatum.
  • FIG. 89 shows a flow of gene expression quantitative analysis by real-time PCR.
  • FIG. 90 is an experimental time chart in the study on gene expression change due to laughter.
  • FIG. 90 is an experimental time chart in the study on gene expression change due to laughter.
  • FIG. 91 is a plot of salivary amylase activity before and after a lecture and before and after a contest in a diabetic group and a non-diabetic group.
  • FIG. 92 is a graph plotting the redox potential (ORP) in saliva before and after a lecture and before and after a lecture in a group with and without diabetes.
  • FIG. 93 is a diagram in which the amount of saliva before and after a lecture and before and after a contest in a diabetic group and a non-diabetic group are plotted.
  • FIG. 94 is a diagram plotting the saliva buffering capacity before and after the lecture and before and after the control in the diabetic group and the non-diabetic group.
  • FIG. 95 is a graph plotting the total emotional disorder (TMD) before and after the lecture and before and after the contest in the diabetic group and the non-diabetic group.
  • FIG. 96 is a plot of anxiety tendencies (STAI) before and after a lecture and before and after a contest in a group with and without diabetes.
  • FIG. 97 is a graph plotting salivary secretory immunoglobulin (s-IgA) before and after the lecture and before and after the lecture in the diabetic group and the non-diabetic group.
  • FIG. 98 is a diagram plotting cortisol in saliva before and after a lecture and before and after a contest in a diabetic group and a non-diabetic group.
  • FIG. 99 is a diagram plotting blood glucose levels before and after the lecture and before and after the contest in the diabetic group and the non-diabetic group.
  • FIG. 100 is a graph plotting the white blood cell count before and after the lecture and before and after the contest in the diabetic group and the non-diabetic group.
  • FIG. 101 is a graph plotting the neutrophil ratio before and after the lecture and before and after the contest in the diabetic group and the non-diabetic group.
  • FIG. 102 is a diagram plotting the lymphocyte ratio before and after the lecture and before and after the contest in the diabetic group and the non-diabetic group.
  • FIG. 103 is a graph plotting the NK cell activity before and after the lecture and before and after the control in the diabetic group and the non-diabetic group.
  • FIG. 104 is a plot of blood cortisol before and after the lecture and before and after the contest in the diabetic and non-diabetic groups.
  • FIG. 105 is a diagram plotting C-reactive protein before and after the lecture and before and after the contest in the diabetic group and non-diabetes group.
  • FIG. 106 is a diagram in which blood glucose levels before and after a lecture and before and after a difference are plotted in a diabetic group and a non-diabetic group.
  • FIG. 107 is a graph plotting salivary amylase activity before and after a lecture and before and after a contest in a group with and without periodontal disease.
  • FIG. 108 is a graph plotting the redox potential in saliva before and after the lecture and before and after the lecture in the group with and without periodontal disease.
  • FIG. 109 is a graph plotting the saliva amount before and after the lecture and before and after the contest in the group with and without periodontal disease.
  • FIG. 110 is a diagram plotting the saliva buffering capacity before and after the lecture and before and after the control in the group with and without periodontal disease.
  • FIG. 111 is a diagram plotting the total emotional disorder (TMD) before and after the lecture and before and after the contest in the group with and without periodontal disease.
  • TMD total emotional disorder
  • FIG. 112 is a plot of anxiety tendencies (STAI) before and after a lecture and before and after a contest in a group with and without periodontal disease.
  • FIG. 113 is a diagram plotting salivary immunoglobulin A in the saliva before and after the lecture and before and after the lecture in the group with and without periodontal disease.
  • FIG. 114 is a plot of salivary cortisol before and after the lecture and before and after the lecture in the group with and without periodontal disease.
  • FIG. 115 is a graph plotting blood glucose levels before and after the lecture and before and after the control in the group with and without periodontal disease.
  • FIG. 116 is a diagram in which the white blood cell count before and after the lecture and before and after the contest in the periodontal disease affected group and the periodontal disease unaffected group is plotted.
  • FIG. 117 is a graph plotting the neutrophil ratio before and after the lecture and before and after the contest in the group with and without periodontal disease.
  • FIG. 118 is a graph plotting the lymphocyte ratio before and after the lecture and before and after the contest in the group with and without periodontal disease.
  • FIG. 119 is a graph plotting the NK cell activity before and after the lecture and before and after the lecture in the group with and without periodontal disease.
  • FIG. 120 is a plot of blood cortisol before and after the lecture and before and after the contest in the group with and without periodontal disease.
  • FIG. 121 is a diagram plotting CRP before and after the lecture and before and after the contest in the periodontal disease affected group and the periodontal disease nonaffected group.
  • FIG. 122 is a diagram in which blood glucose levels before and after a lecture and before and after a difference are plotted in a periodontal disease affected group and a periodontal disease unaffected group.
  • FIG. 123 shows a histogram of genes in which expression changes of 1.5 times or more or 1 / 1.5 times or less were found in analyzes 2 and 4 to 10 of Example 4. The vertical axis represents the Log2 value of the expression change fold, and the horizontal axis represents the sample name.
  • FIG. 124 shows by comparison the relative difference in expression change for each corresponding analysis.
  • FIG. 125 shows a Venn diagram of genes in which expression change of 1.5 times or more was observed by laughter in the non-diabetic group and the diabetic group.
  • FIG. 126 shows a Venn diagram of genes in which expression change of 1.5 times or less was observed by laughter in the non-diabetic group and the diabetic group.
  • FIG. 127 shows a Venn diagram of genes in which expression change of 1.5 times or more was observed by laughter in the non-diabetic / periodontal disease-free group and the non-diabetes / periodontal group.
  • FIG. 128 shows a Venn diagram of genes in which expression change of 1.5 times or less was observed by laughter in the non-diabetic / periodontal disease-free group and the non-diabetic / periodontal group.
  • FIG. 129 shows a Venn diagram of genes in which the expression change of 1.5 times or more was observed by laughter in the non-diabetic / periodontal disease non-affected group and the diabetic / periodontal non-affected group.
  • FIG. 130 shows a Venn diagram of genes in which expression changes of 1 / 1.5 fold or less were observed by laughter in the non-diabetic / periodontal disease non-affected group and the diabetic / periodontal non-affected group.
  • FIG. 131 shows a Venn diagram of genes in which the expression change of 1.5 times or more was observed by laughter in the non-diabetes / periodontal disease affected group and the diabetic / periodontal disease affected group.
  • FIG. 132 shows a Venn diagram of genes in which expression change of 1 / 1.5 fold or less was observed by laughter in the non-diabetic / periodontal disease group and the diabetic / periodontal group.
  • FIG. 133 shows a Venn diagram of genes in which expression changes of 1.5 times or more were observed by laughter in the group with and without diabetes and in the group with and without diabetes.
  • FIG. 134 shows a Venn diagram of genes in which expression changes of 1 / 1.5 times or less were observed by laughter in the group with and without diabetes and in the group with and without diabetes.
  • FIG. 135 shows a Venn diagram of genes in which expression change of 1.5 times or more was observed by laughter in the group not suffering from periodontal disease and the group suffering from periodontal disease.
  • FIG. 136 shows a Venn diagram of genes in which expression change of 1 / 1.5 times or less was observed by laughter in the group not suffering from periodontal disease and the group suffering from periodontal disease.
  • Rat positive emotion marker gene and rat positive emotion evaluation system using the same provides a rat positive emotion evaluation method. Specifically, in the rat positive emotion evaluation method according to the present invention, step a for measuring the expression level of a rat positive emotion marker gene in the rat, and evaluating the positive emotion of the rat based on the expression level measured in step a. Step b.
  • positive emotion / affect refers to, for example, psychological states such as happy, cheerful, pleasant, fun, mild, calm, happiness, joy, love, hope, satisfaction, motivation, It refers to positive emotions such as interest and trust, and any emotions that lead the body and mind to a healthy state or health promotion.
  • Health promotion refers to enhancing the ability of the body and mind to adapt to stimuli from the outside world in the physical and mental aspects of the WHO definition.
  • negative emotion / affect as used in this specification refers to any psychological state such as tension, anxiety, sadness, depression, anger, fear, and embarrassment, negative emotions, and any physical or mental condition that leads to morbidity. Refers to the emotions.
  • a “positive emotion marker gene” is a gene that serves as an index of a subject's positive emotion, and when the subject is in positive emotion, the expression level is significantly increased or decreased. Refers to any gene.
  • the “rat positive emotion marker gene” provided in the present invention has a higher expression level in rats under positive emotion using the generated speech near 50 KHz as an index compared to a rat that does not show such positive emotion characteristics. It is defined as a gene that increases or decreases 1.5 times or more, preferably 2.0 times or more. In other words, the rat positive emotion marker gene is a gene whose expression level increases or decreases by 1.5 times or more, preferably 2.0 times or more in conjunction with the generated speech in the vicinity of 50 KHz of the rat.
  • Tables 1 and 2 below show the rat positive emotion marker genes of the present invention that show an increase or decrease in expression level of 2.0 times or more in positive emotion rats.
  • the “No.” column means the number assigned to each gene
  • the “Target Symbol” column is a general gene notation.
  • the column “Target ⁇ Accession” is a registration ID (accession number) registered in a public database
  • “UniGene” is a nucleic acid sequence database that eliminates duplication of sequence data provided by NCBI.
  • the column “UniGene” is a UiGeneID registered in the database (UniGene).
  • “Up / down” means whether the expression level is increasing (Up) or the expression level is decreasing (Down) in the positive emotional rat.
  • the “biological sample” refers to a sample in which each rat positive emotion marker gene can function as an index of positive emotion in a positive emotion rat.
  • the rat positive emotion marker gene shown in Table 1 is a list of genes that showed a marked increase or decrease in the expression level when a positive stimulus was loaded in a short period of 2 days, and the rat positive emotion marker gene shown in Table 2 It is the list
  • the gene with a significant increase or decrease in the expression level differs between the case of a short-term positive stimulus and the case of a long-term positive stimulus.
  • the rat positive emotion marker gene shown in Table 1 is a marker that can detect transient positive emotions
  • the rat positive emotion marker gene shown in Table 2 is a persistent gene that causes changes in constitution. It is a marker that can detect the result of positive emotion.
  • the expression level of at least one gene selected from the gene group shown in Tables 1 and / or 2 is measured in a specified biological sample.
  • the “designated biological sample” means the sample described in the column of “biological sample” corresponding to each gene shown in Tables 1 and / or 2 (that is, blood, hypothalamus, striatum, oral cavity) Internal cell or salivary gland).
  • the genes listed in Tables 81 to 84 and Tables 45 to 59 described below are genes related to various biological activities among rat positive emotion marker genes found in short-term stimulation and long-term stimulation, respectively.
  • the expression levels of the genes listed in Tables 81 to 84 and / or 45 to 59 are designated. You may measure in the biological origin sample.
  • the gene expression level is not particularly limited as long as the expression level of the target gene can be specifically quantified, and it may be measured by measuring the mRNA level (or cDNA) of the target gene, or the target gene may be encoded. The amount of protein to be measured may be measured.
  • a nucleic acid having a base sequence complementary to the mRNA (or cDNA) can be used for measuring the mRNA amount (or cDNA) of the target gene. Specifically, it is carried out by isolating total mRNA from a rat biological sample and using a nucleic acid containing at least a part (preferably a coding region) of the base sequence of the gene to be measured as a probe or primer.
  • the nucleic acid used for the measurement does not necessarily need to be DNA, and may be RNA, DNA / RNA chimera, other artificial nucleic acid, or the like.
  • nucleic acids can be obtained by methods well known to those skilled in the art, such as cloning of appropriate sequences and cleavage with restriction enzymes, the phosphotriester method (see, for example, Narang et al., 1979, Meth. Enzymol., 68, p90-99), Phosphodiester method (see, for example, Brown et al., 1979, Meth. Enzymol., Vol. 68, p109-151), ethyl phosphoramidite method (see, for example, Beaucage et al., 1981, Tetrahedron Lett., Vol. 22, p1859-1862) ) And the like can be directly synthesized. Moreover, you may synthesize
  • Isolation of total mRNA from a rat biological sample can be performed by a method known to those skilled in the art.
  • total RNA can be obtained from a rat biological sample-disrupted solution according to a conventional method, and mRNA can be recovered using an oligo dT column.
  • any method known to those skilled in the art may be used to measure the amount of mRNA.
  • a method for example, DNA microarray method, RT-PCR, quantitative PCR, Northern blot etc. can be used.
  • RT-PCR quantitative PCR
  • Northern blot or the like.
  • the amount of mRNA can be measured by labeling the nucleic acid probe or primer.
  • the DNA microarray method when measuring the amount of mRNA for a large number of genes such as 100 or more, it is preferable to use the DNA microarray method.
  • the amount of mRNA When measuring the amount of mRNA by the DNA microarray method, it is carried out by bringing the total mRNA as a measurement sample into contact with the support on which the nucleic acid probe is immobilized. In this case, generally, the amount of mRNA can be measured by labeling the mRNA of the measurement sample and detecting the presence or absence of hybridization with each probe by the fluorescence intensity.
  • the hybridization conditions are not particularly limited as long as specific hybridization between each probe and the target mRNA is ensured, but stringent conditions are preferable. Such conditions are known to those skilled in the art, and examples include the conditions described in Sambrook et al. Molecular Cloning: A Laboratory Manual 2nd Ed. Cold Spring Harbor Laboratory Press (1989).
  • the expression level of the target gene is measured by measuring the amount of protein encoded by the gene, a molecule that specifically binds to the protein, for example, an antibody that specifically binds to the protein is added.
  • the antibody is not particularly limited, and a mouse antibody, a rat antibody, a rabbit antibody, a sheep antibody, or the like can be used as appropriate. it can.
  • the antibody may be a polyclonal antibody or a monoclonal antibody, but a monoclonal antibody is preferable in that a homogeneous antibody can be stably produced.
  • Polyclonal and monoclonal antibodies can be prepared by methods well known to those skilled in the art.
  • a hybridoma producing a monoclonal antibody can be basically produced using a known technique as follows. That is, a desired antigen or a cell expressing the desired antigen is used as a sensitizing antigen and immunized according to a normal immunization method, and the resulting immune cell is fused with a known parent cell by a normal cell fusion method. And can be prepared by screening monoclonal antibody-producing cells (hybridomas) by a normal screening method.
  • the hybridoma can be prepared, for example, according to the method of Milstein et al. (Kohler. G. and Milstein, C., Methods Enzymol. (1981) 73: 3-46).
  • the resulting monoclonal antibodies can be used for enzyme-linked immunosorbent assays (ELISA), enzyme immunodot assays, radioimmunoassays, agglutination-based assays, or other well-known immunoassay methods for the quantification of proteins to be measured. Can be used as a test reagent.
  • the monoclonal antibody is preferably labeled. When labeling, for example, an enzyme, a fluorescent substance, a chemiluminescent substance, a radioactive substance, or a staining substance known in the art can be used as the labeling compound.
  • a ligand protein or a receptor protein that can specifically bind to the protein can also be used for the measurement of the protein amount.
  • the test kit for evaluating the positive emotion of the rat comprises the bases of the genes shown in Table 1 and / or 2 (and / or the genes shown in Tables 81 to 84 and / or Tables 45 to 59). Consists of at least one nucleic acid selected from the group consisting of nucleic acids containing at least part of the sequence, or a molecule (for example, an antibody) that specifically binds to a protein encoded by the genes shown in Tables 1 and / or 2 above. It comprises at least one molecule selected from the group.
  • the type of nucleic acid or molecule provided in the test kit of the present invention varies depending on the type of gene to be measured.
  • the test kit may include a support in which the above-described nucleic acid or molecule (or part thereof) is coated.
  • the support include polystyrene, polycarbonate, polypropylene, polyvinyl microtiter plates, test tubes, capillaries, beads, membranes, filters, and the like.
  • step b after measuring the expression level of at least one gene selected from the gene group shown in Table 1 and / or 2 in a specified biological sample, in step b, based on the expression level Evaluate positive emotions in rats.
  • the evaluation in step b can be performed by judging whether the score calculated from the gene expression level measured in step a is different from the reference values for the genes listed in Tables 1 and 2.
  • the score calculated from the expression level is determined whether the “Up / down” column is higher than the reference value for the Up gene, It is determined whether or not the Down gene is lower than the reference value.
  • the reference value refers to, for example, the expression level of a rat positive emotion marker gene in a control rat that does not emit an ultrasonic wave in the vicinity of about 50 KHz, and a significant expression level between the control rat and a rat in positive emotion. It can be a value shown as a relative value with respect to the expression level of any gene X in which no difference is observed. In this case, the score can be calculated by dividing the expression level of the rat positive emotion marker gene measured in step a by the expression level of gene X.
  • Such a reference value may be obtained in advance for each gene listed in Tables 1 and 2, or the reference value in a control rat may be measured in parallel with step a of the present invention.
  • the reference value is preferably set to a reference value ⁇ standard deviation by calculating with a plurality of control rats. Thereby, the influence of the individual difference with respect to a reference value can be excluded.
  • the test kit according to the present invention may further include an antibody against a nucleic acid having the base sequence of gene X or a protein encoded by gene X. Thereby, in order to measure the reference value in the control rat and to calculate the score in the rat to be evaluated, the expression level of gene X can be obtained.
  • the test kit according to the present invention preferably includes a correspondence table of reference values ( ⁇ standard deviation) calculated in advance for each of the positive emotion marker genes shown in Tables 1 and 2. Thereby, calculation of the reference value using the control rat can be omitted.
  • a gene is selected from genes whose expression level is “Up”, and the expression level and gene of the selected gene are selected.
  • the expression level of X is measured and the expression level of the selected gene divided by the expression level of gene X is significantly higher than the reference value calculated in advance for the gene, the rat is positive It can be evaluated as emotion.
  • the above determination is made for a plurality of genes, and is 50% or more, more preferably 60% or more, more preferably 70% or more, more preferably 80% or more, more preferably 90%. % Or more, most preferably 100% of the genes may be evaluated as positive emotions if they are significantly above the reference value.
  • the increase rate or decrease rate of the gene expression level relative to the control may be obtained for each of the selected genes, and the rat may be evaluated as having a positive emotion when the percentage sum exceeds a predetermined value.
  • positive emotion can be evaluated easily and with high reliability.
  • the positive emotion of the rat is evaluated with higher reliability by increasing the types of genes to be measured (for example, 10 to 100 or more). be able to.
  • Human positive emotion marker gene and human positive emotion or health evaluation system using the same The present invention also provides a human positive emotion or health evaluation method. In principle, this method can be performed in the same manner as the rat positive emotion evaluation method. Therefore, the human positive emotion or human health evaluation method of the present invention is based on the step a in which the expression level of a human positive emotion marker gene is measured in a biological sample, for example, blood, and the expression level measured in step a. And b for assessing human positive emotions or health.
  • a “human positive emotion marker gene” refers to a human loaded with a stimulus that induces laughter (also referred to herein as “laughter stimulus”). In comparison, it is defined as a gene whose expression level increases or decreases. In other words, the human positive emotion marker gene is a gene whose expression level increases or decreases in conjunction with human laughter.
  • “Health” refers to “a state of complete physical, mental and social welfare, not simply the absence of illness or illness” according to the WHO definition. Point to. In humans, laughter is known to suppress NK cell activation and postprandial blood glucose levels in diabetic patients. In the present invention, activation of immune functions such as s-IgA, improvement of the stress state seen from salivary amylase and neutrophil as a stress index, reduction of C-reactive protein that increases during infection and inflammation, diabetic patients The suppression of postprandial blood glucose level was confirmed by laughter stimulation, and the results showed that laughter was effective in maintaining and improving health and restoring disease function. These effects of maintaining / promoting health and restoring the function of the disease can be associated with each gene set of positive emotion marker genes. Therefore, the evaluation of human positive emotion based on the expression level of the human positive emotion marker gene can be considered as an evaluation of human health.
  • gene sets 1-8 the gene group consisting of genes that are noted in terms of function and the top 15 genes that show significant increase or decrease in the expression level are called gene sets 1-8, respectively, and are summarized in the table below.
  • Gene Set 1 Table 164; Gene Set 2, Table 172; Gene Set 3, Table 178; Gene Set 4, Table 185; Gene Set 5, Table 155; Gene Set 6, Table 159; Gene Set 7, Table 190; Gene set 8, Table 198.
  • the “No.” column means the number assigned to each gene in each gene set
  • the “Target (Gene) Symbol” column is a general gene notation.
  • the column “Target ⁇ Accession” is a registration ID (accession number) registered in a public database
  • “UniGene” is a nucleic acid sequence database that eliminates duplication of sequence data provided by NCBI.
  • the column “UniGene” is a UiGeneID registered in the database (UniGene).
  • “Up / down” means whether the expression level is increasing (Up) or the expression level is decreasing (Down) in a positive emotional human who has induced laughter.
  • the “subject group” indicates the types of subjects whose gene expression in each gene set was confirmed to increase or decrease: A, non-diabetic and non-periodontal; B, non-diabetic and dental Peripheral disease; C, Diabetes and non-periodontal disease; D, Diabetes and periodontal disease; A + B, Non-diabetes; C + D, Diabetes; A + C, Non-periodontal; B + D, suffering from periodontal disease.
  • Gene sets 1, 2, and 5 are gene sets for positive emotions or health assessment that can be widely used for general people.
  • gene set 1 is particularly positive emotions or health for general people. It is a gene set suitable for performing degree evaluation.
  • Gene sets 3, 4 and 6 are gene sets suitable for diabetics, but can be used to evaluate positive emotions or health of people who have other diseases or who have no diseases.
  • Gene sets 2, 4 and 8 are gene sets suitable for those suffering from periodontal disease, but can be used to evaluate positive emotions or health of people who have other diseases or who have no diseases. . These gene sets can be used for positive emotion or health evaluation, either alone or in combination.
  • the expression level of at least one gene in the gene sets shown in gene sets 1 to 8 is converted into a biological sample such as blood,
  • Measure in The biological sample to be measured refers to tissues, cells, body fluids, excreta and the like derived from living bodies in addition to blood, and the body fluid includes blood-derived samples such as plasma and serum, saliva, milk and the like.
  • Excrements include urine and feces.
  • the cells include leukocytes separated from blood, oral mucosal scraping cells, nasal mucosal cells, and the like, but are not limited thereto, and any cell can be used as long as it can be collected from a living body.
  • the test kit for evaluating human positive emotion or health is a group consisting of nucleic acids containing at least part of the base sequences of genes in the gene sets shown in the gene sets 1 to 8 above. Or at least one selected from the group consisting of molecules (for example, antibodies) that specifically bind to a protein encoded by a gene in the gene set shown in the gene set 1 to 8 above. It has molecules.
  • step b the human positive emotion or health level is evaluated.
  • the evaluation in step b can be performed by judging whether the score calculated from the expression level of the gene measured in step a is different from the reference value for the genes in gene sets 1-8.
  • the reference value refers to, for example, the expression level of a human positive emotion marker gene in a control human that is not loaded with a stimulus that induces laughter, and a significant expression level between the control human and a human in positive emotion. It can be a value shown as a relative value with respect to the expression level of any gene X in which no difference is observed. In this case, the score can be calculated by dividing the expression level of the human positive emotion marker gene measured in step a by the expression level of gene X.
  • Such a reference value may be obtained in advance for each gene described in gene sets 1 to 8, or the reference value in a control human may be measured in parallel with step a of the present invention.
  • the reference value is preferably set to be a reference value ⁇ standard deviation by calculating with a plurality of control humans. Thereby, the influence of the individual difference with respect to a reference value can be excluded.
  • the test kit for evaluating human positive emotion or health according to the present invention may further comprise an antibody against the nucleic acid having the base sequence of gene X or the protein encoded by gene X. it can. Thereby, the expression level of gene X can be determined in order to measure the reference value in the control human and to calculate the score in the human to be evaluated.
  • test kit for evaluating human positive emotion or health is a correspondence table of reference values ( ⁇ standard deviation) calculated in advance for each of the positive emotion marker genes shown in gene sets 1 to 8. It is preferable to contain. Thereby, calculation of the reference value using the control human can be omitted.
  • a gene is selected from genes whose expression level is “Up”, and the expression level of the selected gene and When the expression level of gene X is measured, and the value obtained by dividing the expression level of the selected gene by the expression level of gene X is significantly higher than the reference value calculated in advance for the gene, It can be evaluated that it is a positive emotion or a high degree of health.
  • the above determination is made for a plurality of genes, and is 50% or more, more preferably 60% or more, more preferably 70% or more, more preferably 80% or more, more preferably 90%.
  • the genes may be evaluated as positive emotions or high health if they are significantly above the reference value.
  • the increase rate or decrease rate of the gene expression level relative to the control is obtained for each of a plurality of selected genes, and when the percentage sum exceeds a predetermined value, the human is evaluated as having positive emotions or high health. May be.
  • positive emotion or health can be evaluated simply and with high reliability.
  • the method for evaluating human positive emotion or health according to the present invention by increasing the number of genes to be measured (for example, 10 to 100 or more), human positive can be obtained with higher reliability. Emotion or health can be assessed.
  • rat common gene and human common gene are considered to be usable as a particularly useful positive emotion evaluation marker in a rat and human positive emotion evaluation system and in a human health evaluation system.
  • the rat common gene or human common Genes can be used.
  • a plurality of positive emotion marker genes are used as one gene group, and the gene expression of the gene group Assess positive emotions or health by comprehensively analyzing quantities.
  • the following can be used as the above gene group: gene group listed in Table 1; gene group listed in Table 2; listed in any of Tables 81 to 84 A gene group listed in any of Tables 45 to 59; and a gene group listed in Table 205; or a combination of these gene groups.
  • gene group for example, the following can be used: gene group (or gene set 1) listed in Table 164; table 172 (or gene set) 2); gene group listed in Table 178 (or gene set 3); gene group listed in Table 185 (or gene set 4); gene group listed in Table 155 (or gene set 5); Gene group (or gene set 6) listed in Table 190; gene group listed in Table 190 (or gene set 7); gene group listed in Table 198 (or gene set 8); gene listed in Table 202 Group; or a combination of these genes.
  • the evaluation of positive emotion or health level is performed by measuring the expression level of all genes contained in the gene group as described above using a biological sample. Then, it is performed by evaluating the coincidence rate in the direction of increase / decrease in the expression of each gene.
  • the coincidence rate (%) was obtained by dividing the number of genes significantly increasing or decreasing with respect to the reference value toward the expression direction (Up / Down) indicated for each gene by the total number of genes included in the gene group. It can be obtained by multiplying the value by 100.
  • the coincidence rate (%) is 50% or more, more preferably 60% or more, more preferably 70% or more, more preferably 80% or more, more preferably 90% or more, and most preferably 100%.
  • a subject can be evaluated as having a positive emotion or high health.
  • the above-mentioned coincidence rate after the evaluation object stimulus load is obtained for a plurality of evaluation object stimuli, and the evaluation object stimulus that has produced the highest coincidence rate It can be selected as a positive emotion or a good stimulus that leads to better health.
  • the evaluation of positive emotion or health measures the expression level of all genes contained in the gene group as described above in a biological sample
  • the difference between the measured gene expression level and the expression level of the gene after loading with a Tickling stimulus or laughing stimulus can be performed by comparing all the genes included in the gene group. .
  • the sum of scores obtained by squaring the difference between the measured gene expression level and the gene expression level after loading with a Tickling stimulus or laughing stimulus is obtained according to the following equation.
  • the score after the evaluation target stimulus load is obtained for a plurality of evaluation target stimuli, and the evaluation target stimulus that generated the lowest score is positive emotion. Or it can be selected as a good stimulus leading to health promotion.
  • the evaluation of positive emotion or health level is the above (1-4-1) and (1-4). This can be done by combining the methods of -2). Specifically, the expression level of all genes included in the gene group as described above is measured with a sample derived from a living body, and the reference value is directed toward the expression direction (Up / Down) indicated for each gene. The genes that significantly increased or decreased were selected, the score obtained by squaring the difference between the measured gene expression level and the gene expression level after Tickling stimulation or laughing stimulation loading was obtained for each gene, and the total score was calculated as follows. Get according to the formula.
  • m is significantly increased or decreased with respect to the reference value in the same direction as the gene expression direction after Tickling stimulation or laughing stimulation load among genes in the gene group measured in the biological sample.
  • a i is the expression level of gene i measured in a biological sample;
  • b i is the expression level of gene i after Tickling stimulation or laughing stimulation loading] This score becomes smaller as the difference between the measured gene expression level and the gene expression level after Tickling stimulation or laughing stimulation loading is smaller. Therefore, the degree of positive emotion or the degree of health of the subject can be estimated using this score as an index.
  • the score after the evaluation target stimulus load is obtained for a plurality of evaluation target stimuli, and the evaluation target stimulus that generated the lowest score is positive emotion. Or it can be selected as a good stimulus leading to health promotion.
  • the amount of mutual information of Cullback-Liber is the expression level and expression pattern in the biological sample of the gene group as described above, and the gene after loading with a Tickling stimulus or laughing stimulus (ie, a subject with a positive emotion). It is obtained as a measure of the difference between the expression level and expression pattern of the group.
  • a i is expressed by the following formula:
  • the degree of positive emotion or the degree of health of the subject can be estimated based on the expression level and expression pattern after loading the Tickling stimulus or laughing stimulus.
  • the expression level after the load of Tickling stimulus or laughing stimulus and the amount of mutual information after the stimulus to be evaluated based on the expression pattern is calculated.
  • the mutual information J that is symmetrized with may be used instead of the mutual information of Cullback-Liber. It should be noted that other mutual information, for example, Hellinger mutual information, may be used instead of the Cullback-Librer mutual information.
  • Positive Stimulus Evaluation System is also a method for evaluating whether a given stimulus is a positive stimulus for rats and humans (hereinafter also referred to as “rat positive stimulus evaluation method” and “human positive stimulus evaluation method”, respectively). )I will provide a.
  • positive stimulus refers to any stimulus that leads the subject (ie, rat or human) to positive emotions or leads the subject (ie, human) to health promotion.
  • the “stimulus” used in the present invention refers to a substance that acts on a living body to cause some phenomenon or reaction, and includes an external stimulus and an internal stimulus. Types of stimuli include physical, chemical, biological, and psychological stimuli. These stimuli are one or more of visual, auditory, gustatory, olfactory, vestibular sensation, somatosensory, visceral sensation, and kinematic sensation. Moreover, the stimulus to be evaluated as to whether or not it is a positive stimulus is evaluated not only for the type but also for the stimulus loading pattern such as intermittent or continuous stimulus.
  • Rat positive stimulus evaluation method includes a first step of loading a rat with a stimulus to be evaluated, and a second step of measuring an expression level of at least one rat positive emotion marker gene. And a third step of determining whether the target stimulus is a positive stimulus for the rat based on the measured expression level of the rat positive emotion marker gene.
  • the stimulus to be evaluated is loaded on the rat.
  • the stimulation period is not particularly limited, but when the gene group shown in Table 1 is used as a rat positive emotion marker gene, it is loaded in a short period, and the gene group shown in Table 2 is used as a rat positive emotion marker gene. It is preferable to load for a long time.
  • the term “short period” refers to 1 to several days (for example, 2, 3, 4 or 5 days), and “long period” refers to 3 to 4 weeks.
  • the expression level of at least one of the rat positive emotion marker genes shown in Tables 1 and 2 is measured in a designated biological sample.
  • the expression level of at least one of the genes shown in Tables 81 to 84 and / or 45 to 59 may be measured in addition to or instead of Tables 1 and 2.
  • the expression level of the gene group listed in any of Tables 1, 2, 81 to 84 and 45 to 59 may be measured, and the measurement method is also described above. Street.
  • the evaluation can be performed by determining whether the score calculated from the expression level of the rat positive emotion marker gene measured in the second step is different from the reference value of the gene.
  • the evaluation of the target stimulus in the third step may be performed by comparing the expression level of the rat positive emotion marker gene before and after the load of the target stimulus. That is, if the expression level of the gene increases or decreases after the stimulation load compared to the expression level of the rat positive emotion marker gene measured before the target stimulation load, the stimulation is evaluated as a rat positive stimulation. can do.
  • the rat positive stimulus evaluation method can further include the step of measuring the expression level of the rat positive emotion marker gene before the first step of loading the rat with the stimulus to be evaluated.
  • a positive emotion marker gene to be measured is selected from those that function as a rat positive emotion marker gene in these biological samples. .
  • the evaluation of the target stimulus in the third step may be performed by comparison with a control rat. That is, the expression level of the same positive emotion marker gene is measured in both the rat loaded with the target stimulus and the control rat not loaded with the target stimulus, and the expression level of the gene increases or decreases in the rat loaded with the target stimulus. If so, the target stimulus can be evaluated as a rat positive stimulus.
  • the human positive stimulus evaluation method includes a first step of loading a stimulus to be evaluated to a human and a second step of measuring an expression level of at least one human positive emotion marker gene. And a third step of determining whether the target stimulus is a positive stimulus for a human based on the measured expression level of the human positive emotion marker gene.
  • a stimulus to be evaluated is loaded on a human.
  • the stimulus loading period is not particularly limited, it is more preferable to load in a short period.
  • the term “short term” used in the context of the human positive stimulus evaluation method refers to 1 second to several minutes or several hours.
  • the expression level of at least one of the human positive emotion marker genes shown in gene markers 1 to 8 is measured in a biological sample such as blood.
  • the expression level of at least one human common gene may be measured as a human positive emotion marker gene to be measured, and gene markers 1-8
  • the expression level of any gene group or gene group of human common genes may be comprehensively measured as described above, and the measurement method is also as described above.
  • the third step whether or not the target stimulus is a human positive stimulus is evaluated based on the expression level of the human positive emotion marker gene measured in the second step.
  • the evaluation can be performed by determining whether the score calculated from the expression level of the human positive emotion marker gene measured in the second step is different from the reference value of the gene.
  • the evaluation of the target stimulus in the third step may be performed by comparing the expression level of the human positive emotion marker gene before and after the load of the target stimulus. That is, if the expression level of the gene is increased or decreased after the stimulus load compared to the expression level of the human positive emotion marker gene measured before the target stimulus load, the stimulus is evaluated as a human positive stimulus. can do.
  • the human positive stimulus evaluation method can further include a step of measuring the expression level of the human positive emotion marker gene before the first step of loading a stimulus to be evaluated to a human.
  • the evaluation of the target stimulus in the third step may be performed by comparison with a control human. That is, the expression level of the same positive emotion marker gene is measured in both the human subject loaded with the target stimulus and the control human subject not loaded with the target stimulus, and the expression level of the gene increases or decreases in the human loaded with the target stimulus. If so, the target stimulus can be evaluated as a human positive stimulus.
  • rat common gene and human common gene are considered to be useful as positive emotion evaluation markers particularly useful in rat and human positive emotion evaluation systems.
  • the stimulus to be evaluated is loaded in the rat and the expression level of the common gene of the rat is increased or decreased, it can be evaluated that the target stimulus is a rat positive stimulus, and the same effect is also obtained in humans. It can be inferred as a certain stimulus.
  • stimulation evaluation in rats using a common gene for rats can be used as a search tool for human positive stimuli.
  • the human positive stimulus evaluation method of the present invention comprises the steps of performing the rat positive stimulus evaluation using a rat common gene, and the step of identifying the stimulus evaluated as a positive stimulus as a human positive stimulus. Can be included.
  • rat ticking stimulation and human laughter have a common physiological effect on the living body and gene expression is similar.
  • genes that show similar gene expression behavior after Tickling stimulation and laughing stimulation loading.
  • the human homologous gene of the rat positive emotion marker gene can be expected to change the gene expression in human in response to the applied stimulus. Therefore, a new human positive emotion marker and a human positive stimulus evaluation method can be constructed by conducting a positive emotion evaluation experiment focusing on the human homolog gene. Therefore, the rat positive emotion marker is a search method for a new human positive emotion marker gene.
  • the human homolog gene of a rat positive emotion marker is described in the column of “Homology” in the table of rat gene annotation.
  • Example 1 Establishment of positive stimulation loading method to rat by frequency analysis of generated voice 1-1. Examination of rat breeding method Tickling (see 1-2-1 below) for 6 week old male Wistar male rats (Single Housing) and group housing group (Social Housing, 3 animals) ) was enforced. In order to increase social isolation, the single rearing group has a screen between cages. As a result, high-frequency speech near 50 KHz (physiology & Behavior, 72: 167-173, 2001), which is an indicator of rat positive emotion, was confirmed in the single-bred group, but this voice was not confirmed in the group-bred group. .
  • Tickling stimulus loading method Tickling is a tactile stimulus loading method, which mimics the rodent Rough-and-Tumble Play (J. Panksepp: Behavior Genetics 35, 2005). Specifically, wrap around with the right hand from behind the rat, grab the rat to hold the computer mouse (Dorsal Contact), and tickle the rat's posterior muscle with your finger. Continue flipping immediately, pushing the rat to the floor (Pinning Behavior), tickling the belly violently (in this case, there is no resistance if the hip is put back on the floor so that the rat's front leg floats inside), release. These operations are done within 2 seconds and continue for 15 seconds.
  • the rat was transferred to a clean cage for Tickling (with black felt on the inner surface), left for 15 seconds, and then subjected to a 15-second Tickling stimulation load, which was repeated 4 times, and this was designated as 1 Tickling IV Session. After the 1st session, a 1-minute break was inserted, and the 2nd session (2 minutes) was performed.
  • Fig. 1 shows the time chart of Tickling stimulation load.
  • FIGS. 42, 43, and 44 Measurement of waiting time for approach Immediately after starting stimulation, the next day after starting stimulation, the second week of starting stimulation, and the fourth week of starting stimulation, the approaching waiting time immediately after each treatment to a 4-week-old rat is shown in FIGS. 42, 43, and 44, respectively.
  • Tickling stimulation was effective from the first weaning (3 weeks old) to the 6th week, and when it was individually reared until the 8th week, it was not possible to confirm the voice near 50 KHz that was generated in response to the Tickling stimulation. This was thought to be due to changes in mind and body associated with the growth of rats and excessive chronic stress associated with individual breeding.
  • the Tickling stimulus is a discrimination stimulus.
  • the second week of the stimulus start is the most responsive time, and the voice, which is a positive emotion index, is emitted even during the tickling stimulus break (15 seconds), and the approach waiting time is also shortened.
  • the approach waiting time was shortened in the Light Touch group and the control group at the second week, and that the difference between the three groups was not recognized at the fourth week because the rats became accustomed to the surgeon. .
  • the Tickling stimulus is a stimulus that brings positive emotion to the rat.
  • Example 2 Gene expression analysis experiment Gene expression analysis was performed on rats loaded with Tickling stimulus, rats loaded with Light Touch stimulus, and control rats from 4 weeks of 1-2-2 above (individual breeding immediately after weaning [3 weeks of age]). It was.
  • Cardiac blood sampling and perfusion of the heart The heart was opened under ether anesthesia, and cardiac blood sampling (about 1 mL was collected in a PAXgene dedicated blood collection tube) was performed.
  • RNA stabilizing reagent RNAlater manufactured by QIAGEN
  • RNAlater immersion tissue hypothalamic, striatum, and salivary gland only for gene expression analysis
  • RNeasy Buffer RLT RNeasy Buffer RLT
  • Kit A total RNA preparation was prepared according to the protocol. Samples of each group (Tickling, Light Touch, control) were mixed with equal amounts of total RNA for each site.
  • DNA chip analysis was performed as follows. Equal amounts of RNA preparations fluorescently labeled with each cyanine dye are mixed, and a hybridization reaction is carried out at 65 ° C. for 17 hours on a DNA chip (Agilent (approx. 41,012 oligo DNA), WholeWGenome DNA Microarray). The reaction molecules were washed and removed after the hybridization reaction), and the fluorescence intensity derived from each dye was measured with a confocal laser scanner (Agilent, G2565). After background correction and global normalization, genes with a signal intensity of less than 50 (including transcripts) were excluded and the Cy5 / Cy3 ratio was determined.
  • FIG. 46 shows the signal intensity distribution of each probe on the DNA chip when the difference in gene expression between Tickling / control and Tickling / Light Touch is analyzed by the DNA chip method.
  • the vertical axis represents the signal intensity
  • the horizontal axis represents the number of genes indicating the signal intensity.
  • the tissue samples showed similar signal intensity distributions to each other, but the blood sample had a small number of genes indicating high signal intensity. This is because it is influenced by the globin gene derived from reticulocytes.
  • Table 4 shows the “Target Accession” and “UniGene” of the peripheral blood (Bl) gene in which expression was more than 1.5 times in the Tickling group compared to the gene control group in which expression change was observed .
  • Table 5 shows the “Target Accession” and “UniGene” of the peripheral blood (Bl) gene that was found to be expressed in the Tickling group by less than 1 / 1.5-fold compared to the control group.
  • Table 6 shows the “Target Accession” and “UniGene” of peripheral blood (Bl) genes that have been expressed more than 1.5 times in the Tickling group compared to the Light Touch group.
  • Table 7 shows the “Target Accession” and “UniGene” of peripheral blood (Bl) genes that were found to be expressed in the Tickling group by less than 1 / 1.5-fold compared to the Light Touch group.
  • Table 8 shows the “Target Accession” and “UniGene” of the hypothalamic (Hy) gene that was found to express more than 1.5 times in the Tickling group compared to the control group.
  • Table 9 shows the “Target Accession” and “UniGene” of the hypothalamic (Hy) gene that was found to be expressed in the Tickling group by less than 1 / 1.5-fold compared to the control group.
  • Table 10 shows the “Target Accession” and “UniGene” of hypothalamic genes (Hy) that have been expressed more than 1.5 times in the Tickling group compared to the Light Touch group.
  • Table 11 shows “Target Accession” and “UniGene” of hypothalamic (Hy) genes in which expression in the Tickling group was found to be less than 1 / 1.5 fold compared to the Light Touch group.
  • Table 12 shows the “Target Accession” and “UniGene” of the striatum (St) gene that was expressed more than 1.5 times in the Tickling group compared to the control group.
  • Table 13 shows the “Target Accession” and “UniGene” of the striatum (St) gene that was found to be expressed in the Tickling group by less than 1 / 1.5-fold compared to the control group.
  • Table 14 shows the “Target Accession” and “UniGene” of the striatum (St) gene that was expressed more than 1.5 times in the Tickling group as compared to the Light Touch group.
  • Table 15 shows the “Target Accession” and “UniGene” of the striatum (St) gene in which expression of less than 1 / 1.5 times in the Tickling group compared to the Light Touch group was observed.
  • Table 16 shows the “Target Accession” and “UniGene” of salivary gland (Sg) genes that were found to express more than 1.5 times in the Tickling group compared to the control group.
  • Table 17 shows the “Target Accession” and “UniGene” of salivary gland (Sg) genes that were found to be expressed in the Tickling group by less than 1 / 1.5-fold compared to the control group.
  • Table 18 shows the “Target Accession” and “UniGene” of salivary gland (Sg) genes that were expressed more than 1.5 times in the Tickling group compared to the Light Touch group.
  • Table 19 shows the “Target Accession” and “UniGene” of salivary gland (Sg) genes that were found to be expressed in the Tickling group by less than 1 / 1.5-fold compared to the Light Touch group.
  • Table 20 shows the annotations of peripheral blood (Bl) genes that were expressed more than 2.0 times in the Tickling group compared to the control group.
  • Table 21 shows the annotations of peripheral blood (Bl) genes that showed less than 0.5-fold expression in the Tickling group compared to the control group.
  • Table 22 below shows gene annotations of peripheral blood (Bl) in which expression was more than 2.0 times in the Tickling group compared to the Light Touch group.
  • Table 23 below shows annotations of genes in peripheral blood (Bl) that showed expression of 0.5 times less in the Tickling group than in the Light Touch group.
  • Table 24 shows the annotation of the hypothalamic gene (Hy) in which the expression was more than 2.0 times in the Tickling group compared to the control group.
  • Table 25 shows the annotation of the hypothalamic gene (Hy) in which the expression was less than 0.5-fold in the Tickling group compared to the control group.
  • Table 26 shows the hypothalamic (Hy) gene annotations that have been expressed more than 2.0 times in the Tickling group compared to the Light Touch group.
  • Table 27 shows hypothalamic (Hy) gene annotations that were found to be less than 0.5-fold more expressed in the Tickling group than in the Light Touch group.
  • Table 28 shows the annotation of the striatum (St) gene that was found to express more than 2.0 times in the Tickling group compared to the control group.
  • Table 29 shows the annotation of the striatum (St) gene that was found to be less than 0.5-fold expressed in the Tickling group compared to the control group.
  • Table 30 shows the annotations of the striatum (St) genes that were found to express more than 2.0 times in the Tickling group compared to the Light Touch group.
  • Table 31 shows the annotation of the striatum (St) gene that was found to be less than 0.5-fold expressed in the Tickling group compared to the Light Touch group.
  • Table 32 shows annotations of salivary gland (Sg) genes that were expressed more than 2.0 times in the Tickling group compared to the control group.
  • Table 33 shows annotations of salivary gland (Sg) genes that showed expression less than 0.5-fold in the Tickling group compared to the control group.
  • Table 34 shows the annotation of the salivary gland (Sg) gene that was expressed more than 2.0 times in the Tickling group compared to the Light Touch group.
  • Table 35 shows the annotation of salivary gland (Sg) genes that showed expression of less than 0.5-fold in the Tickling group compared to the Light Touch group.
  • Notable genes include Galp, Slc6a3, Down-regulated Chrna3, Slc5a7, Slc18a3, Avp up-regulated in the hypothalamus by Tickling, Cck down-regulated in the striatum, Up-regulate in salivary glands Amy1, Klks3, Ton, Klk12, Ngfg, down-regulated Fetub, Serpina1, etc.
  • FIG. 48 shows a clustering image of probes in which an expression difference of more than 2 times or less than 0.5 times was recognized. The degree of expression difference is expressed in red for genes up-regulated by Tickling (including transcripts) and green for down-regulated genes (including transcripts). (Displayed on the middle right). In addition, genes with low signal intensity (less than 50) that cannot display differential expression (including transcripts) are shown in gray.
  • FIG. 49 and FIG. 50 show clustering images of the 593 probe and the 171 probe in which the expression difference of more than 1.5 times was observed in the salivary gland tissue of the Tickling group as compared with the control group or the Light Touch group.
  • the number of genes (including transcripts) in which expression differences were observed in both comparisons is shown in the Venn diagram of FIG. 51, but there were 48 genes in which expression differences were commonly recognized.
  • clustering images of the 525 probe and the 166 probe that showed an expression difference of less than 1 / 1.5 times in both comparisons are shown in FIGS. 52 and 53, and the Venn diagram in FIG. 56 genes (including transcripts) showed common expression differences.
  • FIG. 55 and FIG. 56 show clustered images of the 157 probe and the 136 probe in which the expression difference of 1.5 times or more was recognized in the hypothalamic tissue of the Tickling group as compared with the control group or the Light Touch group. Further, the number of genes (including transcripts) in which expression differences were observed in both comparisons is shown in the Venn diagram of FIG. 57, but 39 genes in which expression differences were commonly recognized existed. On the other hand, the clustered images of 57 probes and 185 probes that each showed an expression difference of less than 1 / 1.5 times in both comparisons are shown in FIGS. 58 and 59, and the Venn diagram in FIG. There were 11 genes that showed differential expression in common.
  • FIG. 62 to FIG. FIG. 61 shows the relationship between color shading and P value. The darker the color, the higher the significance assigned to that GO Term, and the larger the circle, the more genes assigned.
  • the genes that are up-regulated in the salivary glands noted in these GO analyzes include 4 genes that are functionally assigned to MAP kinase dephosphorylating enzyme of GO ID: 17017 (Table 45), tissue kallikrein activity (GO ID: 4293) ) Assigned to 5 genes (Table 46), 67 genes (Table 47) assigned as extracellular components (GO ID: 5576, 44421, 5615), and lipid metabolism (GO ID: 6629, 44255) as biological pathways 9 genes (Table 48) and 3 genes (Table 49) assigned to dopamine synthesis (GO ID: 42416) were extracted.
  • Down-regulated genes are functionally assigned as 13 genes assigned to actin binding (GO ID; 3779) (Table 50) and components localized outside the cell (GO ID: 5576, 44421, 5615) 66 genes (Table 51) and 2 genes (Table 52) assigned to metabolism of nucleic acid constituent sugars (GO ID: 9225) as biological pathways were extracted.
  • the genes that are up-regulated in the hypothalamus are functionally 2 genes (Table 53) assigned to hormone activity (GO ID: 5184, 5179) and 3 genes assigned to monoamine transporter (GO ID: 8504) (Table 54), 4 genes assigned to receptor binding (GO ID: 5102) (Table 55), 6 genes assigned to eating behavior (GO ID: 7631) as biological pathways (Table 56), biogenic amine synthesis (Table 56) Three genes (Table 57) assigned to GO ID: 42401, 42426, 42416, 9309) and 1 gene (Table 58) assigned to biogenic amine metabolism (GO ID: 9308, 6584, 6576) were extracted. As genes to be down-regulated, 21 genes (Table 59) that were functionally assigned to cation binding (GO ID; 5509, 43169) were extracted.
  • Tickling stimulation was applied to rats over a long period of 4 weeks, so we mainly observed differences in gene expression in each tissue associated with changes in constitution rather than immediate response to stimulation. It is estimated that In other words, in response to temporary stimuli from the outside world, it was predicted that each tissue cooperates through the neuro-endocrine-immune system in order to maintain homeostasis, so that the expression level of the gene is linked. Since the stimuli act on the original functions of each tissue, the results are thought to be expressed as changes in the constitution, and few genes change in conjunction with each other.
  • hypothalamus is the center of life support, compared to other tissues in GO analysis, extracted genes were assigned to more GO Term.
  • An interesting result is that in the analysis between the control group and the Tickling group, genes that are down-regulated by Tickling are assigned to a wide range of terms, whereas genes that are up-regulated are assigned to limited terms. is there.
  • genes that are up-regulated by Tickling are assigned to a wide range of terms, whereas genes that are down-regulated are assigned to a narrow range of terms. .
  • the biological reaction reacts macroscopically with a gene assigned to a non-specific Term, and finely regulated by a gene assigned to a specific Term that undergoes the reverse regulation. It is that you are.
  • the group of genes down-regulated in the Tickling group compared to the Light Touch group belongs to the term related to anion binding, intracellular stimulation transmission via Ca ions in the hypothalamus by Tickling, abnormal excitation by cholinergic nerves Is thought to be non-specifically controlled.
  • the genes that are up-regulated belong to a term having a limited action involved in eating behavior and biogenic amine synthesis / metabolism, and are characterized by working specifically.
  • Galp galanin-like peptide
  • Pomc opiomelanocortin precursor
  • Pmch melanin-concentrating hormone
  • Agouti-related protein Agouti-related protein
  • Hcrt hypocretin
  • Npy neuropeptide Y
  • salivary glands In the analysis of salivary glands, the expression of many genes fluctuates due to Tickling, and it is suggested that saliva components deduced from these results may be a novel stress indicator.
  • Tonin Tonin
  • Klks2 submandibular gland kallikrein
  • Klk12 glandular caliculein
  • Ngfg nerve growth factor
  • serine down-regulated compared to the control group Protease inhibitor (Serpina1) and fetubin
  • genes encoding many structural proteins have been up-regulated, suggesting the possibility that these components can also be used as stress indicators.
  • genes that show differential expression in the Tickling group compared to the Light Touch group are positioned as the first candidate for the healthy responsible gene. Focusing on the up-regulated gene assigned to the specific GO Term rather than the down-regulated gene assigned to the non-specific GO Term, the above 6 genes involved in eating behavior and the dopamine transporter (Slc6a3 ), Tyrosine hydroxylase (Th) is a candidate for this, but as future issues, it is awaited to analyze the relationship between short-term Tickling and expression changes in peripheral cells.
  • Specific saliva components that meet this selection criteria include Tonin (Ton), submandibular gland kallikrein (Klks2), glandular kallikrein (Klk12), nerve growth factor (Ngfg), serine protease inhibitor (Serpina1), Many secreted proteins in saliva, including fetub, are candidates. If the time changes in the expression of these genes after a stress load are examined in advance, it is possible to determine whether the living body is in a stress state or in the recovery phase based on the saliva component concentrations derived from multiple salivary gland genes at a certain point in time. Become. In addition, it is possible to determine the type of stress by pattern analysis of a plurality of saliva component concentrations specified by this experiment, and the application range is wide.
  • Tickling is a stimulus that expresses positive emotions to rats by verifying the analysis of generated speech near 50 KHz, and the genes that change expression by Tickling (4 weeks) are expressed in blood, brain tissue (hypothalamic, hypothalamus, Striatum) and salivary glands.
  • Tickling stimulation Genes for rats loaded with Tickling stimulation and rats loaded with Light Touch stimulation (control) for 2 days after short-term (2 days) Tickling stimulation 6 weeks of age (individually reared immediately after weaning [3 weeks of age]) Expression analysis was performed.
  • tickling stimulation follow the method described in 1-2-2 above. Transfer the rat to a clean cage for Tickling (with black felt on the inner surface), leave it for 15 seconds, and repeat the operation of tickling stimulation load for 15 seconds 4 times. A 1-tickling session was set, and a 1-minute break was placed after the end of the 1-session, and a second Tickling (2 minutes) was performed.
  • Tickling stimulus loading time chart is as shown in FIG.
  • the Light Touch stimulation was applied by gently touching the rat back once every 3 seconds instead of Tickling after moving the rat to the Tickling clean cage (described above) and leaving it for 15 seconds.
  • the stimulus load time was the same as the Tickling stimulus load (FIG. 1).
  • Fig. 73 and Fig. 74 show the frequency component analysis charts of rat-generated speech on the second day of Tickling load and the second day of Light Touch load, respectively.
  • the voice in the vicinity of 50KHz (60KHz) was able to be confirmed at the time of Tickling stimulation, this voice was not able to be confirmed with the Light Touch stimulation as a control, 1-2-5. The result of was reproduced.
  • RNA preparation from oral mucosal scraping cells and brain tissue RNAlater-immersed tissue were homogenized in RNeasy Buffer RLT (TissueLyzer (QIAGEN)
  • RNeasy Buffer RLT TissueLyzer (QIAGEN)
  • the total RNA preparation was prepared according to the following Kit protocol.
  • Oral mucosal scratched cells in each group were mixed with total RNA, and brain tissue was mixed with equal volume.
  • DNA chip analysis was performed as follows. Equal amounts of RNA preparations fluorescently labeled with each cyanine dye are mixed, and a hybridization reaction is carried out at 65 ° C. for 17 hours on a DNA chip (Agilent (approx. 41,012 oligo DNA), WholeWGenome DNA Microarray). The reaction molecules were washed and removed after the hybridization reaction), and the fluorescence intensity derived from each dye was measured with a confocal laser scanner (Agilent, G2565). After background correction and global normalization, genes with a signal intensity of less than 50 (including transcripts) were excluded and the Cy5 / Cy3 ratio was determined.
  • T Tickling L: Light Touch Bl: Blood Bc: Oral mucosal scraping cells Hy: hypothalamus St: Striatum 2-2-4-1.
  • Tickling L Light Touch
  • Bl Blood Bc: Oral mucosal scraping cells
  • the vertical axis shows the signal intensity
  • the horizontal axis shows the number of genes indicating the signal intensity, but the brain tissue samples showed similar signal intensity distributions, but the blood sample and the oral mucosal scratched cell sample showed a high signal intensity. Fewer genes were shown.
  • Table 60 shows “Target Accession” and “UniGene” of peripheral blood (Bl) genes whose expression was observed more than 1.5 times in the Tickling group as compared to the gene Light Touch group in which expression change was observed .
  • Table 61 shows the “Target Accession” and “UniGene” of peripheral blood (Bl) genes whose expression was found to be less than 1 / 1.5-fold in the Tickling group compared to the Light Touch group.
  • Table 62 shows the “Target Accession” and “UniGene” of hypothalamic genes (Hy) in which expression of 1.5 times more was observed in the Tickling group than in the Light Touch group.
  • Table 63 shows the “Target Accession” and “UniGene” of hypothalamic (Hy) genes that were found to be expressed in the Tickling group by less than 1 / 1.5-fold compared to the Light Touch group.
  • Table 64 shows the “Target Accession” and “UniGene” of the striatum (St) gene that was expressed more than 1.5 times in the Tickling group compared to the Light Touch group.
  • Table 65 shows the “Target Accession” and “UniGene” of the striatum (St) gene that was found to be expressed in the Tickling group by less than 1 / 1.5-fold compared to the Light Touch group.
  • Table 66 shows the “Target Accession” and “UniGene” of the genes of oral cells (Bc) that have been expressed more than 1.5 times in the Tickling group as compared to the Light Touch group.
  • Table 67 shows the “Target Accession” and “UniGene” of genes of oral cells (Bc) that were found to be expressed in the Tickling group in an expression of less than 1 / 1.5-fold compared to the Light Touch group.
  • Table 68 shows annotations of genes in peripheral blood (Bl) that were expressed more than 2.0 times in the Tickling group compared to the Light Touch group.
  • Table 69 below shows gene annotations of peripheral blood (Bl) in which expression of less than 0.5-fold was observed in the Tickling group compared to the Light Touch group.
  • Table 70 shows annotations of hypothalamic genes (Hy) that have been expressed more than 2.0 times in the Tickling group compared to the Light Touch group.
  • Table 71 shows hypothalamic (Hy) gene annotations in which expression of less than 0.5-fold was observed in the Tickling group compared to the Light Touch group.
  • Table 72 shows the annotation of the striatum (St) gene that was found to express more than 2.0 times in the Tickling group compared to the Light Touch group.
  • Table 73 shows the annotation of the striatum (St) gene in which the expression was less than 0.5-fold in the Tickling group compared to the Light Touch group.
  • Table 74 shows annotations of genes of oral cells (Bc) that have been expressed more than 2.0 times in the Tickling group compared to the Light Touch group.
  • Table 75 shows gene annotations of oral cells (Bc) in which expression in the Tickling group was less than 0.5 times that of the Light Touch group.
  • Notable genes include Gsta5, Aldh1a1, Yc2, Gsta2, Cdig1l, Selenbp1, Down-regulated Cdkn1c, Gda, Slc6a3 up-regulated in the hypothalamus, lines There are Avp, Neurod2, Vip, Slc17a7, Cck, Sstr1, Cort, Nrn1, Prss, and Down-regulated Ttr that are up-regulated in the striatum.
  • FIG. 77 shows a clustering image of 868 probes in which an expression difference of more than double or 1 / 1.5 times or less was observed
  • FIG. 78 shows a clustering image of 88 probes in which an expression difference of 2 times or more or 0.5 times or less was recognized.
  • the degree of expression difference is expressed in red for genes up-regulated by Tickling (including transcripts) and green for down-regulated genes (including transcripts). (Displayed on the middle right).
  • genes with low signal intensity (less than 50) that cannot display differential expression are shown in gray.
  • genes that change in both directions are prominent, especially in the latter, which contain many genes with a large fluctuation range (more than 2 times or less than 0.5 times). It was.
  • the expression variation in the hypothalamus was slow in both directions, whereas in the striatum, there were many up-regulated genes, and the variation range was more than twice as many.
  • the association between the probe on the DNA chip and the gene was performed based on information provided by Agilent and NCBI Entrez (http://www.ncbi.nlm.nih.gov/). GeneID and GO are mapped using BiNGO (http://www.psb.ugent.be/cbd/papers/BiNGO/), and the hierarchy of GO is Cytoscape (http://www.cytoscape.org/ ). For each GO Term, the frequency of occurrence in the extracted gene group is higher than the frequency of occurrence in all genes on the Agilent Whole Rat Genome Microarray. Tested at ⁇ 0.1. GO Term determined to be significant by the test was defined as GO characteristic of the extracted gene group.
  • Tables 77 to 80 show the GO analysis results of the genes (see Table 76) extracted from each sample.
  • FIGS. 84 to 88 show GO hierarchy diagrams drawn using Cytoscape for each analysis in which a GO Term determined to be significant exists.
  • FIG. 83 shows the relationship between color shading and P value. The darker the color, the higher the significance assigned to that GO Term, and the larger the circle, the more genes assigned.
  • genes with increased expression and genes with decreased expression are both present during long-term Tickling stimulation, and there are 641 and 321 genes whose expression changes (increase or decrease), respectively, and 148 and 32 genes during short-term Tickling stimulation are large.
  • the striatum was characterized in that there were many genes (140 genes) whose expression increased upon short-term Tickling stimulation.
  • genes whose expression changes in the forward direction over a short period of time there are many genes whose expression changes in the reverse direction in blood and striatum, and in short-term Tickling stimulation
  • genes of interest that have the potential to be positioned as health promotion gene candidates include Gsta5, Aldh1a1, Yc2, Gsta2, Cdig1l, and Selenbp1 that have increased expression in oral mucosal scraping cells.
  • the former 5 encode enzymes and the like involved in oral detoxification
  • Selenbp1 is a gene encoding a selenium carrier involved in taste.
  • genes with decreased expression include Cdkn1c, which encodes a CDK inhibitor that regulates the cell cycle in response to cell damage caused by external stimuli, Hpd, which encodes an enzyme that regulates amino acid metabolism, and guanine, an indicator of liver damage.
  • Hpd which encodes an enzyme that regulates amino acid metabolism
  • guanine an indicator of liver damage.
  • Gda that encodes deaminase, but it is unclear at this time what its expression change means.
  • Slc6a3 which encodes a dopamine transporter involved in the transmission of “positive” stimuli, is up-regulated in the same way as long-term Tickling stimulation, and the expression level continues to increase from the very beginning of Tickling stimulation. it was thought.
  • the expression of the Cga gene is increasing, but considering that this gene regulates hormone production related to females, the significance of increasing in male rats requires macroscopic consideration.
  • the most commonly expressed genes are Avp and Vip involved in blood pressure regulation, Neurod2 and Nrn1 involved in nerve growth and differentiation, Slc17a7 involved in neurotransmission, Cck involved in feeding regulation, Alzheimer's disease Sstr1, Cort, which is involved in the improvement of memory ability by lowering the level of ⁇ -amyloid protein related to, and the Prss gene involved in learning and memory were listed as candidates for health promoting genes.
  • Ttr which inhibits fiber formation of ⁇ -amyloid protein, is decreased in the striatum, but it may be related to the function of Ttr main body (carrier function as prealbumin).
  • the GO Term to which genes up-regulated in the striatum are significantly assigned include signal transmission-related, neuropeptide hormone activity, blood pressure regulation-related, dietary behavior-related, biological rhythm-related Term, but health promoting genes What should be noted as a candidate is a gene assigned to the latter four terms.
  • genes assigned to neuropeptide hormone activity include Grp, Adcyap1, and Nppc. Nppc, along with Nppa, Avp, and Vip assigned to blood pressure regulation-related terms, regulates blood circulation functions. Can be a candidate.
  • An adrenergic receptor gene is also assigned to a term related to blood pressure regulation. It is worth noting that Bdnf was extracted in Term related to eating behavior in addition to Cck and Pmch extracted by GO analysis in the long-term Tickling stimulation experiment. In addition, the significant assignment of genes to biological rhythm-related GO Term is also notable as a short-term stimulation effect of Tickling, and the Ptgds gene, which is particularly suggested to be related to sleep, is an animal's ⁇ motivation '' There is a possibility that it may lead to health promotion as a prescription for behavior.
  • a model experimental system was developed to search the central nervous system and other genes (groups) whose expression changes in response to positive stimulation using generalized rats.
  • the stimulation period is initially set to 4 weeks to ensure the stimulation effect. planned.
  • genes whose expression changes in the hypothalamus and salivary glands could be identified, but long-term repeated stimulation was accompanied by changes in the constitution of the rat.
  • Tickling stimulation was given to a 4-week-old rat, the rat uttered a voice in the vicinity of 50 KHz from the first day of stimulation, and the peak of the voice was 2 weeks. It was considered advantageous.
  • short-term (2 days) stimulation as expected, gene expression changes in the striatum were mainly observed.
  • test materials present embodiment the hypothalamus 4 weeks old (weaning immediately [3 weeks old from individual rearing) Load 4 weeks positive stimulus from rat (Tickling 4 individuals, Light Touch 2 individuals), the (2 Total RNA prepared in the same manner as in -2-2) was used.
  • RNA purity and intactness assay The total RNA concentration and A260 / 280 ratio were determined for each individual (before mixing the total RNA preparation).
  • the reverse transcription reaction was performed using High® Capacity® cDNA® Reverse® Transcription® Kit with with RNase® IH (Applied Biosystems). Regarding the reaction solution composition, the total amount of RNA was 5 ⁇ g, and for other reaction solutions, the amount described in the instructions was added to make the total volume 100 ⁇ l. The reverse transcription reaction was performed at the time and temperature described in the product instructions.
  • Example 4 In this example, based on the results of model experiments using rats, positive stimulation (laughing) is loaded on humans, and genes that vary in expression in blood are analyzed. I did research.
  • Subjects Diabetes patients were those whose Fair Control HbA1c value ranged from 6.5 to 7.9%. Recruitment of target subjects was requested by diabetics for diabetic patients, and healthy subjects were recruited through public recruitment of newspapers.
  • Periodontal disease index 1 As an index of periodontal disease, WHO-standard Community Periodontal Index of Treatment Needs (CPITN) method was used (Table 94). A predetermined number of teeth were measured, and the CPITN value showing the maximum value among them was used as the CPITN value for each subject. The group of subjects with CPITN values of 3 or more had a periodontal disease (periodontal disease affected group), the group of subjects with a CPITN value of 2 or less had no periodontal disease (periodontal disease non-affected group) It was.
  • Diabetes index HbA1C value was used as a blood glucose control index of diabetic patients.
  • blood sampled at C1 shown in the timing diagram of (4-1-2) below was used.
  • Subject profile Subject profiles are shown in Table 95.
  • the HbA1C and CPITN values in the diabetic patient group are 7.2 ⁇ 0.6% and 3.4 ⁇ 0.7, respectively, and the healthy group is 5.1 ⁇ 0.3% and 2.7 ⁇ 0.6.
  • the CPITN value in the diabetic patient group is compared with that in the healthy group. Significantly higher (p ⁇ 0.05). This resulted in the verification of the clinical trend that diabetics are more susceptible to periodontal disease.
  • Table 96 shows the analysis results of the non-diabetic and periodontal disease non-affected group (hereinafter referred to as group A) .
  • group A non-diabetic and periodontal disease non-affected group
  • Table 97 shows the analysis results of a group of non-diabetic and periodontal disease patients (hereinafter referred to as group B) .
  • group B non-diabetic and periodontal disease patients
  • Table 98 shows the analysis analysis results of the group with diabetes and non-periodontal disease (hereinafter referred to as group C) .
  • group C The blood glucose level for 2 hours after meal increased to 145.5 ⁇ 12.0 mg / dL in the lecture and 94.0 ⁇ 1.4 mg / dL in the conte, and the increase was remarkably suppressed to 51.5 mg / dL by the comte appreciation.
  • the amylase activity in saliva which is an index of stress, decreased after the lecture and decreased significantly after the contest.
  • the white blood cell count did not show any difference before and after the lecture, but it increased after the contest.
  • the neutrophil ratio which is a stress index, did not show a difference before and after the lecture, but decreased after contesting.
  • the C-reactive protein that increased during infection and inflammation did not show any difference between before and after the lecture, but decreased after contest.
  • Table 99 shows the analysis results of the group with diabetes and periodontal disease (hereinafter referred to as group D) .
  • group D The blood glucose level for 2 hours after meal increased to 147.0 ⁇ 55.3 mg / dL in the lecture and 135.9 ⁇ 58.0 mg / dL in the conte, and the increase was suppressed by 11.1 mg / dL by the comte appreciation.
  • the TMD value total emotional disorder
  • s-IgA secretory immunoglobulin A in saliva
  • the C-reactive protein that increases during infection and inflammation decreased both after the lecture and after the contest.
  • the 2-hour postprandial blood glucose level increased to 146.85 ⁇ 51.8mg / dL in lectures and 130.9 ⁇ 56.0 / mg / dL in comtes, and the increase was suppressed by 15.9mg / dL by comte appreciation.
  • the TMD values total emotional disorder calculated from the POMS 6 emotional index did not show a significant difference before and after the lecture, but decreased to a significant (p ⁇ 0.05) after watching the contest.
  • the redox potential in saliva decreased after the lecture and after the contest.
  • the leukocyte count increased significantly (p ⁇ 0.05) after the lecture and after the contest.
  • TMD levels total emotional disorder
  • saliva increased
  • immune function was activated by increasing s-IgA.
  • Health maintenance and enhancement by appreciation was recognized.
  • TMD values did not show a significant difference before and after the lecture, but decreased to a significant (p ⁇ 0.05) after control.
  • the redox potential in saliva decreased significantly (p ⁇ 0.05) both after the lecture and after the contest.
  • the leukocyte count increased significantly (p ⁇ 0.05) both after the lecture and after the contest.
  • s-IgA increased after the lecture and after the contest.
  • NK cell activity increased after lecture and after contest.
  • C-reactive protein decreased after lecture and after contest.
  • TMD values did not show a significant difference before and after the lecture, but decreased after contest.
  • s-IgA decreased after the lecture, but increased after the contest.
  • Salivary amylase activity decreased after control.
  • the redox potential in saliva decreased after the lecture and after the contest.
  • the white blood cell count was not significantly different between before and after the lecture, but increased significantly (p ⁇ 0.05) after contest.
  • C-reactive protein was not significantly different between before and after the lecture, but decreased significantly after control (p ⁇ 0.05).
  • TMD levels total affective disorder
  • salivary amylase activity as a stress index decreased
  • s-IgA increased, resulting in immune function.
  • 4-1-4-1 experimental method 4-1-4-1-1. Collection of sample to be analyzed Collection of blood Samples were collected in a PAXgene blood collection tube, left at room temperature for 4 hours and at 4 ° C overnight, and then stored at -30 ° C.
  • RNA preparation and assay from analysis sample 1 Preparation of total RNA from blood sample Total RNA preparation was prepared using PAXgene Blood RNA Kit (manufactured by QIAGEN).
  • DNA chip analysis method Using the total RNA sample obtained from the blood sample of (4-1-4-1-1) above as the starting material, cDNA synthesis was performed using Oligo (dT) 24 with T7RNA Polymerase Promoter sequence as a primer. Furthermore, mRNA was amplified with T7 RNA polymerase. By using Cyanine [Cy] 3-CTP (before or before lecture) or Cy5-CTP (after or after lecture) as the substrate for the amplification reaction at this time, fluorescently labeled cRNA was obtained for DNA chip analysis. Provided. DNA chip analysis was performed as follows.
  • RNA samples fluorescently labeled with each cyanine dye are mixed, and hybridization reaction is performed at 65 ° C for 17 hours on a DNA chip (Agilent (approximately 41,000 oligo DNAs), Whole Human Genome DNA Microarray 4 ⁇ 44K).
  • the unreacted molecules were washed and removed after the hybridization reaction, and the fluorescence intensity derived from each dye was measured with a confocal laser scanner (Agilent, G2565). After background correction and global normalization, the Cy5 / Cy3 ratio was determined as a change in gene expression.
  • Samples analyzed were those with high and low periodontal disease score (CPITN value) in the diabetes affected group (upper 3 and lower 2), periodontal disease score (CPITN value) in the non-diabetic group
  • the subjects were 11 subjects with a high and low subjects (top 3 and low 3).
  • Three types of cDNA reaction solution subject sample: cDNA sample synthesized by reverse transcription reaction from total RNA of the subject sample for standard quantification using a standard curve. Standard: After laughter of subject number 114 for creating a standard curve A dilution series of the cDNA sample “114 P2-3” of (Negative Control: a sample to which water was added instead of cDNA).
  • a PCR reaction solution was prepared by adding a TaqMan probe and primer set reaction solution and a cDNA reaction solution to TaqMan Universal PCR Master Mix. Three wells were used for each sample, and the reliability of data analysis was improved by taking an average (replicate) of 2 wells or more in which the fluorescence signal could be observed.
  • PCR conditions are 2 minutes at 50 ° C (activation of UNG [AmpErase uracil-N-glycosylase] to prevent re-PCR amplification from PCR products containing dU), 10 minutes at 95 ° C (AmpliTaq Gold After the pre-reaction of (registered trademark DNA polymerase activation), repeat the cycle of 15 seconds at 95 ° C (denaturation of cDNA) and 1 minute at 60 ° C (primer and probe annealing, extension reaction) 40 times. I went.
  • dilution series total RNA equivalent: 50 ng, 10 ng, 2 ng, 0.4 ng, 0.08 ng, 0.016 ng, 0.0032 ng
  • a line was created by the following method to obtain a quantitative value for each sample.
  • a calibration curve is created by plotting the fluorescence signal ( ⁇ Rn) corresponding to the amplified PCR product against the number of cycles of the PCR reaction, and the baseline is in the region where the growth curve is exponentially amplified.
  • Threshold Cycle (CT) value
  • CT Threshold Cycle
  • Table 105 shows the results showing the number of genes whose expression changes were 1.5 times or more or 1 / 1.5 times or less in gene analysis 2, 4 to 10 in which expression changes were observed.
  • FIG. 123 shows a histogram of genes in which expression changes of 1.5 times or more or 1 / 1.5 times or less are observed in histograms and Venn diagram analysis 2, 4 to 10.
  • the vertical axis represents the Log2 value of the expression change fold
  • the horizontal axis represents the sample name.
  • analysis 6 before and after laughing in group B
  • analysis 8 before and after laughing in group D
  • analysis 10 before and after laughing in group B + D
  • the relative difference in expression change for each corresponding analysis is shown in FIG.
  • the vertical axis represents the log2 value of the expression change fold
  • the horizontal axis represents the analysis combination.
  • the difference in the expression change of each gene is large before and after the lecture and before and after laughing, but it is relatively small in the diabetic group. Moreover, the difference in expression change was large between the A + C group and the B + D group, and the A group and the B group were also large. Moreover, in the comparison between the C group and the D group, the change width of the gene whose expression was decreased was relatively large.
  • FIG. 125 shows a Venn diagram of genes in which an expression change of 1.5 times or more was observed in the A + B group and the C + D group. There were 928 up-regulated genes in the A + B group alone, 163 in the C + D group alone, and 41 in both groups.
  • FIG. 126 shows a Venn diagram of genes in which expression changes of 1 / 1.5 times or less were observed. There were 804 down-regulated genes in the A + B group alone, 353 in the C + D group alone, and 63 in both groups.
  • FIG. 127 shows a Venn diagram of genes in which expression changes of 1.5 times or more were observed in the A group and the B group.
  • FIG. 128 shows a Venn diagram of genes in which an expression change of 1 / 1.5 times or less was observed.
  • FIG. 129 shows a Venn diagram of genes that showed an expression change of 1.5 times or more in the A group and the C group.
  • FIG. 130 shows a Venn diagram of genes in which expression changes of 1 / 1.5-fold or less were observed.
  • FIG. 128 shows a Venn diagram of genes in which an expression change of 1 / 1.5 times or less was observed.
  • FIG. 128 shows a Venn diagram of genes in which an expression change of 1 / 1.5 times or less was observed.
  • FIG. 129 shows a Venn diagram of genes that showed an expression change of 1.5 times or
  • FIG. 131 shows a Venn diagram of genes in which expression changes of 1.5 times or more were observed in the B group and the D group. There were 819 up-regulated genes in group B alone, 531 in group D alone, and 103 in both groups.
  • FIG. 132 shows a Venn diagram of genes in which an expression change of 1 / 1.5 times or less was observed. There were 771 down-regulated genes in group B alone, 881 in group D alone, and 256 in both groups.
  • FIG. 133 shows a Venn diagram of genes in which expression changes of 1.5 times or more were observed in the C group and the D group. There were 129 genes up-regulated in group C alone, 589 in group D alone, and 45 in both groups.
  • FIG. 134 shows a Venn diagram of genes in which an expression change of 1 / 1.5 times or less was observed. There were 481 down-regulated genes in group C alone, 984 in group D alone, and 153 in both groups.
  • FIG. 135 shows a Venn diagram of genes in which an expression change of 1.5 times or more was observed in the A + C group and the B + D group. There were 361 genes that were up-regulated only in the A + C group, 2,538 in the B + D group alone, and 54 in both groups.
  • FIG. 136 shows a Venn diagram of genes in which an expression change of 1 / 1.5-fold or less was observed. There were 103 down-regulated genes in the A + C group alone, 1,944 in the B + D group alone, and 54 in both groups.
  • the probe on the DNA chip is associated with the GO term based on the probe information provided by Agilent and the GeneID information of NCBI Entrez (http://www.ncbi.nlm.nih.gov/).
  • the association between GO and GO was done using BiNGO (http://www.psb.ugent.be/cbd/papers/BiNGO/).
  • the appearance frequency of the gene assigned to each GO term was determined by Fisher's exact test to determine whether the extracted gene was significantly larger than the DNA chip-mounted gene, and the test was performed with a significance level of p-Value ⁇ 0.05.
  • a GO term determined to be significant by the test was defined as a GO characteristic of the extracted gene group. Tables 108 to 123 show GO terms determined to be significant when p-Value ⁇ 0.01.
  • Term that is attracting attention in biological pathways is related to antigen receptor signaling pathway (GO ID: 50854, 50857) in the analysis of genes up-regulated by laughter in non-diabetic group (A + B group) 3 genes assigned (Table 124), 5 genes assigned to glucose transport (GO ID: 15758) (Table 125), 5 assigned to lipoprotein metabolism (GO ID: 42157) in analysis of down-regulated genes There were 4 genes (Table 127) assigned to genes (Table 126), pH adjustment (GO ID: 6885). In the diabetic group (C + D group), in the analysis of genes down-regulated by laughter, there were 8 genes (Table 128) assigned to programmed cell death control (GO ID: 12502).
  • group A non-diabetic and periodontal disease non-affected group
  • 1 gene assigned to anti-cell death GO ID: 0006916
  • gene set 1 No. 3 1 gene assigned to IgE receptor activity (GO ID: 0019767)
  • gene assigned to response defense GO: 0006952 (defense response)
  • gene set 1 No. 5 1 gene assigned to response defense GO: 0006952 (defense response)
  • serotonin transport related 1 gene (gene set 1 No. 7) assigned to GO222ID: 6837 or 15222 or 5335), 1 gene (gene set 1 No.
  • group B non-diabetic and periodontal disease group
  • 6 genes (Table 129) assigned to cytokine / chemokine signaling (GO ID: 19221), lipo 2 genes assigned to protein catabolism (GO ID: 42159, 45192) (Table 130), 10 genes assigned to lipid transport (GO ID: 6869) (Table 131), insulin receptor signaling related (GO ID: 8286 or There were 6 genes (Table 132) assigned to 46627) and 3 genes (Table 133) assigned to interleukin 6 biosynthesis related (GO ID: 45408, 42226).
  • the up-regulated gene is the gene PROK2 (gene set 1 No. 3) assigned to anti-cell death (GO ID: 0006916) in GO analysis, and IgE receptor activity (GO ID: 0019767) Assigned to the gene FCER1A (Gene set 1 No. 4), the gene CD69 (Gene set 1 No. 5) assigned to the response defense GO: 0006952 (defense response), and the serotonin transport related (GO ID: 6837 or 15222 or 5335) Gene SLC6A4 (gene set 1 No. 7), gene GGCX (gene set 1 No. 8) assigned to ⁇ -glutamate carboxylase activity (GOID: 0008488), gene HBA2 (gene set 1 No.
  • genes that are down-regulated include cancer-related genes JUN, TP53, RAB5B, RAB8A, RAB12, RAB20, RAB30 (gene set 1 No. 11-17), genes assigned to innate immune responses (GOID: 0045087) VISA, CD55 (gene set 1 No. 18-19), gene IGHG1 (gene set 1 No. 20) assigned to immune response (GOID: 0006955), gene CD84 (gene set 1 No. 1) assigned to response defense (GO: 0006952) 22), gene NFAT5 (gene set 1 No. 25) assigned to transcription factors (GOIID: 0003700), and gene TNFAIP3 (gene set 1 No. 27) assigned to cell death (GOID: 0006915).
  • group B in the analysis of genes that are down-regulated by laughter, the genes SOCS1, CCR1, CSF2RB, STAT5A, which are assigned to cytokine chemokine signaling (GO ID: 19221), STAT3, TNFRSF1B (Table 129), genes assigned to lipoprotein catabolism (GO ID: 42159, 45192), SNX17, SCARF1 (Table 130), lipid transport assigned to lipid transport (GO ID: 6869) (GO ID: 6869) LDLR, STARD3, CLN8, APOL1, ATP10A, PSAP, OSBP2, SLC27A1, APOL6 (Table 131), SOCS1, STXBP4, SOCS3, AKT1, assigned to insulin receptor signaling related (GO ID: 8286 or 46627)
  • CEBPB and NLRP12 Table 133 assigned to MLLT7, INSIGF (Table 132), and interleukin 6 biosynthesis related (GO ID: 45408, 42226).
  • the three genes PTPRC, PAWR, TRAT1 (Table 124), Down-regulate assigned to antigen receptor signaling pathway-related (GO ID: 50854, 50857) 5 genes STXBP4, SLC2A5, SLC2A3, SORBS1, AKT1 (Table 126), genes APOL6, ATG10 assigned to lipoprotein metabolism (GO ID: 42157) assigned to glucose transport (GO ID: 15758) , SNX17, PIGS, FNTB (Table 126), and genes SLC9A5, LOC133308, CLN5, and CLN3 (Table 127) assigned to pH adjustment (GO ID: 6885).
  • Periodontal disease is said to be the sixth complication of diabetes, and the increased vulnerability of peripheral blood vessels due to diabetes causes the onset and exacerbation of periodontal disease, and tumor necrosis factor (TNF) in periodontal disease, which is a chronic inflammatory disease. It has been reported that production of cytokines such as - ⁇ ) increases insulin resistance and becomes an exacerbation factor of diabetes. Furthermore, the correlation between diabetes and periodontal disease has been known a lot in recent years.
  • polymorphonuclear leukocyte function decreases, Abnormal collagen metabolism in periodontal connective tissue, inflammation due to accumulation of advanced glycation end products (AGE) in gingival tissues, delayed wound healing, etc. are observed.
  • AGE advanced glycation end products
  • genes contained in these GO terms include HLA-A, HLA-B, HLA-C, HLA-E, HLA-H, CD1D, CD74, VISA, CASP8, CFLAR, APOL, LTBR, CTSA, CTSB, LOC391803,
  • genes related to immunity / inflammation / self-protection such as KRTAP3-3 and SESN2
  • genes related to lipid secretion such as STAT5A and STAT5B
  • genes related to lipid metabolism such as FFAR2.
  • group D diabetes and periodontal disease group
  • group D virus response protection in GO analysis
  • interferon biosynthesis related GO ID: 45356
  • TLR7, TLR8 and BCL2 assigned to interleukin 8 biosynthesis
  • TLR7, TLR8 and BCL2 assigned to interleukin 8 biosynthesis
  • PPARA PPARA
  • NR5A1, C1QTNF I-kappaB assigned to metabolic control
  • ERC1 assigned to phosphate group transfer
  • Group B non-diabetic / periodontal diseased group
  • LDCS, STARD3, CLN8, APOL1, ATP10A, PSAP, SLC27A1, APOL6, SOCS1, STXBP4, SOCS3, AKT1, MLLT7, INSIGF assigned to insulin receptor signaling related GO ID: 8286 or 46627) .
  • Table 156 to 158 show genes that are notable in terms of function among the genes whose variation in gene expression was recognized by laughter in analysis 4 above, and the top 15 genes that have particularly large variation in expression. Is shown as a table for gene set 6.
  • Table 165 to 171 show the genes that should be noted in terms of function and the top 15 genes that have the most significant fluctuations in expression among the genes whose fluctuations in gene expression were recognized by laughter in Analysis 6 above. Is shown as a table of gene set 2.
  • Table 173 to 177 show the genes that should be noted in terms of function and the top 15 genes with the most significant fluctuation in expression among the genes whose fluctuations in gene expression were recognized by laughter in Analysis 7 above. Is shown as a table of gene set 3.
  • Table 179 to 184 show the genes that should be noted in terms of function, and the top 15 genes that have the most significant fluctuations in expression among the genes whose fluctuations in gene expression were recognized by laughter in Analysis 8 above. Is shown as a table of gene set 4.
  • Tables 200 and 201 show 40 types of annotations of human genes (human common genes) whose expression changes in the same direction as when rats were loaded with a short-term positive stimulus (Tickling stimulus), and Table 202 shows a summary thereof.
  • Tables 203 and 204 show annotations of 40 types of short-term positive stimulation (tickling stimulation) loaded rat genes (rat common genes) whose expression was changed in the same direction as the genes whose expression was changed by human laughter, and Table 205 Here is a summary.

Abstract

Disclosed is a system for evaluating positive affect or the degree of health, which relies on a gene marker that enables the evaluation of positive affect or the degree of health in a subject conveniently and with high reliability.  Specifically disclosed is a method for evaluating positive affect or the degree of health in a rat or a human body, which comprises the following steps (a) and (b): (a) measuring the level of expression of a rat positive affect marker gene or a human positive affect marker gene in the rat or the human body; and (b) evaluating the positive affect or the degree of health in the rat or the human body based on the level of expression measured in step (a).

Description

陽性感情マーカー遺伝子及びその利用方法Positive emotion marker gene and method of use thereof
　本発明は、陽性感情の客観的な指標となる遺伝子マーカー及びその利用方法に関する。 The present invention relates to a genetic marker that is an objective index of positive emotion and a method of using the same.
　社会の複雑化に伴い、ストレス及びそれに起因する人体への健康障害が社会問題として提起されつつある。快・不快の心理状態の推定方法としては、心理テスト、心拍・体温解析、脳波解析やfMRI、脳内情報伝達物質の推定などが開発されているが、実施難易度と得られる情報の信頼性はトレードオフの関係にあり、簡便かつ信頼性の高い方法はない。 As society becomes more complex, stress and health problems resulting from it are being raised as social problems. Psychological tests, heart rate and body temperature analysis, electroencephalogram analysis and fMRI, estimation of information transmitters in the brain, etc. have been developed as methods for estimating pleasant and unpleasant psychological states. Are in a trade-off relationship, and there is no simple and reliable method.
　近年、ゲノム情報の解明や遺伝子診断技術の進歩により、ストレスのように人が感じている心理状態や、発癌、生活習慣病などの身体状態を、遺伝子の発現状態から推定することが可能になってきている。しかし、これらの推定技術は、全て陰性の心理状態・身体状態に関するものであり、陽性の心理・身体状態、即ち、快感情やリラックスといった心理状態、身体活性化や健康といった身体状態に関する推定技術はほとんどない。笑いがNK細胞の活性化（特許文献１）や糖尿病患者の食後血糖値を抑えることは知られているが（非特許文献１）、NK細胞や血糖値だけで個体レベルでの評価を実施することはできない。 In recent years, elucidation of genomic information and advances in genetic diagnosis technology have made it possible to estimate the psychological state that humans feel like stress, and the physical conditions such as carcinogenesis and lifestyle-related diseases from the gene expression state. It is coming. However, these estimation techniques are all related to negative psychological states / physical states, and positive psychological / physical states, that is, psychological states such as pleasant feelings and relaxation, estimation techniques related to physical states such as physical activation and health are rare. It is known that laughter activates NK cells (Patent Document 1) and suppresses postprandial blood glucose levels in diabetic patients (Non-Patent Document 1), but assessment is performed at the individual level using only NK cells and blood glucose levels. It is not possible.
　陰性の心理状態や身体状態は、モデル動物などを用いた実験系の確立が容易であり、対照との比較により、その遺伝子発現の差の解析も比較的容易である。一方、陽性の心理状態や身体状態は、個人の感覚的なものであるため、定義づけが困難であり、モデル動物を用いた実験系も長く開発されなかった。したがって、陽性の心理状態にあると、健康になれるといった漠然とした思いはあっても、これまで研究の対象にはならなかった。
特開平8-266632 Journal of Psychosomatic Research 62 (2007) 703-706: Laughter regulates gene expression in patient with type 2 dabetes For negative psychological and physical conditions, it is easy to establish an experimental system using model animals, etc., and by comparison with controls, it is relatively easy to analyze the difference in gene expression. On the other hand, positive psychological states and physical states are difficult to define because they are individual sensations, and experimental systems using model animals have not been developed for a long time. Therefore, even if there is a vague feeling that you can be healthy if you are in a positive psychological state, it has not been the subject of research so far.
JP-A-8-266632 Journal of Psychosomatic Research 62 (2007) 703-706: Laughter regulates gene expression in patient with
　このように、現在までに陽性の心理状態（以下、「陽性感情」とも称する）を遺伝子発現レベルで客観的に推定する試みはなされていない。 Thus, no attempt has been made so far to objectively estimate a positive psychological state (hereinafter also referred to as “positive emotion”) at the gene expression level.
　いったん陽性感情の客観的な指標たる遺伝子マーカーを見出すことができれば、かかる指標に基づき、所与の刺激が被験体を陽性感情又は健康増進に導くものであるか否かを評価することができ、快適さを追求する製品開発の分野での利用が期待できる。 Once a genetic marker that is an objective indicator of positive emotions can be found, based on such indicators, it can be assessed whether a given stimulus is one that leads the subject to positive emotions or health promotion, It can be used in the field of product development that pursues comfort.
　したがって、本発明は、被験体の陽性感情又は健康度を簡便且つ高い信頼性をもって評価することができる遺伝子マーカーを提供すること、及び該遺伝子マーカーに基づく陽性感情又は健康度の評価系を提供することを目的とする。 Therefore, the present invention provides a genetic marker that can easily and reliably evaluate the positive emotion or health level of a subject, and provides a positive emotion or health evaluation system based on the genetic marker. For the purpose.
　ラットにおいて、50KHz近傍の超音波は、遊び行動、交尾及び報酬事象の際に発せられる感情指標であり、22KHz近傍の超音波は嫌悪事象、薬物離脱の際に発せられる感情指標であることが報告されている（J. Burgdorf et al., Breeding for 50-kHz Positive Affective Vocalization in Rats. Behavior Genetics, Vol.35 No.1, January 2005）。そこで、本発明者らは、50KHz近傍の発生音声を指標とした陽性感情下にあるラットにおいて遺伝子発現量の網羅的解析を行ったところ、かかる陽性感情ラットで発現量が有意に増大している遺伝子(以下、「ラット陽性感情マーカー遺伝子」とも称する)が存在することを見出した。なお、本明細書で使用する50KHz近傍とは、45～65KHz、好ましくは45～55KHzの音域を指す。 In rats, it is reported that ultrasound near 50 KHz is an emotional index emitted during play behavior, mating and reward events, and ultrasound near 22 KHz is an emotional index emitted during aversive events and drug withdrawal. (J. Burgdorf et al., Breeding for 50-kHz Positive Affective Vocalization in Rats. Behavior Genetics, Vol.35 No.1, January 2005). Therefore, the present inventors conducted a comprehensive analysis of gene expression levels in rats under positive emotion using the generated speech near 50 KHz as an index, and the expression level was significantly increased in such positive emotion rats. It was found that a gene (hereinafter also referred to as “rat positive emotion marker gene”) exists. As used herein, the vicinity of 50 KHz refers to a sound range of 45 to 65 KHz, preferably 45 to 55 KHz.
　さらに本発明者らは、笑いを誘発させた陽性感情下にあるヒトにおいて遺伝子発現量の網羅的解析を行ったところ、かかる陽性感情のヒトで発現量が有意に増大している遺伝子(以下、「ヒト陽性感情マーカー遺伝子」とも称する)が存在することを見出した。 Furthermore, the present inventors conducted a comprehensive analysis of gene expression levels in humans under positive emotions that induced laughter, and found that genes whose expression levels were significantly increased in humans with such positive emotions (hereinafter, It was found that there is also a “human positive emotion marker gene”.
　本発明は上記知見を基に完成されたものであり、以下の特徴を包含するものである。 The present invention has been completed based on the above findings, and includes the following features.
　すなわち、本発明は、ラット又はヒトに評価対象である刺激を負荷する第1のステップと、少なくとも1種のラット陽性感情マーカー遺伝子又はヒト陽性感情マーカー遺伝子の発現量を生体由来試料において測定する第2のステップと、測定した前記遺伝子の発現量に基づいて、対象刺激が陽性刺激であるか否かを評価する第3のステップとを含む、所与の刺激がラット又はヒトに対する陽性刺激であるか否かを評価する方法に関する。 That is, the present invention is a first step of loading a rat or human with a stimulus to be evaluated, and a first step of measuring the expression level of at least one rat positive emotion marker gene or human positive emotion marker gene in a biological sample. The given stimulus is a positive stimulus for a rat or a human, including the second step and a third step of evaluating whether the target stimulus is a positive stimulus based on the measured expression level of the gene. It relates to a method for evaluating whether or not.
　上記方法では、第2のステップにおいて、測定対象の遺伝子の塩基配列の少なくとも一部を含む核酸を固定化した支持体を用いて、当該遺伝子の発現量を測定することができる。 In the above method, in the second step, the expression level of the gene can be measured using a support on which a nucleic acid containing at least a part of the base sequence of the gene to be measured is immobilized.
　本発明はまた、ラット陽性感情マーカー遺伝子又はヒト陽性感情マーカー遺伝子の塩基配列の少なくとも一部を含む核酸からなる群から選ばれる少なくとも1種の核酸、又はラット陽性感情マーカー遺伝子又はヒト陽性感情マーカー遺伝子によってコードされるタンパク質に対して特異的に結合する分子からなる群から選択される少なくとも1種の分子を含む、ラット又はヒトの陽性感情、又はヒトの健康度を評価するための検査キットに関する。 The present invention also provides at least one nucleic acid selected from the group consisting of nucleic acids comprising at least part of the base sequence of a rat positive emotion marker gene or a human positive emotion marker gene, or a rat positive emotion marker gene or a human positive emotion marker gene The present invention relates to a test kit for evaluating a positive emotion of a rat or a human or a human health level, comprising at least one molecule selected from the group consisting of molecules that specifically bind to a protein encoded by
　本発明に係る検査キットにおいて、前記分子は抗体、リガンドタンパク質又は受容体タンパク質であることができる。 In the test kit according to the present invention, the molecule may be an antibody, a ligand protein, or a receptor protein.
　本発明に係る検査キットにおいて、前記核酸又は分子は支持体に固定されていてもよい。 In the test kit according to the present invention, the nucleic acid or molecule may be fixed to a support.
　本発明によれば、陽性感情マーカー遺伝子を指標とするラット又はヒトの陽性感情又は健康度の評価系、及びラット又はヒトにおいて陽性感情評価を行うための検査キット、及びヒトにおいて健康度の評価を行うための検査キットが提供される。 According to the present invention, a rat or human positive emotion or health evaluation system using a positive emotion marker gene as an index, a test kit for performing positive emotion evaluation in a rat or human, and a human health evaluation A test kit for performing is provided.
図1は、実施例で採用したTickling刺激負荷時程図を示す。FIG. 1 shows a time chart of the Tickling stimulus load employed in the example. 図2は、刺激開始日におけるTickling(+)4週令ラット発生音声の周波数成分解析図である。FIG. 2 is a frequency component analysis diagram of Tickling (+) 4-week-old rat-generated speech on the stimulation start date. 図3は、刺激開始日におけるTickling(-)4週令ラット発生音声の周波数成分解析図である。FIG. 3 is a frequency component analysis diagram of Tickling (−) 4-week-old rat-generated speech on the stimulation start date. 図4は、刺激開始日におけるLight Touch(+)4週令ラット発生音声の周波数成分解析図である。FIG. 4 is a frequency component analysis diagram of Light Touch (+) 4-week-old rat-generated speech on the stimulation start date. 図5は、刺激開始日におけるLight Touch(-)4週令ラット発生音声の周波数成分解析図である。FIG. 5 is a frequency component analysis diagram of Light Touch (−) 4-week-old rat-generated speech on the stimulation start date. 図6は、刺激開始日における対照(+)4週令ラット発生音声の周波数成分解析図である。FIG. 6 is a frequency component analysis diagram of control (+) 4-week-old rat-generated speech on the stimulation start date. 図7は、刺激開始日における対照(-)4週令ラット発生音声の周波数成分解析図である。FIG. 7 is a frequency component analysis diagram of control (−) 4-week-old rat-generated speech on the stimulation start date. 図8は、刺激開始日におけるTickling(+)6週令ラット発生音声の周波数成分解析図である。FIG. 8 is a frequency component analysis diagram of Tickling (+) 6-week-old rat-generated speech on the stimulation start date. 図9は、刺激開始日におけるTickling(-)6週令ラット発生音声の周波数成分解析図である。FIG. 9 is a frequency component analysis diagram of Tickling (−) 6-week-old rat-generated speech on the stimulation start date. 図10は、刺激開始日におけるTickling(+)8週令ラット発生音声の周波数成分解析図である。FIG. 10 is a frequency component analysis diagram of Tickling (+) 8-week-old rat-generated speech on the stimulation start date. 図11は、刺激開始日におけるTickling(-)8週令ラット発生音声の周波数成分解析図である。FIG. 11 is a frequency component analysis diagram of Tickling (−) 8-week-old rat-generated speech on the stimulation start date. 図12は、刺激開始翌日におけるTickling(+)4週令ラット発生音声の周波数成分解析図である。FIG. 12 is a frequency component analysis diagram of Tickling (+) 4-week-old rat-generated speech on the day following the start of stimulation. 図13は、刺激開始翌日におけるTickling(-)4週令ラット発生音声の周波数成分解析図である。FIG. 13 is a frequency component analysis diagram of Tickling (−) 4-week-old rat-generated speech the day after the start of stimulation. 図14は、刺激開始翌日におけるLight Touch(+)4週令ラット発生音声の周波数成分解析図である。FIG. 14 is a frequency component analysis diagram of Light Touch (+) 4-week-old rat-generated speech on the day after the start of stimulation. 図15は、刺激開始翌日におけるLight Touch(-)4週令ラット発生音声の周波数成分解析図である。FIG. 15 is a frequency component analysis diagram of Light Touch (−) 4-week-old rat-generated speech the day after the start of stimulation. 図16は、刺激開始翌日における対照(+)4週令ラット発生音声の周波数成分解析図である。FIG. 16 is a frequency component analysis diagram of control (+) 4-week-old rat-generated speech on the day following the start of stimulation. 図17は、刺激開始翌日における対照(-)4週令ラット発生音声の周波数成分解析図である。FIG. 17 is a frequency component analysis diagram of control (−) 4-week-old rat-generated speech on the day following the start of stimulation. 図18は、刺激開始翌日におけるTickling(+)6週令ラット発生音声の周波数成分解析図である。FIG. 18 is a frequency component analysis diagram of Tickling (+) 6-week-old rat-generated speech on the day following the start of stimulation. 図19は、刺激開始翌日におけるTickling(-)6週令ラット発生音声の周波数成分解析図である。FIG. 19 is a frequency component analysis diagram of Tickling (−) 6-week-old rat-generated speech the day after the start of stimulation. 図20は、刺激開始翌日におけるTickling(+)8週令ラット発生音声の周波数成分解析図である。FIG. 20 is a frequency component analysis diagram of Tickling (+) 8-week-old rat-generated speech the day after the start of stimulation. 図21は、刺激開始翌日におけるTickling(-)8週令ラット発生音声の周波数成分解析図である。FIG. 21 is a frequency component analysis diagram of Tickling (−) 8-week-old rat-generated speech on the day following the start of stimulation. 図22は、刺激開始2週目におけるTickling(+)4週令ラット発生音声の周波数成分解析図である。FIG. 22 is a frequency component analysis diagram of Tickling (+) 4-week-old rat-generated speech in the second week after the start of stimulation. 図23は、刺激開始2週目におけるTickling(-)4週令ラット発生音声の周波数成分解析図である。FIG. 23 is a frequency component analysis diagram of Tickling (−) 4-week-old rat-generated speech in the second week after the start of stimulation. 図24は、刺激開始2週目におけるLight Touch(+)4週令ラット発生音声の周波数成分解析図である。FIG. 24 is a frequency component analysis diagram of Light Touch (+) 4-week-old rat-generated speech in the second week after the start of stimulation. 図25は、刺激開始2週目におけるLight Touch(-)4週令ラット発生音声の周波数成分解析図である。FIG. 25 is a frequency component analysis diagram of Light Touch (−) 4-week-old rat-generated speech in the second week after the start of stimulation. 図26は、刺激開始2週目における対照(+)4週令ラット発生音声の周波数成分解析図である。FIG. 26 is a frequency component analysis diagram of control (+) 4-week-old rat-generated speech at the second week after the start of stimulation. 図27は、刺激開始2週目における対照(-)4週令ラット発生音声の周波数成分解析図である。FIG. 27 is a frequency component analysis diagram of control (−) 4-week-old rat-generated speech at the second week after the start of stimulation. 図28は、刺激開始2週目におけるTickling(+)6週令ラット発生音声の周波数成分解析図である。FIG. 28 is a frequency component analysis diagram of Tickling (+) 6-week-old rat-generated speech in the second week after the start of stimulation. 図29は、刺激開始2週目におけるTickling(-)6週令ラット発生音声の周波数成分解析図である。FIG. 29 is a frequency component analysis diagram of Tickling (−) 6-week-old rat-generated speech in the second week after the start of stimulation. 図30は、刺激開始2週目におけるTickling(+)8週令ラット発生音声の周波数成分解析図である。FIG. 30 is a frequency component analysis diagram of Tickling (+) 8-week-old rat-generated speech in the second week after the start of stimulation. 図31は、刺激開始2週目におけるTickling(-)8週令ラット発生音声の周波数成分解析図である。FIG. 31 is a frequency component analysis diagram of Tickling (−) 8-week-old rat-generated speech in the second week after the start of stimulation. 図32は、刺激開始4週目におけるTickling(+)4週令ラット発生音声の周波数成分解析図である。FIG. 32 is a frequency component analysis diagram of Tickling (+) 4-week-old rat-generated speech at the fourth week after the start of stimulation. 図33は、刺激開始4週目におけるTickling(-)4週令ラット発生音声の周波数成分解析図である。FIG. 33 is a frequency component analysis diagram of Tickling (−) 4-week-old rat-generated speech at the fourth week after the start of stimulation. 図34は、刺激開始4週目におけるLight Touch(+)4週令ラット発生音声の周波数成分解析図である。FIG. 34 is a frequency component analysis diagram of Light Touch (+) 4-week-old rat-generated speech in the fourth week after the start of stimulation. 図35は、刺激開始4週目におけるLight Touch(-)4週令ラット発生音声の周波数成分解析図である。FIG. 35 is a frequency component analysis diagram of Light Touch (−) 4-week-old rat-generated speech at the fourth week after the start of stimulation. 図36は、刺激開始4週目における対照(+)4週令ラット発生音声の周波数成分解析図である。FIG. 36 is a frequency component analysis diagram of control (+) 4-week-old rat-generated speech at 4 weeks after the start of stimulation. 図37は、刺激開始4週目における対照(-)4週令ラット発生音声の周波数成分解析図である。FIG. 37 is a frequency component analysis diagram of control (−) 4-week-old rat-generated speech at 4 weeks after the start of stimulation. 図38は、刺激開始4週目におけるTickling(+)6週令ラット発生音声の周波数成分解析図である。FIG. 38 is a frequency component analysis diagram of Tickling (+) 6-week-old rat-generated speech at the fourth week after the start of stimulation. 図39は、刺激開始4週目におけるTickling(-)6週令ラット発生音声の周波数成分解析図である。FIG. 39 is a frequency component analysis diagram of Tickling (−) 6-week-old rat-generated speech at 4 weeks after the start of stimulation. 図40は、刺激開始4週目におけるTickling(+)8週令ラット発生音声の周波数成分解析図である。FIG. 40 is a frequency component analysis diagram of Tickling (+) 8-week-old rat-generated speech at 4 weeks after the start of stimulation. 図41は、刺激開始4週目におけるTickling(-)8週令ラット発生音声の周波数成分解析図である。FIG. 41 is a frequency component analysis diagram of Tickling (−) 8-week-old rat-generated speech at the fourth week after the start of stimulation. 図42は、刺激開始直後における４週令ラットへの各処置直後の接近待機時間を示す。FIG. 42 shows the waiting time for approach immediately after each treatment on a 4-week-old rat immediately after the start of stimulation. 図43は、刺激開始翌日における４週令ラットへの各処置直後の接近待機時間を示す。FIG. 43 shows the waiting time for approach immediately after each treatment on a 4-week-old rat on the day following the start of stimulation. 図44は、刺激開始２週目における４週令ラットへの各処置直後の接近待機時間を示す。FIG. 44 shows the approach waiting time immediately after each treatment on a 4-week-old rat in the second week after the start of stimulation. 図45は、刺激開始４週目における４週令ラットへの各処置直後の接近待機時間を示す。FIG. 45 shows the approach waiting time immediately after each treatment on a 4-week-old rat at 4 weeks after the start of stimulation. 図46は、Tickling／対照間、Tickling／Light Touch間の遺伝子発現の差をDNAチップ法にて解析した際の、DNAチップ上の各プローブの信号強度の分布を示す。FIG. 46 shows the signal intensity distribution of each probe on the DNA chip when the difference in gene expression between Tickling / control and Tickling / Light Touch was analyzed by the DNA chip method. 図47は、いずれか２組の解析においてCy3およびCy5の信号強度が50以上あったプローブをクラスタリングした像である。FIG. 47 is an image obtained by clustering probes having signal intensities of Cy3 and Cy5 of 50 or more in any two sets of analysis. 図48は、少なくとも２組の解析において２倍超あるいは0.5倍未満の発現差を認めたプローブのクラスタリング像である。FIG. 48 is a clustering image of probes in which an expression difference of more than 2 times or less than 0.5 times was observed in at least two sets of analyses. 図49は、対照群と比較してTickling群の唾液腺組織で1.5倍超の発現差を認めた593プローブのクラスタリング像である。FIG. 49 is a clustered image of 593 probes in which an expression difference of more than 1.5 times was observed in the salivary gland tissue of the Tickling group compared to the control group. 図50は、Light Touch群と比較してTickling群の唾液腺組織で1.5倍超の発現差を認めた171プローブのクラスタリング像である。FIG. 50 is a clustered image of 171 probes in which an expression difference of more than 1.5 times was observed in the salivary gland tissue of the Tickling group compared to the Light Touch group. 図51は、対照群あるいはLight Touch群と比較してTickling群において発現差を認めた遺伝子(トランスクリプトを含む)の数を示したベン図である。FIG. 51 is a Venn diagram showing the number of genes (including transcripts) in which an expression difference was observed in the Tickling group compared to the control group or the Light Touch group. 図52は、対照群と比較してTickling群の唾液腺組織で1/1.5倍未満の発現差を認めた525プローブのクラスタリング像である。FIG. 52 is a clustering image of 525 probes in which an expression difference of less than 1 / 1.5 times was observed in the salivary gland tissue of the Tickling group compared to the control group. 図53は、Light Touch群と比較してTickling群の唾液腺組織で1/1.5倍未満の発現差を認めた166プローブのクラスタリング像である。FIG. 53 is a clustered image of 166 probes in which an expression difference of less than 1 / 1.5 times was observed in the salivary gland tissue of the Tickling group compared to the Light Touch group. 図54は、対照群あるいはLight Touch群と比較してTickling群において発現差を認めた唾液腺組織の遺伝子の数を示すベン図である。FIG. 54 is a Venn diagram showing the number of salivary gland tissue genes in which a difference in expression was found in the Tickling group compared to the control group or the Light Touch group. 図55は、対照群と比較してTickling群の視床下部組織で1.5倍超の発現差を認めた157プローブのクラスタリング像である。FIG. 55 is a clustered image of 157 probes in which an expression difference of more than 1.5 times was observed in the hypothalamic tissue of the Tickling group compared to the control group. 図56は、Light Touch群と比較してTickling群の視床下部組織で1.5倍超の発現差を認めた136プローブのクラスタリング像である。FIG. 56 is a clustered image of 136 probes in which an expression difference of more than 1.5 times was observed in the hypothalamic tissue of the Tickling group compared to the Light Touch group. 図57は、対照群あるいはLight Touch群と比較してTickling群において発現差を認めた視床下部の遺伝子(トランスクリプトを含む)の数を示すベン図である。FIG. 57 is a Venn diagram showing the number of hypothalamic genes (including transcripts) in which expression differences were observed in the Tickling group compared to the control group or the Light Touch group. 図58は、対照群と比較してTickling群の視床下部で1/1.5倍未満の発現差を認めた57プローブのクラスタリング像である。FIG. 58 is a clustered image of 57 probes in which an expression difference of less than 1 / 1.5 times was observed in the hypothalamus of the Tickling group compared to the control group. 図59は、Light Touch群と比較してTickling群の視床下部組織で1/1.5倍未満の発現差を認めた185プローブのクラスタリング像である。FIG. 59 is a clustered image of 185 probes in which an expression difference of less than 1 / 1.5-fold was observed in the hypothalamic tissue of the Tickling group compared to the Light Touch group. 図60は、対照群あるいはLight Touch群と比較してTickling群において1/1.5倍未満の発現差を認めた視床下部の遺伝子の数を示すベン図である。FIG. 60 is a Venn diagram showing the number of hypothalamic genes in which a difference in expression of less than 1 / 1.5-fold was observed in the Tickling group compared to the control group or the Light Touch group. 図61は、GO階層図の色の濃淡とP値を示す。FIG. 61 shows the color shading and P value of the GO hierarchy diagram. 図62は、対照群と比較してTickling群で発現が増加した脳及び血液の遺伝子のGO階層図である。FIG. 62 is a GO hierarchy diagram of brain and blood genes whose expression was increased in the Tickling group compared to the control group. 図63は、Light Touch群と比較してTickling群で発現が増加した脳及び血液の遺伝子のGO階層図である。FIG. 63 is a GO hierarchy diagram of brain and blood genes whose expression is increased in the Tickling group compared to the Light Touch group. 図64は、Light Touch群と比較してTickling群で発現が減少した脳及び血液の遺伝子のGO階層図である。FIG. 64 is a GO hierarchy diagram of brain and blood genes whose expression is decreased in the Tickling group compared to the Light Touch group. 図65は、対照群と比較してTickling群で発現が増加した唾液腺の遺伝子のGO階層図である。FIG. 65 is a GO hierarchy diagram of salivary gland genes whose expression was increased in the Tickling group compared to the control group. 図66は、対照群と比較してTickling群で発現が減少した唾液腺の遺伝子のGO階層図である。FIG. 66 is a GO hierarchy diagram of salivary gland genes whose expression was decreased in the Tickling group compared to the control group. 図67は、Light Touch群と比較してTickling群で発現が増加した唾液腺の遺伝子のGO階層図である。FIG. 67 is a GO hierarchy diagram of salivary gland genes whose expression is increased in the Tickling group compared to the Light Touch group. 図68は、Light Touch群と比較してTickling群で発現が減少した唾液腺の遺伝子のGO階層図である。FIG. 68 is a GO hierarchy diagram of salivary gland genes whose expression is decreased in the Tickling group compared to the Light Touch group. 図69は、対照群と比較してTickling群で発現が増加した視床下部の遺伝子のGO階層図である。FIG. 69 is a GO hierarchy diagram of hypothalamic genes whose expression was increased in the Tickling group compared to the control group. 図70は、対照群と比較してTickling群で発現が減少した視床下部の遺伝子のGO階層図である。FIG. 70 is a GO hierarchy diagram of hypothalamic genes whose expression was decreased in the Tickling group compared to the control group. 図71は、Light Touch群と比較してTickling群で発現が増加した視床下部の遺伝子のGO階層図である。FIG. 71 is a GO hierarchy diagram of hypothalamic genes whose expression is increased in the Tickling group compared to the Light Touch group. 図72は、Light Touch群と比較してTickling群で発現が減少した視床下部の遺伝子のGO階層図である。FIG. 72 is a GO hierarchy diagram of hypothalamic genes whose expression is decreased in the Tickling group compared to the Light Touch group. 図73は、Tickling負荷２日目のラット発生音声の周波数成分解析図である。FIG. 73 is a frequency component analysis diagram of rat-generated speech on the second day of Tickling load. 図74は、Light Touch負荷開始目のラット発生音声の周波数成分解析図である。FIG. 74 is a frequency component analysis diagram of rat-generated speech at the start of Light Touch load. 図75は、Tickling／Light Touch間の遺伝子発現の差をDNAチップ法にて解析した際の、DNAチップ上の各プローブの信号強度の分布を示す。FIG. 75 shows the signal intensity distribution of each probe on the DNA chip when the difference in gene expression between Tickling / Light Touch was analyzed by the DNA chip method. 図76は、いずれかの解析においてCy3およびCy5の信号強度が50以上あったプローブをクラスタリングした像である。FIG. 76 is an image obtained by clustering probes in which the signal intensity of Cy3 and Cy5 is 50 or more in any analysis. 図77は、いずれかの解析において1.5倍以上あるいは1/1.5倍以下の発現差を認めた868プローブのクラスタリング像である。FIG. 77 is a clustered image of 868 probes in which an expression difference of 1.5 times or more or 1 / 1.5 times or less was recognized in any analysis. 図78は、２倍以上あるいは0.5倍以下の発現差を認めた88プローブのクラスタリング像である。FIG. 78 is a clustering image of 88 probes in which an expression difference of 2 times or more or 0.5 times or less was recognized. 図79は、いずれかの解析において1.5倍以上あるいは1/1.5倍以下の発現差を認めた4,397プローブのクラスタリング像（プローブの並びは長期刺激を基準)である。FIG. 79 is a clustering image of 4,397 probes that recognized an expression difference of 1.5 times or more or 1 / 1.5 times or less in any analysis (probe alignment is based on long-term stimulation). 図80は、いずれかの解析において1.5倍以上あるいは1/1.5倍以下の発現差を認めた4,397プローブのクラスタリング像(プローブの並びは短期刺激を基準)である。FIG. 80 is a clustering image of 4,397 probes that recognized an expression difference of 1.5 times or more or 1 / 1.5 times or less in any analysis (probe alignment is based on short-term stimulation). 図81は、いずれかの解析において２倍以上あるいは0.5倍以下の発現差を認めた865プローブのクラスタリング像(プローブの並びは長期刺激を基準)である。FIG. 81 is a clustered image of 865 probes in which an expression difference of 2 times or more or 0.5 times or less was recognized in any analysis (probe alignment is based on long-term stimulation). 図82は、いずれかの解析において２倍以上あるいは0.5倍以下の発現差を認めた865プローブのクラスタリング像(プローブの並びは短期刺激を基準)である。FIG. 82 is a clustered image of 865 probes in which an expression difference of 2 times or more or 0.5 times or less was recognized in any analysis (probe alignment is based on short-term stimulation). 図83は、GO階層図の色の濃淡とP値を示す。FIG. 83 shows the color shading and P value of the GO hierarchy diagram. 図84は、脳と擦過細胞で共通して発現増加した遺伝子のGO階層図である。FIG. 84 is a GO hierarchy diagram of genes whose expression is commonly increased in the brain and scratched cells. 図85は、視床下部で発現増加した遺伝子のGO階層図である。FIG. 85 is a GO hierarchy diagram of genes whose expression is increased in the hypothalamus. 図86は、視床下部で発現が減少した遺伝子のGO階層図である。FIG. 86 is a GO hierarchy diagram of genes with decreased expression in the hypothalamus. 図87は、線条体で発現増加した遺伝子のGO階層図である。FIG. 87 is a GO hierarchy diagram of genes whose expression is increased in the striatum. 図88は、線条体で発現が減少した遺伝子のGO階層図である。FIG. 88 is a GO hierarchy diagram of genes with decreased expression in the striatum. 図89は、リアルタイムPCRによる遺伝子発現定量解析のフローを示す。FIG. 89 shows a flow of gene expression quantitative analysis by real-time PCR. 図90は、笑いによる遺伝子発現の変化に関する研究における実験時程図である。FIG. 90 is an experimental time chart in the study on gene expression change due to laughter. 図91は、糖尿病罹病者群及び糖尿病非罹病者群における、講義前後、コント前後の唾液中アミラーゼ活性をプロットした図である。FIG. 91 is a plot of salivary amylase activity before and after a lecture and before and after a contest in a diabetic group and a non-diabetic group. 図92は、糖尿病罹病者群及び糖尿病非罹病者群における、講義前後、コント前後の唾液中酸化還元電位(ORP)をプロットした図である。FIG. 92 is a graph plotting the redox potential (ORP) in saliva before and after a lecture and before and after a lecture in a group with and without diabetes. 図93は、糖尿病罹病者群及び糖尿病非罹病者群における、講義前後、コント前後の唾液量をプロットした図である。FIG. 93 is a diagram in which the amount of saliva before and after a lecture and before and after a contest in a diabetic group and a non-diabetic group are plotted. 図94は、糖尿病罹病者群及び糖尿病非罹病者群における、講義前後、コント前後の唾液緩衝能をプロットした図である。FIG. 94 is a diagram plotting the saliva buffering capacity before and after the lecture and before and after the control in the diabetic group and the non-diabetic group. 図95は、糖尿病罹病者群及び糖尿病非罹病者群における、講義前後、コント前後の総合感情障害度(TMD)をプロットした図である。FIG. 95 is a graph plotting the total emotional disorder (TMD) before and after the lecture and before and after the contest in the diabetic group and the non-diabetic group. 図96は、糖尿病罹病者群及び糖尿病非罹病者群における、講義前後、コント前後の不安傾向(STAI)をプロットした図である。FIG. 96 is a plot of anxiety tendencies (STAI) before and after a lecture and before and after a contest in a group with and without diabetes. 図97は、糖尿病罹病者群及び糖尿病非罹病者群における、講義前後、コント前後の唾液中分泌型イムノグロブリン(s-IgA)をプロットした図である。FIG. 97 is a graph plotting salivary secretory immunoglobulin (s-IgA) before and after the lecture and before and after the lecture in the diabetic group and the non-diabetic group. 図98は、糖尿病罹病者群及び糖尿病非罹病者群における、講義前後、コント前後の唾液中コルチゾールをプロットした図である。FIG. 98 is a diagram plotting cortisol in saliva before and after a lecture and before and after a contest in a diabetic group and a non-diabetic group. 図99は、糖尿病罹病者群及び糖尿病非罹病者群における、講義前後、コント前後の血糖値をプロットした図である。FIG. 99 is a diagram plotting blood glucose levels before and after the lecture and before and after the contest in the diabetic group and the non-diabetic group. 図100は、糖尿病罹病者群及び糖尿病非罹病者群における、講義前後、コント前後の白血球数をプロットした図である。FIG. 100 is a graph plotting the white blood cell count before and after the lecture and before and after the contest in the diabetic group and the non-diabetic group. 図101は、糖尿病罹病者群及び糖尿病非罹病者群における、講義前後、コント前後の好中球比をプロットした図である。FIG. 101 is a graph plotting the neutrophil ratio before and after the lecture and before and after the contest in the diabetic group and the non-diabetic group. 図102は、糖尿病罹病者群及び糖尿病非罹病者群における、講義前後、コント前後のリンパ球比をプロットした図である。FIG. 102 is a diagram plotting the lymphocyte ratio before and after the lecture and before and after the contest in the diabetic group and the non-diabetic group. 図103は、糖尿病罹病者群及び糖尿病非罹病者群における、講義前後、コント前後のNK細胞活性をプロットした図である。FIG. 103 is a graph plotting the NK cell activity before and after the lecture and before and after the control in the diabetic group and the non-diabetic group. 図104は、糖尿病罹病者群及び糖尿病非罹病者群における、講義前後、コント前後の血中コルチゾールをプロットした図である。FIG. 104 is a plot of blood cortisol before and after the lecture and before and after the contest in the diabetic and non-diabetic groups. 図105は、糖尿病罹病者群及び糖尿病非罹病者群における、講義前後、コント前後のC反応性蛋白をプロットした図である。FIG. 105 is a diagram plotting C-reactive protein before and after the lecture and before and after the contest in the diabetic group and non-diabetes group. 図106は、糖尿病罹病者群及び糖尿病非罹病者群における、血糖値の講義前後差及びコント前後差をプロットした図である。FIG. 106 is a diagram in which blood glucose levels before and after a lecture and before and after a difference are plotted in a diabetic group and a non-diabetic group. 図107は、歯周病罹病者群及び歯周病非罹病者群における、講義前後、コント前後の唾液アミラーゼ活性をプロットした図である。FIG. 107 is a graph plotting salivary amylase activity before and after a lecture and before and after a contest in a group with and without periodontal disease. 図108は、歯周病罹病者群及び歯周病非罹病者群における、講義前後、コント前後の唾液中酸化還元電位をプロットした図である。FIG. 108 is a graph plotting the redox potential in saliva before and after the lecture and before and after the lecture in the group with and without periodontal disease. 図109は、歯周病罹病者群及び歯周病非罹病者群における、講義前後、コント前後の唾液量をプロットした図である。FIG. 109 is a graph plotting the saliva amount before and after the lecture and before and after the contest in the group with and without periodontal disease. 図110は、歯周病罹病者群及び歯周病非罹病者群における、講義前後、コント前後の唾液緩衝能をプロットした図である。FIG. 110 is a diagram plotting the saliva buffering capacity before and after the lecture and before and after the control in the group with and without periodontal disease. 図111は、歯周病罹病者群及び歯周病非罹病者群における、講義前後、コント前後の総合感情障害度(TMD)をプロットした図である。FIG. 111 is a diagram plotting the total emotional disorder (TMD) before and after the lecture and before and after the contest in the group with and without periodontal disease. 図112は、歯周病罹病者群及び歯周病非罹病者群における、講義前後、コント前後の不安傾向(STAI)をプロットした図である。FIG. 112 is a plot of anxiety tendencies (STAI) before and after a lecture and before and after a contest in a group with and without periodontal disease. 図113は、歯周病罹病者群及び歯周病非罹病者群における、講義前後、コント前後の唾液中分泌型イムノグロブリンAをプロットした図である。FIG. 113 is a diagram plotting salivary immunoglobulin A in the saliva before and after the lecture and before and after the lecture in the group with and without periodontal disease. 図114は、歯周病罹病者群及び歯周病非罹病者群における、講義前後、コント前後の唾液中コルチゾールをプロットした図である。FIG. 114 is a plot of salivary cortisol before and after the lecture and before and after the lecture in the group with and without periodontal disease. 図115は、歯周病罹病者群及び歯周病非罹病者群における、講義前後、コント前後の血糖値をプロットした図である。FIG. 115 is a graph plotting blood glucose levels before and after the lecture and before and after the control in the group with and without periodontal disease. 図116は、歯周病罹病者群及び歯周病非罹病者群における、講義前後、コント前後の白血球数をプロットした図である。FIG. 116 is a diagram in which the white blood cell count before and after the lecture and before and after the contest in the periodontal disease affected group and the periodontal disease unaffected group is plotted. 図117は、歯周病罹病者群及び歯周病非罹病者群における、講義前後、コント前後の好中球比をプロットした図である。FIG. 117 is a graph plotting the neutrophil ratio before and after the lecture and before and after the contest in the group with and without periodontal disease. 図118は、歯周病罹病者群及び歯周病非罹病者群における、講義前後、コント前後のリンパ球比をプロットした図である。FIG. 118 is a graph plotting the lymphocyte ratio before and after the lecture and before and after the contest in the group with and without periodontal disease. 図119は、歯周病罹病者群及び歯周病非罹病者群における、講義前後、コント前後のNK細胞活性をプロットした図である。FIG. 119 is a graph plotting the NK cell activity before and after the lecture and before and after the lecture in the group with and without periodontal disease. 図120は、歯周病罹病者群及び歯周病非罹病者群における、講義前後、コント前後の血中コルチゾールをプロットした図である。FIG. 120 is a plot of blood cortisol before and after the lecture and before and after the contest in the group with and without periodontal disease. 図121は、歯周病罹病者群及び歯周病非罹病者群における、講義前後、コント前後のCRPをプロットした図である。FIG. 121 is a diagram plotting CRP before and after the lecture and before and after the contest in the periodontal disease affected group and the periodontal disease nonaffected group. 図122は、歯周病罹病者群及び歯周病非罹病者群における、血糖値の講義前後差及びコント前後差をプロットした図である。FIG. 122 is a diagram in which blood glucose levels before and after a lecture and before and after a difference are plotted in a periodontal disease affected group and a periodontal disease unaffected group. 図123は、実施例4の解析2、4～10において、1.5倍以上あるいは1/1.5倍以下の発現変化を認めた遺伝子のヒストグラムを示す。縦軸に発現変化倍数のLog2値を表し、横軸にサンプル名を表した。FIG. 123 shows a histogram of genes in which expression changes of 1.5 times or more or 1 / 1.5 times or less were found in analyzes 2 and 4 to 10 of Example 4. The vertical axis represents the Log2 value of the expression change fold, and the horizontal axis represents the sample name. 図124は、対応する解析ごとの発現変化の相対差を比較により示す。FIG. 124 shows by comparison the relative difference in expression change for each corresponding analysis. 図125は、糖尿病非罹病者群と糖尿病罹病者群で笑いにより1.5倍以上の発現変化を認めた遺伝子のベン図を示す。FIG. 125 shows a Venn diagram of genes in which expression change of 1.5 times or more was observed by laughter in the non-diabetic group and the diabetic group. 図126は、糖尿病非罹病者群と糖尿病罹病者群で笑いにより1.5倍以下の発現変化を認めた遺伝子のベン図を示す。FIG. 126 shows a Venn diagram of genes in which expression change of 1.5 times or less was observed by laughter in the non-diabetic group and the diabetic group. 図127は、糖尿病非罹病・歯周病非罹病者群と糖尿病非罹病・歯周病罹病者群で笑いにより1.5倍以上の発現変化を認めた遺伝子のベン図を示す。FIG. 127 shows a Venn diagram of genes in which expression change of 1.5 times or more was observed by laughter in the non-diabetic / periodontal disease-free group and the non-diabetes / periodontal group. 図128は、糖尿病非罹病・歯周病非罹病者群と糖尿病非罹病・歯周病罹病者群で笑いにより1.5倍以下の発現変化を認めた遺伝子のベン図を示す。FIG. 128 shows a Venn diagram of genes in which expression change of 1.5 times or less was observed by laughter in the non-diabetic / periodontal disease-free group and the non-diabetic / periodontal group. 図129は、糖尿病非罹病・歯周病非罹病者群と糖尿病罹病・歯周病非罹病者群で笑いにより1.5倍以上の発現変化を認めた遺伝子のベン図を示す。FIG. 129 shows a Venn diagram of genes in which the expression change of 1.5 times or more was observed by laughter in the non-diabetic / periodontal disease non-affected group and the diabetic / periodontal non-affected group. 図130は、糖尿病非罹病・歯周病非罹病者群と糖尿病罹病・歯周病非罹病者群で笑いにより1/1.5倍以下の発現変化を認めた遺伝子のベン図を示す。FIG. 130 shows a Venn diagram of genes in which expression changes of 1 / 1.5 fold or less were observed by laughter in the non-diabetic / periodontal disease non-affected group and the diabetic / periodontal non-affected group. 図131は、糖尿病非罹病・歯周病罹病者群と糖尿病罹病・歯周病罹病者群で笑いにより1.5倍以上の発現変化を認めた遺伝子のベン図を示す。FIG. 131 shows a Venn diagram of genes in which the expression change of 1.5 times or more was observed by laughter in the non-diabetes / periodontal disease affected group and the diabetic / periodontal disease affected group. 図132は、糖尿病非罹病・歯周病罹病者群と糖尿病罹病・歯周病罹病者群で笑いにより1/1.5倍以下の発現変化を認めた遺伝子のベン図を示す。FIG. 132 shows a Venn diagram of genes in which expression change of 1 / 1.5 fold or less was observed by laughter in the non-diabetic / periodontal disease group and the diabetic / periodontal group. 図133は、糖尿病罹病・歯周病非罹病者群と糖尿病罹病・歯周病罹病者群で笑いにより1.5倍以上の発現変化を認めた遺伝子のベン図を示す。FIG. 133 shows a Venn diagram of genes in which expression changes of 1.5 times or more were observed by laughter in the group with and without diabetes and in the group with and without diabetes. 図134は、糖尿病罹病・歯周病非罹病者群と糖尿病罹病・歯周病罹病者群で笑いにより1/1.5倍以下の発現変化を認めた遺伝子のベン図を示す。FIG. 134 shows a Venn diagram of genes in which expression changes of 1 / 1.5 times or less were observed by laughter in the group with and without diabetes and in the group with and without diabetes. 図135は、歯周病非罹病者群と歯周病罹病者群で笑いにより1.5倍以上の発現変化を認めた遺伝子のベン図を示す。FIG. 135 shows a Venn diagram of genes in which expression change of 1.5 times or more was observed by laughter in the group not suffering from periodontal disease and the group suffering from periodontal disease. 図136は、歯周病非罹病者群と歯周病罹病者群で笑いにより1/1.5倍以下の発現変化を認めた遺伝子のベン図を示す。FIG. 136 shows a Venn diagram of genes in which expression change of 1 / 1.5 times or less was observed by laughter in the group not suffering from periodontal disease and the group suffering from periodontal disease.
　本明細書は本願の優先権の基礎である日本国特許出願2008-170854号の明細書及び/又は図面に記載される内容を包含する。 This specification includes the contents described in the specification and / or drawings of Japanese Patent Application No. 2008-170854, which is the basis of the priority of the present application.
１－１．ラット陽性感情マーカー遺伝子及びそれを用いたラット陽性感情評価系
　本発明は、ラットの陽性感情評価方法を提供する。具体的に、本発明に係るラットの陽性感情評価方法は、ラットにおいてラット陽性感情マーカー遺伝子の発現量を測定するステップaと、ステップaで測定した発現量に基づいて、ラットの陽性感情を評価するステップbとを含んでいる。 1-1. Rat positive emotion marker gene and rat positive emotion evaluation system using the same The present invention provides a rat positive emotion evaluation method. Specifically, in the rat positive emotion evaluation method according to the present invention, step a for measuring the expression level of a rat positive emotion marker gene in the rat, and evaluating the positive emotion of the rat based on the expression level measured in step a. Step b.
　本明細書で使用する「陽性感情」（positive emotion/affect）とは、例えば、嬉しい、快活、愉快、楽しい、温和、穏やか、といった心理状態や、幸せ、喜び、愛、希望、満足、意欲、興味、信頼などのプラスの感情、心身を健康状態又は健康増進に導く任意の感情を指す。健康増進とは、WHO定義のうち、身体的、精神的なものにおいて、外界からの刺激に対する心身の適応能力を高めることをいう。一方、本明細書で使用する「陰性感情」（negative emotion/affect）とは、緊張、不安、悲しみ、うつ、怒り、恐怖、困惑といった心理状態やマイナスの感情、心身を病的状態に導く任意の感情を指す。 As used herein, “positive emotion / affect” refers to, for example, psychological states such as happy, cheerful, pleasant, fun, mild, calm, happiness, joy, love, hope, satisfaction, motivation, It refers to positive emotions such as interest and trust, and any emotions that lead the body and mind to a healthy state or health promotion. Health promotion refers to enhancing the ability of the body and mind to adapt to stimuli from the outside world in the physical and mental aspects of the WHO definition. On the other hand, “negative emotion / affect” as used in this specification refers to any psychological state such as tension, anxiety, sadness, depression, anger, fear, and embarrassment, negative emotions, and any physical or mental condition that leads to morbidity. Refers to the emotions.
　本明細書で使用する「陽性感情マーカー遺伝子」とは、被験体の陽性感情の指標となる遺伝子であって、被験体が陽性感情にある場合に、発現量が有意に増加するか又は減少する任意の遺伝子を指す。 As used herein, a “positive emotion marker gene” is a gene that serves as an index of a subject's positive emotion, and when the subject is in positive emotion, the expression level is significantly increased or decreased. Refers to any gene.
　特に本発明で提供する「ラット陽性感情マーカー遺伝子」は、50KHz近傍の発生音声を指標とした陽性感情下にあるラットにおいて、そのような陽性感情特性を示さないラットに比較して、発現量が1.5倍以上、好ましくは2.0倍以上増加するか又は減少する遺伝子として定義される。言い換えれば、ラット陽性感情マーカー遺伝子は、ラットの50KHz近傍の発生音声と連動して発現量が1.5倍以上、好ましくは2.0倍以上増減する遺伝子である。 In particular, the “rat positive emotion marker gene” provided in the present invention has a higher expression level in rats under positive emotion using the generated speech near 50 KHz as an index compared to a rat that does not show such positive emotion characteristics. It is defined as a gene that increases or decreases 1.5 times or more, preferably 2.0 times or more. In other words, the rat positive emotion marker gene is a gene whose expression level increases or decreases by 1.5 times or more, preferably 2.0 times or more in conjunction with the generated speech in the vicinity of 50 KHz of the rat.
　陽性感情ラットにおいて2.0倍以上の発現量の増減を示す本発明のラット陽性感情マーカー遺伝子を下記の表1、表2に示す。
Figure JPOXMLDOC01-appb-T000001
 Tables 1 and 2 below show the rat positive emotion marker genes of the present invention that show an increase or decrease in expression level of 2.0 times or more in positive emotion rats.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-I000001
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-I000001
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-I000002
Figure JPOXMLDOC01-appb-I000002
Figure JPOXMLDOC01-appb-I000003
Figure JPOXMLDOC01-appb-I000003
Figure JPOXMLDOC01-appb-I000004
Figure JPOXMLDOC01-appb-I000004
Figure JPOXMLDOC01-appb-I000005
Figure JPOXMLDOC01-appb-I000005
Figure JPOXMLDOC01-appb-I000006
Figure JPOXMLDOC01-appb-I000006
Figure JPOXMLDOC01-appb-I000007
Figure JPOXMLDOC01-appb-I000007
Figure JPOXMLDOC01-appb-I000008
Figure JPOXMLDOC01-appb-I000008
Figure JPOXMLDOC01-appb-I000009
Figure JPOXMLDOC01-appb-I000009
　表1及び2において、「No.」の欄は各遺伝子に割り振った番号を意味し、「Target Symbol」の欄は一般的な遺伝子表記である。また、「Target Accession」の欄は公共のデータベースに登録されている登録ID（アクセッション番号）であり、「UniGene」とは、NCBIが提供する配列データの重複をなくした核酸配列データベースであり、「UniGene」の欄は、データベース（UniGene）に登録されているUiGeneIDである。さらに、「Up/down」は、陽性感情ラットにおいて発現量が増加（Up）しているか、発現量が減少（Down）しているかを意味している。さらに「生体由来試料」は、陽性感情ラットにおいて各ラット陽性感情マーカー遺伝子が陽性感情の指標として機能することができる試料を示す。 In Tables 1 and 2, the “No.” column means the number assigned to each gene, and the “Target Symbol” column is a general gene notation. In addition, the column “Target 登録 Accession” is a registration ID (accession number) registered in a public database, and “UniGene” is a nucleic acid sequence database that eliminates duplication of sequence data provided by NCBI. The column “UniGene” is a UiGeneID registered in the database (UniGene). Furthermore, “Up / down” means whether the expression level is increasing (Up) or the expression level is decreasing (Down) in the positive emotional rat. Furthermore, the “biological sample” refers to a sample in which each rat positive emotion marker gene can function as an index of positive emotion in a positive emotion rat.
　表1に示すラット陽性感情マーカー遺伝子は、2日間の短期間で陽性刺激を負荷した際に発現量の顕著な増減を認めた遺伝子のリストであり、表2に示すラット陽性感情マーカー遺伝子は、陽性刺激を4週間の長期間で負荷した際に発現量の顕著な増減を認めた遺伝子のリストである。表1及び2から分かるとおり、短期間の陽性刺激による場合と、長期間の陽性刺激による場合とで、発現量の顕著な増減が認められる遺伝子が異なる。 The rat positive emotion marker gene shown in Table 1 is a list of genes that showed a marked increase or decrease in the expression level when a positive stimulus was loaded in a short period of 2 days, and the rat positive emotion marker gene shown in Table 2 It is the list | wrist of the gene which recognized the remarkable increase / decrease in the expression level when positive stimulation was loaded for a long period of 4 weeks. As can be seen from Tables 1 and 2, the gene with a significant increase or decrease in the expression level differs between the case of a short-term positive stimulus and the case of a long-term positive stimulus.
　すなわち、表1に示すラット陽性感情マーカー遺伝子は、一過性の陽性感情を検出することができるマーカーであり、表2に示すラット陽性感情マーカー遺伝子は、体質の変化を生じさせるような持続的な陽性感情の結果を検出することができるマーカーである。 That is, the rat positive emotion marker gene shown in Table 1 is a marker that can detect transient positive emotions, and the rat positive emotion marker gene shown in Table 2 is a persistent gene that causes changes in constitution. It is a marker that can detect the result of positive emotion.
　本発明に係るラットの陽性感情評価方法においては、先ずステップaにおいて、表1及び/又は2に示す遺伝子群から選択される少なくとも1種の遺伝子の発現量を、指定の生体由来試料において測定する。ここで、「指定の生体由来試料」とは、表1及び/又は2に示す各遺伝子に対応する「生体由来試料」の欄に記載の試料（すなわち、血液、視床下部、線条体、口腔内細胞又は唾液腺）を指す。なお、下記に記載の表81～84及び表45～59に列挙する遺伝子は、それぞれ短期刺激、長期刺激において見出されたラット陽性感情マーカー遺伝子の中でも、種々の生物学的活性に関連する遺伝子であり、ステップaにおいては、上記表1及び/又は表2に示す遺伝子群に加えて、又はこれに代えて、表81～84及び/又は45～59に列挙する遺伝子の発現量を、指定の生体由来試料において測定してもよい。 In the rat positive emotion evaluation method according to the present invention, first, in step a, the expression level of at least one gene selected from the gene group shown in Tables 1 and / or 2 is measured in a specified biological sample. . Here, the “designated biological sample” means the sample described in the column of “biological sample” corresponding to each gene shown in Tables 1 and / or 2 (that is, blood, hypothalamus, striatum, oral cavity) Internal cell or salivary gland). The genes listed in Tables 81 to 84 and Tables 45 to 59 described below are genes related to various biological activities among rat positive emotion marker genes found in short-term stimulation and long-term stimulation, respectively. In step a, in addition to or instead of the genes shown in Table 1 and / or Table 2, the expression levels of the genes listed in Tables 81 to 84 and / or 45 to 59 are designated. You may measure in the biological origin sample.
　遺伝子発現量の測定は、対象の遺伝子の発現量を特異的に定量できる方法であれば特に制限はなく、対象遺伝子のmRNA量(又はcDNA)を測定することによってもよいし、対象遺伝子がコードするタンパク質量を測定することによってもよい。 The gene expression level is not particularly limited as long as the expression level of the target gene can be specifically quantified, and it may be measured by measuring the mRNA level (or cDNA) of the target gene, or the target gene may be encoded. The amount of protein to be measured may be measured.
　対象遺伝子のmRNA量（又はcDNA）の測定には、該mRNA(又はcDNA)に対して相補的な塩基配列を有する核酸を使用することができる。具体的には、ラット生体由来試料から全mRNAを単離し、測定対象の遺伝子の塩基配列の少なくとも一部（好ましくはコード領域）を含む核酸をプローブ又はプライマーとして用いることによって行う。測定に使用する核酸は必ずしもDNAである必要はなく、RNA、DNA/RNAキメラ、他の人工核酸などであってもよい。かかる核酸は、当業者に周知の方法、例えば適当な配列のクローニング及び制限酵素による切断、ホスホトリエステル法（例えばNarangら，1979年，Meth.Enzymol.，第68巻，p90～99参照）、ホスホジエステル法（例えばBrownら，1979年、Meth.Enzymol.，第68巻，p109～151参照）、エチルホスホアミダイト法（例えばBeaucageら，1981年，Tetrahedron Lett.，第22巻，p1859～1862参照）などの方法により、直接合成することができる。また市販の自動DNA合成装置を使用することによって合成してもよい。 A nucleic acid having a base sequence complementary to the mRNA (or cDNA) can be used for measuring the mRNA amount (or cDNA) of the target gene. Specifically, it is carried out by isolating total mRNA from a rat biological sample and using a nucleic acid containing at least a part (preferably a coding region) of the base sequence of the gene to be measured as a probe or primer. The nucleic acid used for the measurement does not necessarily need to be DNA, and may be RNA, DNA / RNA chimera, other artificial nucleic acid, or the like. Such nucleic acids can be obtained by methods well known to those skilled in the art, such as cloning of appropriate sequences and cleavage with restriction enzymes, the phosphotriester method (see, for example, Narang et al., 1979, Meth. Enzymol., 68, p90-99), Phosphodiester method (see, for example, Brown et al., 1979, Meth. Enzymol., Vol. 68, p109-151), ethyl phosphoramidite method (see, for example, Beaucage et al., 1981, Tetrahedron Lett., Vol. 22, p1859-1862) ) And the like can be directly synthesized. Moreover, you may synthesize | combine by using a commercially available automatic DNA synthesizer.
　ラット生体由来試料からの全mRNAの単離は、当業者に公知の手法によって行うことができる。例えば、ラット生体由来試料の破壊液から定法に従って全RNAを取得し、オリゴdTカラムにてmRNAを回収することができる。 Isolation of total mRNA from a rat biological sample can be performed by a method known to those skilled in the art. For example, total RNA can be obtained from a rat biological sample-disrupted solution according to a conventional method, and mRNA can be recovered using an oligo dT column.
　mRNA量の測定には、当業者に公知のいずれの方法を用いてもよい。そのような方法として例えば、DNAマイクロアレイ法、RT-PCR、定量PCR、ノーザンブロット等を使用することができる。一般的には、1個～数種類の遺伝子に関してmRNA量を測定する場合には、RT-PCR、定量PCR、ノーザンブロット等を使用することが好ましい。この場合、上記核酸プローブ又はプライマーを標識することにより、mRNA量を測定することができる。一方、100種以上といった多数の遺伝子についてmRNA量を測定する場合には、DNAマイクロアレイ法を使用することが好ましい。DNAマイクロアレイ法でmRNA量を測定する場合には、上記核酸プローブを固定化した支持体に、測定試料である全mRNAを接触させることによって行う。この場合、一般的には、測定試料のmRNAを標識し、各プローブとのハイブリダイゼーションの有無を蛍光強度によって検出することにより、mRNA量を測定することができる。なお、ハイブリダイゼーション条件は、各プローブと対象のmRNAとの特異的なハイブリダイゼーションが担保される条件であれば特に制限されないが、ストリンジェントな条件であることが好ましい。そのような条件は当業者に公知であり、例えばSambrookらMolecular Cloning: A Laboratory Manual 2nd Ed. Cold Spring Harbor Laboratory Press (1989)等に記載の条件が挙げられる。 Any method known to those skilled in the art may be used to measure the amount of mRNA. As such a method, for example, DNA microarray method, RT-PCR, quantitative PCR, Northern blot etc. can be used. Generally, when measuring the amount of mRNA for one to several kinds of genes, it is preferable to use RT-PCR, quantitative PCR, Northern blot or the like. In this case, the amount of mRNA can be measured by labeling the nucleic acid probe or primer. On the other hand, when measuring the amount of mRNA for a large number of genes such as 100 or more, it is preferable to use the DNA microarray method. When measuring the amount of mRNA by the DNA microarray method, it is carried out by bringing the total mRNA as a measurement sample into contact with the support on which the nucleic acid probe is immobilized. In this case, generally, the amount of mRNA can be measured by labeling the mRNA of the measurement sample and detecting the presence or absence of hybridization with each probe by the fluorescence intensity. The hybridization conditions are not particularly limited as long as specific hybridization between each probe and the target mRNA is ensured, but stringent conditions are preferable. Such conditions are known to those skilled in the art, and examples include the conditions described in Sambrook et al. Molecular Cloning: A Laboratory Manual 2nd Ed. Cold Spring Harbor Laboratory Press (1989).
　一方、対象遺伝子の発現量の測定を該遺伝子がコードするタンパク質量を測定することによって行う場合には、該タンパク質に対して特異的に結合する分子、例えば該タンパク質に特異的に結合する抗体を使用することができる。なお、測定対象の遺伝子がコードするタンパク質を抗原とし、当該抗原に特異的に結合する限り、前記抗体としては特に制限はなく、マウス抗体、ラット抗体、ウサギ抗体、ヒツジ抗体等を適宜用いることができる。抗体は、ポリクローナル抗体であってもモノクローナル抗体であってもよいが、均質な抗体を安定に生産できる点でモノクローナル抗体が好ましい。ポリクローナル抗体およびモノクローナル抗体は当業者に周知の方法により作製することができる。 On the other hand, when the expression level of the target gene is measured by measuring the amount of protein encoded by the gene, a molecule that specifically binds to the protein, for example, an antibody that specifically binds to the protein is added. Can be used. As long as the protein encoded by the gene to be measured is an antigen and specifically binds to the antigen, the antibody is not particularly limited, and a mouse antibody, a rat antibody, a rabbit antibody, a sheep antibody, or the like can be used as appropriate. it can. The antibody may be a polyclonal antibody or a monoclonal antibody, but a monoclonal antibody is preferable in that a homogeneous antibody can be stably produced. Polyclonal and monoclonal antibodies can be prepared by methods well known to those skilled in the art.
　モノクローナル抗体を産生するハイブリドーマは、基本的には公知技術を使用し、以下のようにして作製できる。すなわち、所望の抗原や所望の抗原を発現する細胞を感作抗原として使用して、これを通常の免疫方法にしたがって免疫し、得られる免疫細胞を通常の細胞融合法によって公知の親細胞と融合させ、通常のスクリーニング法により、モノクローナルな抗体産生細胞（ハイブリドーマ）をスクリーニングすることによって作製できる。ハイブリドーマの作製は、たとえば、ミルステインらの方法（Kohler. G. and Milstein, C., Methods Enzymol. (1981) 73: 3-46 ）等に準じて行うことができる。 A hybridoma producing a monoclonal antibody can be basically produced using a known technique as follows. That is, a desired antigen or a cell expressing the desired antigen is used as a sensitizing antigen and immunized according to a normal immunization method, and the resulting immune cell is fused with a known parent cell by a normal cell fusion method. And can be prepared by screening monoclonal antibody-producing cells (hybridomas) by a normal screening method. The hybridoma can be prepared, for example, according to the method of Milstein et al. (Kohler. G. and Milstein, C., Methods Enzymol. (1981) 73: 3-46).
　得られたモノクローナル抗体は、測定対象のタンパク質の定量用に、エンザイム－リンクイムノソルベントアッセイ（ELISA）、酵素イムノドットアッセイ、ラジオイムノアッセイ、凝集に基づいたアッセイ、あるいは他のよく知られているイムノアッセイ法で検査試薬として用いることができる。また、モノクローナル抗体は標識化されることが好ましい。標識化を行う際、標識化合物としては例えば当分野で公知の酵素、蛍光物質、化学発光物質、放射性物質、染色物質などを使用することができる。 The resulting monoclonal antibodies can be used for enzyme-linked immunosorbent assays (ELISA), enzyme immunodot assays, radioimmunoassays, agglutination-based assays, or other well-known immunoassay methods for the quantification of proteins to be measured. Can be used as a test reagent. The monoclonal antibody is preferably labeled. When labeling, for example, an enzyme, a fluorescent substance, a chemiluminescent substance, a radioactive substance, or a staining substance known in the art can be used as the labeling compound.
　その他、タンパク質量の測定には、該タンパク質に特異的に結合可能なリガンドタンパク質又は受容体タンパク質を使用することもできる。 In addition, a ligand protein or a receptor protein that can specifically bind to the protein can also be used for the measurement of the protein amount.
　一方、本発明に係るラットの陽性感情を評価するための検査キットは、上記表1及び/若しくは2に示す遺伝子（並びに/又は表81～84及び/若しくは表45～59に示す遺伝子）の塩基配列の少なくとも一部を含む核酸からなる群から選ばれる少なくとも1種の核酸、或いは、上記表1及び/又は2に示す遺伝子によってコードされるタンパク質に特異的に結合する分子（例えば抗体）からなる群から選ばれる少なくとも1種の分子を備える。本発明の検査キットが備える核酸又は分子の種類は、測定対象の遺伝子の種類に応じて変化する。 On the other hand, the test kit for evaluating the positive emotion of the rat according to the present invention comprises the bases of the genes shown in Table 1 and / or 2 (and / or the genes shown in Tables 81 to 84 and / or Tables 45 to 59). Consists of at least one nucleic acid selected from the group consisting of nucleic acids containing at least part of the sequence, or a molecule (for example, an antibody) that specifically binds to a protein encoded by the genes shown in Tables 1 and / or 2 above. It comprises at least one molecule selected from the group. The type of nucleic acid or molecule provided in the test kit of the present invention varies depending on the type of gene to be measured.
　例えば、当該検査キットは、支持体に上述した核酸又は分子(若しくはその一部)を被覆したものを挙げることができる。支持体としてはポリスチレンやポリカーボネート、ポリプロピレン、ポリビニール製のマイクロタイタープレート、試験管、キャピラリー、ビーズ、膜、フィルターなどが挙げられる。 For example, the test kit may include a support in which the above-described nucleic acid or molecule (or part thereof) is coated. Examples of the support include polystyrene, polycarbonate, polypropylene, polyvinyl microtiter plates, test tubes, capillaries, beads, membranes, filters, and the like.
　以上のようにして、上記表1及び/又は2に示す遺伝子群から選択される少なくとも1種の遺伝子の発現量を指定の生体由来試料において測定した後、ステップbにおいて、当該発現量に基づいて、ラットの陽性感情の評価を行う。 As described above, after measuring the expression level of at least one gene selected from the gene group shown in Table 1 and / or 2 in a specified biological sample, in step b, based on the expression level Evaluate positive emotions in rats.
　ステップbにおける評価は、表1、2に挙げる遺伝子についての基準値に対して、ステップaで測定した遺伝子の発現量から算出されるスコアが異なるかを判断することによって行うことができる。 The evaluation in step b can be performed by judging whether the score calculated from the gene expression level measured in step a is different from the reference values for the genes listed in Tables 1 and 2.
　具体的には、表1、2に挙げる遺伝子のうち、発現量から算出されるスコアが、「Up/down」の欄がUpの遺伝子については基準値より高くなっているか否かを判断し、Downの遺伝子については基準値より低くなっている否かを判断する。 Specifically, among the genes listed in Tables 1 and 2, the score calculated from the expression level is determined whether the “Up / down” column is higher than the reference value for the Up gene, It is determined whether or not the Down gene is lower than the reference value.
　上記「基準値」は、表1、2に挙げる陽性感情マーカー遺伝子ごとに設定される。本発明において、基準値は、例えば、約50KHz近傍の超音波を発していない対照ラットにおけるラット陽性感情マーカー遺伝子の発現量を、対照ラットと陽性感情にあるラットとの間で発現量の有意な差が認められない任意の遺伝子Xの発現量に対する相対値として示した値とすることができる。この場合、上記スコアは、ステップaで測定したラット陽性感情マーカー遺伝子の発現量を、遺伝子Xの発現量で除算することによって算出することができる。かかる基準値は、表1、2に記載の各遺伝子について予め求めておいてもよいし、対照ラットにおける基準値の測定を本発明のステップaと平行して行ってもよい。基準値は、複数の対照ラットで算出することにより、基準値±標準偏差とすることが好ましい。これにより基準値に対する個体差の影響を排除することができる。またこれに関連して、本発明に係る検査キットは、上記遺伝子Xの塩基配列を有する核酸又は遺伝子Xがコードするタンパク質に対する抗体をさらに含むことができる。これにより、対照ラットにおいて基準値を測定するために、及び評価対象のラットにおいて上記スコアを算出するために、遺伝子Xの発現量を求めることができる。また、本発明に係る検査キットは、表1、2に示す陽性感情マーカー遺伝子のそれぞれについて予め算出された基準値（±標準偏差）の対応表を含むことが好ましい。これにより、対照ラットを用いた基準値の算出を省略することができる。 The above “reference value” is set for each positive emotion marker gene listed in Tables 1 and 2. In the present invention, the reference value refers to, for example, the expression level of a rat positive emotion marker gene in a control rat that does not emit an ultrasonic wave in the vicinity of about 50 KHz, and a significant expression level between the control rat and a rat in positive emotion. It can be a value shown as a relative value with respect to the expression level of any gene X in which no difference is observed. In this case, the score can be calculated by dividing the expression level of the rat positive emotion marker gene measured in step a by the expression level of gene X. Such a reference value may be obtained in advance for each gene listed in Tables 1 and 2, or the reference value in a control rat may be measured in parallel with step a of the present invention. The reference value is preferably set to a reference value ± standard deviation by calculating with a plurality of control rats. Thereby, the influence of the individual difference with respect to a reference value can be excluded. In this connection, the test kit according to the present invention may further include an antibody against a nucleic acid having the base sequence of gene X or a protein encoded by gene X. Thereby, in order to measure the reference value in the control rat and to calculate the score in the rat to be evaluated, the expression level of gene X can be obtained. The test kit according to the present invention preferably includes a correspondence table of reference values (± standard deviation) calculated in advance for each of the positive emotion marker genes shown in Tables 1 and 2. Thereby, calculation of the reference value using the control rat can be omitted.
　ここで、評価の一例を挙げると、例えば、表1、2に記載されるラット陽性感情マーカー遺伝子のうち、発現量が「Up」の遺伝子から遺伝子を選択し、選択した遺伝子の発現量及び遺伝子Xの発現量を測定し、この選択した遺伝子の発現量を遺伝子Xの発現量で除算した値が、該遺伝子について予め算出しておいた基準値を有意に上回っている場合に、ラットは陽性感情であると評価することができる。なお、かかる評価の信頼度を高めるために、複数の遺伝子について上記判断を行い、50％以上、より好ましくは60％以上、より好ましくは70％以上、より好ましくは80％以上、より好ましくは90％以上、最も好ましくは100％の遺伝子において基準値を有意に上回っている場合に、ラットは陽性感情であると評価することとしてもよい。あるいは、対照に対する遺伝子発現量の増加率又は減少率を、選択した複数の遺伝子のそれぞれについて求め、その百分比合計が所定の数値を超える場合に、ラットは陽性感情であると評価してもよい。 Here, as an example of evaluation, for example, among the rat positive emotion marker genes described in Tables 1 and 2, a gene is selected from genes whose expression level is “Up”, and the expression level and gene of the selected gene are selected. When the expression level of X is measured and the expression level of the selected gene divided by the expression level of gene X is significantly higher than the reference value calculated in advance for the gene, the rat is positive It can be evaluated as emotion. In order to increase the reliability of such evaluation, the above determination is made for a plurality of genes, and is 50% or more, more preferably 60% or more, more preferably 70% or more, more preferably 80% or more, more preferably 90%. % Or more, most preferably 100% of the genes may be evaluated as positive emotions if they are significantly above the reference value. Alternatively, the increase rate or decrease rate of the gene expression level relative to the control may be obtained for each of the selected genes, and the rat may be evaluated as having a positive emotion when the percentage sum exceeds a predetermined value.
　本発明に係るラットの陽性感情評価方法及び検査キットによれば、陽性感情を簡便且つ高い信頼性をもって評価することができる。特に、本発明に係るラットの陽性感情評価方法において、測定対象の遺伝子の種類を増やすこと（例えば10種～100種又はそれ以上）によって、より優れた信頼度をもって、ラットの陽性感情を評価することができる。 According to the positive emotion evaluation method and test kit for rats according to the present invention, positive emotion can be evaluated easily and with high reliability. In particular, in the rat positive emotion evaluation method according to the present invention, the positive emotion of the rat is evaluated with higher reliability by increasing the types of genes to be measured (for example, 10 to 100 or more). be able to.
１－２．ヒト陽性感情マーカー遺伝子及びそれを用いたヒトの陽性感情又は健康度の評価系
　本発明はまた、ヒトの陽性感情又は健康度の評価方法を提供する。この方法は、原理的には、上記ラット陽性感情評価方法と同様にして行うことができる。したがって、本発明のヒト陽性感情又はヒト健康度の評価方法は、ヒト陽性感情マーカー遺伝子の発現量を生体由来試料、例えば血液、において測定するステップaと、ステップaで測定した発現量に基づいて、ヒトの陽性感情又は健康度を評価するステップbとを含んでいる。 1-2. Human positive emotion marker gene and human positive emotion or health evaluation system using the same The present invention also provides a human positive emotion or health evaluation method. In principle, this method can be performed in the same manner as the rat positive emotion evaluation method. Therefore, the human positive emotion or human health evaluation method of the present invention is based on the step a in which the expression level of a human positive emotion marker gene is measured in a biological sample, for example, blood, and the expression level measured in step a. And b for assessing human positive emotions or health.
　本明細書で使用する「ヒト陽性感情マーカー遺伝子」は、笑いを誘発する刺激（本明細書中、「笑い刺激」ともいう）を負荷したヒトにおいて、そのような刺激を負荷していないヒトに比較して、発現量が増加するか又は減少する遺伝子として定義される。言い換えれば、ヒト陽性感情マーカー遺伝子は、ヒトの笑いと連動して発現量が増減する遺伝子である。 As used herein, a “human positive emotion marker gene” refers to a human loaded with a stimulus that induces laughter (also referred to herein as “laughter stimulus”). In comparison, it is defined as a gene whose expression level increases or decreases. In other words, the human positive emotion marker gene is a gene whose expression level increases or decreases in conjunction with human laughter.
　また「健康」とは、WHO定義より、「完全な肉体的、精神的及び社会的福祉の状態であり、単に疾病又は病弱の存在しないことではない」を指し、「健康度」とはその程度を指す。ヒトにおいて、笑いがNK細胞の活性化や糖尿病患者の食後血糖値を抑えることが知られている。本発明では、s-IgAなどの免疫機能の活性化、ストレス指標である唾液アミラーゼ、好中球などから見られるストレス状態の改善、感染や炎症時に増加するC反応性蛋白の減少、糖尿病患者の食後血糖値抑制などを、笑い刺激により確認し、笑いが健康維持・増進や、疾病の機能回復に効果があるという結果を得た。これらの健康維持・増進や、疾病の機能回復の効果と、陽性感情マーカー遺伝子の各遺伝子セットとは、対応付けすることができている。そのため、ヒト陽性感情マーカー遺伝子の発現量に基づくヒト陽性感情の評価は、ヒトの健康度の評価と考えることもできる。 “Health” refers to “a state of complete physical, mental and social welfare, not simply the absence of illness or illness” according to the WHO definition. Point to. In humans, laughter is known to suppress NK cell activation and postprandial blood glucose levels in diabetic patients. In the present invention, activation of immune functions such as s-IgA, improvement of the stress state seen from salivary amylase and neutrophil as a stress index, reduction of C-reactive protein that increases during infection and inflammation, diabetic patients The suppression of postprandial blood glucose level was confirmed by laughter stimulation, and the results showed that laughter was effective in maintaining and improving health and restoring disease function. These effects of maintaining / promoting health and restoring the function of the disease can be associated with each gene set of positive emotion marker genes. Therefore, the evaluation of human positive emotion based on the expression level of the human positive emotion marker gene can be considered as an evaluation of human health.
　ヒト陽性感情マーカー遺伝子のうち、機能面で注目される遺伝子と、顕著な発現量の増減を示す遺伝子の上位15遺伝子とからなる遺伝子群は、それぞれ遺伝子セット1～8と称し、下記表に要約されている：遺伝子セット1、表164；遺伝子セット2、表172；遺伝子セット3、表178；遺伝子セット4、表185；遺伝子セット5、表155；遺伝子セット6、表159；遺伝子セット7、表190；遺伝子セット8、表198。これらの表中、「No.」の欄は各遺伝子セット中の各遺伝子に割り振った番号を意味し、「Target(Gene) Symbol」の欄は一般的な遺伝子表記である。また、「Target Accession」の欄は公共のデータベースに登録されている登録ID（アクセッション番号）であり、「UniGene」とは、NCBIが提供する配列データの重複をなくした核酸配列データベースであり、「UniGene」の欄は、データベース（UniGene）に登録されているUiGeneIDである。さらに、「Up/down」は、笑いを誘導した陽性感情下のヒトにおいて発現量が増加（Up）しているか、発現量が減少（Down）しているかを意味している。 Among the human positive emotion marker genes, the gene group consisting of genes that are noted in terms of function and the top 15 genes that show significant increase or decrease in the expression level are called gene sets 1-8, respectively, and are summarized in the table below. Gene Set 1, Table 164; Gene Set 2, Table 172; Gene Set 3, Table 178; Gene Set 4, Table 185; Gene Set 5, Table 155; Gene Set 6, Table 159; Gene Set 7, Table 190; Gene set 8, Table 198. In these tables, the “No.” column means the number assigned to each gene in each gene set, and the “Target (Gene) Symbol” column is a general gene notation. In addition, the column “Target 登録 Accession” is a registration ID (accession number) registered in a public database, and “UniGene” is a nucleic acid sequence database that eliminates duplication of sequence data provided by NCBI. The column “UniGene” is a UiGeneID registered in the database (UniGene). Furthermore, “Up / down” means whether the expression level is increasing (Up) or the expression level is decreasing (Down) in a positive emotional human who has induced laughter.
　また「被験者群」は、各遺伝子セットに含まれる遺伝子の発現の増減が確認された被験者の種類を示している：A、糖尿病非罹病かつ歯周病非罹病者；B、糖尿病非罹病かつ歯周病罹病者；C、糖尿病罹病かつ歯周病非罹病者；D、糖尿病罹病かつ歯周病罹病者；A＋B、糖尿病非罹病者；C＋D、糖尿病罹病者；A＋C、歯周病非罹病者；B＋D、歯周病罹病者。 The “subject group” indicates the types of subjects whose gene expression in each gene set was confirmed to increase or decrease: A, non-diabetic and non-periodontal; B, non-diabetic and dental Peripheral disease; C, Diabetes and non-periodontal disease; D, Diabetes and periodontal disease; A + B, Non-diabetes; C + D, Diabetes; A + C, Non-periodontal; B + D, suffering from periodontal disease.
　遺伝子セット1、2、5は、広く一般の人に対して利用できる陽性感情又は健康度評価のための遺伝子セットであり、その中でも特に遺伝子セット1は広く一般の人に対して陽性感情又は健康度評価を行うのに適した遺伝子セットである。遺伝子セット3、4、6は、糖尿病罹病者に適した遺伝子セットであるが、他の疾病に罹病している人、疾病に罹病していない人の陽性感情又は健康度評価に利用できる。遺伝子セット2、4、8は、歯周病罹病者に適した遺伝子セットであるが、他の疾病に罹病している人、疾病に罹病していない人の陽性感情又は健康度評価に利用できる。これらの遺伝子セットは、単独でも、それぞれを組み合わせても陽性感情又は健康度評価に利用できる。 Gene sets 1, 2, and 5 are gene sets for positive emotions or health assessment that can be widely used for general people. Among them, gene set 1 is particularly positive emotions or health for general people. It is a gene set suitable for performing degree evaluation. Gene sets 3, 4 and 6 are gene sets suitable for diabetics, but can be used to evaluate positive emotions or health of people who have other diseases or who have no diseases. Gene sets 2, 4 and 8 are gene sets suitable for those suffering from periodontal disease, but can be used to evaluate positive emotions or health of people who have other diseases or who have no diseases. . These gene sets can be used for positive emotion or health evaluation, either alone or in combination.
　本発明に係るヒトの陽性感情又は健康度の評価方法においては、先ずステップaにおいて、遺伝子セット1～8に示す遺伝子セット中の少なくとも1種の遺伝子の発現量を、生体由来試料、例えば血液、において測定する。測定する生体由来試料は、血液のほか、生体由来の組織、細胞、体液、排泄物等をいい、体液には血漿、血清等の血液由来試料、唾液、乳等が含まれる。排泄物には尿、便等が含まれる。細胞には血液から分離した白血球、口腔内粘膜擦過細胞、鼻粘膜細胞等が含まれるが、これらに限定されるものではなく、生体から採取できるものであれば試料として利用できる。 In the method for evaluating human positive emotion or health according to the present invention, first, in step a, the expression level of at least one gene in the gene sets shown in gene sets 1 to 8 is converted into a biological sample such as blood, Measure in The biological sample to be measured refers to tissues, cells, body fluids, excreta and the like derived from living bodies in addition to blood, and the body fluid includes blood-derived samples such as plasma and serum, saliva, milk and the like. Excrements include urine and feces. The cells include leukocytes separated from blood, oral mucosal scraping cells, nasal mucosal cells, and the like, but are not limited thereto, and any cell can be used as long as it can be collected from a living body.
　遺伝子発現量の測定は、上記ラット陽性感情評価方法の節に記載されるようにして行えばよい。この関連で、本発明に係るヒトの陽性感情又は健康度を評価するための検査キットは、上記遺伝子セット1～8に示す遺伝子セット中の遺伝子の塩基配列の少なくとも一部を含む核酸からなる群から選ばれる少なくとも1種の核酸、或いは、上記遺伝子セット1～8に示す遺伝子セット中の遺伝子によってコードされるタンパク質に特異的に結合する分子（例えば抗体）からなる群から選ばれる少なくとも1種の分子を備えている。 The measurement of the gene expression level may be performed as described in the section of the rat positive emotion evaluation method. In this connection, the test kit for evaluating human positive emotion or health according to the present invention is a group consisting of nucleic acids containing at least part of the base sequences of genes in the gene sets shown in the gene sets 1 to 8 above. Or at least one selected from the group consisting of molecules (for example, antibodies) that specifically bind to a protein encoded by a gene in the gene set shown in the gene set 1 to 8 above. It has molecules.
　次いで、上記測定結果に基づき、ステップbにおいて、ヒトの陽性感情又は健康度の評価を行う。 Next, based on the measurement result, in step b, the human positive emotion or health level is evaluated.
　ステップbにおける評価は、遺伝子セット1～8中の遺伝子についての基準値に対して、ステップaで測定した遺伝子の発現量から算出されるスコアが異なるかを判断することによって行うことができる。 The evaluation in step b can be performed by judging whether the score calculated from the expression level of the gene measured in step a is different from the reference value for the genes in gene sets 1-8.
　具体的には、遺伝子セット1～8に挙げる遺伝子のうち、発現量から算出されるスコアが、「Up/down」の欄がUpの遺伝子については基準値より高くなっているか否かを判断し、Downの遺伝子については基準値より低くなっている否かを判断する。 Specifically, among the genes listed in gene sets 1 to 8, it is determined whether or not the score calculated from the expression level is higher than the reference value for genes whose Up / down column is Up. It is determined whether the Down gene is lower than the reference value.
　上記「基準値」は、遺伝子セット1～8に挙げる陽性感情マーカー遺伝子ごとに設定される。本発明において、基準値は、例えば、笑いを誘発する刺激を負荷していない対照ヒトにおけるヒト陽性感情マーカー遺伝子の発現量を、対照ヒトと陽性感情にあるヒトとの間で発現量の有意な差が認められない任意の遺伝子Xの発現量に対する相対値として示した値とすることができる。この場合、上記スコアは、ステップaで測定したヒト陽性感情マーカー遺伝子の発現量を、遺伝子Xの発現量で除算することによって算出することができる。かかる基準値は、遺伝子セット1～8に記載の各遺伝子について予め求めておいてもよいし、対照ヒトにおける基準値の測定を本発明のステップaと平行して行ってもよい。基準値は、複数の対照ヒトで算出することにより、基準値±標準偏差とすることが好ましい。これにより基準値に対する個体差の影響を排除することができる。またこれに関連して、本発明に係るヒトの陽性感情又は健康度を評価するための検査キットは、上記遺伝子Xの塩基配列を有する核酸又は遺伝子Xがコードするタンパク質に対する抗体をさらに含むことができる。これにより、対照ヒトにおいて基準値を測定するために、及び評価対象のヒトにおいて上記スコアを算出するために、遺伝子Xの発現量を求めることができる。また、本発明に係るヒトの陽性感情又は健康度を評価するための検査キットは、遺伝子セット1～8に示す陽性感情マーカー遺伝子のそれぞれについて予め算出された基準値（±標準偏差）の対応表を含むことが好ましい。これにより、対照ヒトを用いた基準値の算出を省略することができる。 The above “reference value” is set for each positive emotion marker gene listed in gene sets 1-8. In the present invention, the reference value refers to, for example, the expression level of a human positive emotion marker gene in a control human that is not loaded with a stimulus that induces laughter, and a significant expression level between the control human and a human in positive emotion. It can be a value shown as a relative value with respect to the expression level of any gene X in which no difference is observed. In this case, the score can be calculated by dividing the expression level of the human positive emotion marker gene measured in step a by the expression level of gene X. Such a reference value may be obtained in advance for each gene described in gene sets 1 to 8, or the reference value in a control human may be measured in parallel with step a of the present invention. The reference value is preferably set to be a reference value ± standard deviation by calculating with a plurality of control humans. Thereby, the influence of the individual difference with respect to a reference value can be excluded. In this connection, the test kit for evaluating human positive emotion or health according to the present invention may further comprise an antibody against the nucleic acid having the base sequence of gene X or the protein encoded by gene X. it can. Thereby, the expression level of gene X can be determined in order to measure the reference value in the control human and to calculate the score in the human to be evaluated. In addition, the test kit for evaluating human positive emotion or health according to the present invention is a correspondence table of reference values (± standard deviation) calculated in advance for each of the positive emotion marker genes shown in gene sets 1 to 8. It is preferable to contain. Thereby, calculation of the reference value using the control human can be omitted.
　ここで、評価の一例を挙げると、例えば、遺伝子セット1～8に記載されるヒト陽性感情マーカー遺伝子のうち、発現量が「Up」の遺伝子から遺伝子を選択し、選択した遺伝子の発現量及び遺伝子Xの発現量を測定し、この選択した遺伝子の発現量を遺伝子Xの発現量で除算した値が、該遺伝子について予め算出しておいた基準値を有意に上回っている場合に、ヒトは陽性感情である又は健康度が高いと評価することができる。なお、かかる評価の信頼度を高めるために、複数の遺伝子について上記判断を行い、50％以上、より好ましくは60％以上、より好ましくは70％以上、より好ましくは80％以上、より好ましくは90％以上、最も好ましくは100％の遺伝子において基準値を有意に上回っている場合に、ヒトは陽性感情である又は健康度が高いと評価することとしてもよい。あるいは、対照に対する遺伝子発現量の増加率又は減少率を、選択した複数の遺伝子のそれぞれについて求め、その百分比合計が所定の数値を超える場合に、ヒトは陽性感情である又は健康度が高いと評価してもよい。 Here, as an example of evaluation, for example, among human positive emotion marker genes described in gene sets 1 to 8, a gene is selected from genes whose expression level is “Up”, and the expression level of the selected gene and When the expression level of gene X is measured, and the value obtained by dividing the expression level of the selected gene by the expression level of gene X is significantly higher than the reference value calculated in advance for the gene, It can be evaluated that it is a positive emotion or a high degree of health. In order to increase the reliability of such evaluation, the above determination is made for a plurality of genes, and is 50% or more, more preferably 60% or more, more preferably 70% or more, more preferably 80% or more, more preferably 90%. % Or more, and most preferably 100% of the genes may be evaluated as positive emotions or high health if they are significantly above the reference value. Alternatively, the increase rate or decrease rate of the gene expression level relative to the control is obtained for each of a plurality of selected genes, and when the percentage sum exceeds a predetermined value, the human is evaluated as having positive emotions or high health. May be.
　本発明に係るヒトの陽性感情又は健康度の評価方法及び検査キットによれば、陽性感情又は健康度を簡便且つ高い信頼性をもって評価することができる。特に、本発明に係るヒトの陽性感情又は健康度の評価方法において、測定対象の遺伝子の種類を増やすこと（例えば10種～100種又はそれ以上）によって、より優れた信頼度をもって、ヒトの陽性感情又は健康度を評価することができる。 According to the human positive emotion or health evaluation method and test kit according to the present invention, positive emotion or health can be evaluated simply and with high reliability. In particular, in the method for evaluating human positive emotion or health according to the present invention, by increasing the number of genes to be measured (for example, 10 to 100 or more), human positive can be obtained with higher reliability. Emotion or health can be assessed.
１－３．ラット共通遺伝子及びヒト共通遺伝子
　ヒトにおけるTicking刺激およびユーモア刺激により誘発される笑いには、共通性があることが分かってきている。（文献　Biological Psychology 30(1990)141-150, 笑いの科学 2008/05/12 vol.1）。 1-3. Rat common genes and human common genes It has been found that laughter induced by Ticking and humor stimuli in humans has a commonality. (Literature Biological Psychology 30 (1990) 141-150, Science of Laughter 2008/05/12 vol.1).
　また、ラットにおいて、Ticking刺激により50kHz近傍の発生音声を誘発する行為が、ヒトの笑いの原型ではないかと提案している。（文献　Psychology & Behaivior 79 (2003)533-547）これらのことから、ラットのTicking刺激とヒトの笑いとの関係については、共通する生体への生理作用があり、遺伝子発現が類似していることが推測できる。 Also, in rats, it is proposed that the act of inducing a generated voice near 50kHz by Ticking stimulation is the prototype of human laughter. (Reference: Psychology & hai Behaivior 79 (2003) 533-547) From these facts, the relationship between rat ticking stimuli and human laughter has a common physiological effect on the living body and similar gene expression. Can be guessed.
　そこで本願発明者らは、上記ラット陽性感情マーカー遺伝子及び上記ヒト陽性感情マーカー遺伝子間で、それぞれTickling刺激及び笑い刺激負荷後に、同様の遺伝子発現挙動を示す遺伝子が存在するかどうかを調べたところ、40種の共通遺伝子が存在することを見出した（本明細書中、それぞれラット共通遺伝子、ヒト共通遺伝子と称する）。かかるラット共通遺伝子及びヒト共通遺伝子は、ラット及びヒトの陽性感情評価系で、及びヒトの健康度評価系で、特に有用な陽性感情評価マーカーとして利用できると考えられる。 Therefore, the present inventors examined whether there is a gene exhibiting the same gene expression behavior after the Tickling stimulus and the laughing stimulus load between the rat positive emotion marker gene and the human positive emotion marker gene, respectively. It was found that 40 kinds of common genes exist (in the present specification, they are referred to as rat common gene and human common gene, respectively). Such a rat common gene and a human common gene are considered to be usable as a particularly useful positive emotion evaluation marker in a rat and human positive emotion evaluation system and in a human health evaluation system.
　したがって、本発明のラット又はヒトの陽性感情評価方法、又はヒトの健康度評価方法において、上記ヒト陽性感情マーカー遺伝子又はラット陽性感情マーカー遺伝子に加えて又はこれに代えて、ラット共通遺伝子又はヒト共通遺伝子を使用することができる。 Therefore, in the rat or human positive emotion evaluation method or human health evaluation method of the present invention, in addition to or in place of the human positive emotion marker gene or rat positive emotion marker gene, the rat common gene or human common Genes can be used.
　これらヒト共通遺伝子及びラット共通遺伝子は、それぞれ下記表202及び205に要約されている。 These human common gene and rat common gene are summarized in Tables 202 and 205 below, respectively.
１－４．他の評価手法
　本発明の陽性感情又は健康度の評価方法において、好ましくは、評価の信頼度を高めるために、複数の陽性感情マーカー遺伝子を1つの遺伝子群として使用し、該遺伝子群の遺伝子発現量を網羅的に解析することによって、陽性感情又は健康度を評価する。 1-4. Other Evaluation Methods In the positive emotion or health level evaluation method of the present invention, preferably, in order to increase the reliability of the evaluation, a plurality of positive emotion marker genes are used as one gene group, and the gene expression of the gene group Assess positive emotions or health by comprehensively analyzing quantities.
　ラット陽性感情評価方法において、上記遺伝子群として、例えば次のものを使用することができる：表1に列挙される遺伝子群；表2に列挙される遺伝子群；表81～84のいずれかに列挙される遺伝子群；表45～59のいずれかに列挙される遺伝子群；及び表205に列挙される遺伝子群；又はこれらの遺伝子群の組合せ。 In the rat positive emotion evaluation method, for example, the following can be used as the above gene group: gene group listed in Table 1; gene group listed in Table 2; listed in any of Tables 81 to 84 A gene group listed in any of Tables 45 to 59; and a gene group listed in Table 205; or a combination of these gene groups.
　またヒト陽性感情又はヒト健康度の評価方法において、上記遺伝子群として、例えば次のものを使用することができる：表164に列挙される遺伝子群(又は遺伝子セット1)；表172(又は遺伝子セット2)；表178に列挙される遺伝子群(又は遺伝子セット3)；表185に列挙される遺伝子群(又は遺伝子セット4)；表155に列挙される遺伝子群(又は遺伝子セット5)；表159に列挙される遺伝子群(又は遺伝子セット6)；表190に列挙される遺伝子群(又は遺伝子セット7)；表198に列挙される遺伝子群(又は遺伝子セット8)；表202に列挙される遺伝子群；又はこれらの遺伝子群の組合せ。 Further, in the method for evaluating human positive emotion or human health, as the above gene group, for example, the following can be used: gene group (or gene set 1) listed in Table 164; table 172 (or gene set) 2); gene group listed in Table 178 (or gene set 3); gene group listed in Table 185 (or gene set 4); gene group listed in Table 155 (or gene set 5); Gene group (or gene set 6) listed in Table 190; gene group listed in Table 190 (or gene set 7); gene group listed in Table 198 (or gene set 8); gene listed in Table 202 Group; or a combination of these genes.
１－４－１．発現方向の一致率の評価
　本発明の陽性感情又は健康度の評価方法において、陽性感情又は健康度の評価は、上記のような遺伝子群に含まれる全遺伝子の発現量を生体由来試料にて測定し、各遺伝子の発現増減の方向の一致率を評価することによって行われる。一致率(％)は、各遺伝子について示されている発現方向(Up/Down)に向かって基準値に対して有意に増減している遺伝子数を、遺伝子群に含まれる遺伝子の総数で割った値に100を乗じることによって求めることができる。 1-4-1. Evaluation of coincidence rate of expression direction In the positive emotion or health level evaluation method of the present invention, the evaluation of positive emotion or health level is performed by measuring the expression level of all genes contained in the gene group as described above using a biological sample. Then, it is performed by evaluating the coincidence rate in the direction of increase / decrease in the expression of each gene. The coincidence rate (%) was obtained by dividing the number of genes significantly increasing or decreasing with respect to the reference value toward the expression direction (Up / Down) indicated for each gene by the total number of genes included in the gene group. It can be obtained by multiplying the value by 100.
　この場合、例えば、一致率(％)が50％以上、より好ましくは60％以上、より好ましくは70％以上、より好ましくは80％以上、より好ましくは90％以上、最も好ましくは100％である場合に、被験体は陽性感情である又は健康度が高いと評価することができる。 In this case, for example, the coincidence rate (%) is 50% or more, more preferably 60% or more, more preferably 70% or more, more preferably 80% or more, more preferably 90% or more, and most preferably 100%. In some cases, a subject can be evaluated as having a positive emotion or high health.
　あるいは、かかる評価手法を後述する陽性刺激評価系にて使用する場合には、評価対象刺激負荷後の上記一致率を複数の評価対象刺激について求め、最も高い一致率を生じさせた評価対象刺激を陽性感情又は健康増進に導く良好な刺激として選択することができる。 Alternatively, when such an evaluation method is used in a positive stimulus evaluation system, which will be described later, the above-mentioned coincidence rate after the evaluation object stimulus load is obtained for a plurality of evaluation object stimuli, and the evaluation object stimulus that has produced the highest coincidence rate It can be selected as a positive emotion or a good stimulus that leads to better health.
１－４－２．発現強度のスコア化
　本発明の陽性感情又は健康度の評価方法において、陽性感情又は健康度の評価は、上記のような遺伝子群に含まれる全遺伝子の発現量を生体由来試料にて測定し、測定した遺伝子の発現量とTickling刺激又は笑い刺激負荷後の(すなわち陽性感情の被験体の)該遺伝子の発現量との差を、遺伝子群に含まれる全遺伝子について比較することによって行うことができる。具体的には、測定した遺伝子発現量とTickling刺激又は笑い刺激負荷後の遺伝子発現量との差を二乗したスコアの総和を次式に従って取得する。
Figure JPOXMLDOC01-appb-M000001
 1-4-2. Expression strength scoring In the positive emotion or health evaluation method of the present invention, the evaluation of positive emotion or health measures the expression level of all genes contained in the gene group as described above in a biological sample, The difference between the measured gene expression level and the expression level of the gene after loading with a Tickling stimulus or laughing stimulus (i.e., in a subject with a positive emotion) can be performed by comparing all the genes included in the gene group. . Specifically, the sum of scores obtained by squaring the difference between the measured gene expression level and the gene expression level after loading with a Tickling stimulus or laughing stimulus is obtained according to the following equation.
Figure JPOXMLDOC01-appb-M000001
[式中、nは遺伝子群に含まれる遺伝子の総数であり；aiは生体由来試料にて測定された遺伝子iの発現量であり；biはTickling刺激又は笑い刺激負荷後の遺伝子iの発現量である]
　このスコアは、測定された遺伝子発現量と、Tickling刺激又は笑い刺激負荷後の遺伝子発現量との差が小さいほど小さくなる。したがって、被験体の陽性感情の程度又は健康度をこのスコアを指標にして推定することができる。 [Wherein n is the total number of genes contained in the gene group; a i is the expression level of gene i measured in the biological sample; b i is the gene i after Tickling stimulation or laughing stimulation loading] Expression level]
This score becomes smaller as the difference between the measured gene expression level and the gene expression level after Tickling stimulation or laughing stimulation loading is smaller. Therefore, the degree of positive emotion or the degree of health of the subject can be estimated using this score as an index.
　例えば、かかる評価手法を後述する陽性刺激評価系にて使用する場合には、評価対象刺激負荷後の上記スコアを複数の評価対象刺激について求め、最も低いスコアを生じさせた評価対象刺激を陽性感情又は健康増進に導く良好な刺激として選択することができる。 For example, when such an evaluation method is used in a positive stimulus evaluation system, which will be described later, the score after the evaluation target stimulus load is obtained for a plurality of evaluation target stimuli, and the evaluation target stimulus that generated the lowest score is positive emotion. Or it can be selected as a good stimulus leading to health promotion.
１－４－３．発現方向の一致率の評価と発現強度のスコア化の組合せ
　本発明の陽性感情又は健康度の評価方法において、陽性感情又は健康度の評価は、上記(1－4－1)と(1－4－2)の手法を組合わせることによって行うことができる。具体的には、上記のような遺伝子群に含まれる全遺伝子の発現量を生体由来試料にて測定し、各遺伝子について示されている発現方向(Up/Down)に向かって基準値に対して有意に増減している遺伝子を選別し、測定した遺伝子発現量とTickling刺激又は笑い刺激負荷後の遺伝子発現量との差を二乗したスコアを選別した各遺伝子について求め、これを総和したスコアを次式に従って取得する。
Figure JPOXMLDOC01-appb-M000002
 1-4-3. Combination of evaluation of coincidence rate of expression direction and scoring of expression intensity In the method of evaluating positive emotion or health level of the present invention, the evaluation of positive emotion or health level is the above (1-4-1) and (1-4). This can be done by combining the methods of -2). Specifically, the expression level of all genes included in the gene group as described above is measured with a sample derived from a living body, and the reference value is directed toward the expression direction (Up / Down) indicated for each gene. The genes that significantly increased or decreased were selected, the score obtained by squaring the difference between the measured gene expression level and the gene expression level after Tickling stimulation or laughing stimulation loading was obtained for each gene, and the total score was calculated as follows. Get according to the formula.
Figure JPOXMLDOC01-appb-M000002
[式中、mは、生体由来試料にて測定された遺伝子群中の遺伝子のうち、Tickling刺激又は笑い刺激負荷後の遺伝子発現方向と同方向に向かって基準値に対して有意に増減している遺伝子の数であり；aiは生体由来試料にて測定された遺伝子iの発現量であり；biはTickling刺激又は笑い刺激負荷後の遺伝子iの発現量である]
　このスコアは、測定された遺伝子発現量と、Tickling刺激又は笑い刺激負荷後の遺伝子発現量との差が小さいほど小さくなる。したがって、被験体の陽性感情の程度又は健康度をこのスコアを指標にして推定することができる。 [In the formula, m is significantly increased or decreased with respect to the reference value in the same direction as the gene expression direction after Tickling stimulation or laughing stimulation load among genes in the gene group measured in the biological sample. A i is the expression level of gene i measured in a biological sample; b i is the expression level of gene i after Tickling stimulation or laughing stimulation loading]
This score becomes smaller as the difference between the measured gene expression level and the gene expression level after Tickling stimulation or laughing stimulation loading is smaller. Therefore, the degree of positive emotion or the degree of health of the subject can be estimated using this score as an index.
　例えば、かかる評価手法を後述する陽性刺激評価系にて使用する場合には、評価対象刺激負荷後の上記スコアを複数の評価対象刺激について求め、最も低いスコアを生じさせた評価対象刺激を陽性感情又は健康増進に導く良好な刺激として選択することができる。 For example, when such an evaluation method is used in a positive stimulus evaluation system, which will be described later, the score after the evaluation target stimulus load is obtained for a plurality of evaluation target stimuli, and the evaluation target stimulus that generated the lowest score is positive emotion. Or it can be selected as a good stimulus leading to health promotion.
１－４－４．カルバック・ライブラー（Kullback-Leibler）相互情報量
　本発明の陽性感情又は健康度の評価方法において、陽性感情又は健康度の評価は、カルバック・ライブラー(Kullback-Leibler)相互情報量に基づいて行うことができる。本発明において、カルバック・ライブラー相互情報量は、上記のような遺伝子群の生体由来試料における発現量及び発現パターンと、Tickling刺激又は笑い刺激負荷後の(すなわち陽性感情の被験体の)前記遺伝子群の発現量及び発現パターンとの差の尺度として取得される。具体的には、aiを次式：
Figure JPOXMLDOC01-appb-M000003
 1-4-4. Mutual information amount of Kullback-Leibler In the method of evaluating positive emotion or health level of the present invention, the evaluation of positive emotion or health level is performed based on the mutual information amount of Kullback-Leibler. be able to. In the present invention, the amount of mutual information of Cullback-Liber is the expression level and expression pattern in the biological sample of the gene group as described above, and the gene after loading with a Tickling stimulus or laughing stimulus (ie, a subject with a positive emotion). It is obtained as a measure of the difference between the expression level and expression pattern of the group. Specifically, a i is expressed by the following formula:
Figure JPOXMLDOC01-appb-M000003
に従い正規化することで、
Figure JPOXMLDOC01-appb-M000004
 By normalizing according to
Figure JPOXMLDOC01-appb-M000004
　カルバック・ライブラー相互情報量は、その値が小さいほど、比較される2群間の遺伝子発現量及び遺伝子発現パターンがより近似していることを示す。したがって、被験体の陽性感情の程度又は健康度は、Tickling刺激又は笑い刺激負荷後の発現量及び発現パターンを基準にして推定することができる。 The smaller the value of the Cullback-Liber mutual information, the closer the gene expression level and gene expression pattern between the two groups to be compared. Therefore, the degree of positive emotion or the degree of health of the subject can be estimated based on the expression level and expression pattern after loading the Tickling stimulus or laughing stimulus.
　例えば、かかる評価手法を後述する陽性刺激評価系にて使用する場合には、Tickling刺激又は笑い刺激負荷後の発現量及び発現パターンを基準とした評価対象刺激後のカルバック・ライブラー相互情報量を、複数の評価対象刺激について求め、最も小さいカルバック・ライブラー相互情報量を生じさせた評価対象刺激を陽性感情又は健康増進に導く良好な刺激として選択することができる。 For example, when such an evaluation method is used in a positive stimulus evaluation system, which will be described later, the expression level after the load of Tickling stimulus or laughing stimulus and the amount of mutual information after the stimulus to be evaluated based on the expression pattern is calculated. Thus, it is possible to obtain a plurality of evaluation target stimuli and select the evaluation target stimulus that generates the smallest amount of mutual information between the Cullback and the library as a positive stimulus or a good stimulus that leads to health promotion.
　なお、カルバック・ライブラー相互情報量を、下記式によって定義してもよい。
Figure JPOXMLDOC01-appb-M000005
 Note that the amount of mutual information between the Cullback and the library may be defined by the following equation.
Figure JPOXMLDOC01-appb-M000005
　さらに、下記式:
Figure JPOXMLDOC01-appb-M000006
 Furthermore, the following formula:
Figure JPOXMLDOC01-appb-M000006
と対称化した相互情報量Jをカルバック・ライブラー相互情報量の代わりに使ってもよい。なお、カルバック・ライブラー相互情報量ではなく、その他の相互情報量、例えば、Hellinger相互情報量など、を使ってもよい。 The mutual information J that is symmetrized with may be used instead of the mutual information of Cullback-Liber. It should be noted that other mutual information, for example, Hellinger mutual information, may be used instead of the Cullback-Librer mutual information.
２．陽性刺激評価系
　本発明はまた、所与の刺激がラット及びヒトに対する陽性刺激であるか否かを評価する方法（以下、それぞれ「ラット陽性刺激評価方法」及び「ヒト陽性刺激評価方法」とも称する）を提供する。 2. Positive Stimulus Evaluation System The present invention is also a method for evaluating whether a given stimulus is a positive stimulus for rats and humans (hereinafter also referred to as “rat positive stimulus evaluation method” and “human positive stimulus evaluation method”, respectively). )I will provide a.
　本明細書で使用する「陽性刺激」とは、被験体（すなわちラット又はヒト）を陽性感情に導くか又は被験体(すなわちヒト)を健康増進に導く、任意の刺激を指す。また本発明で使用する「刺激」とは、生体に作用してなんらかの現象や反応を起こさせるものを指し、外的な刺激と、内的な刺激を含む。刺激の種類には、物理的、化学的、生物的、心理的な刺激が挙げられる。これらの刺激は、視覚的、聴覚的、味覚的、嗅覚的、前庭感覚的、体性感覚的、内臓感覚的、運動感覚的刺激の１種以上である。また、陽性刺激であるか否かの評価対象となる刺激は、その種類だけではなく、断続的又は継続的な刺激といった刺激の負荷様式についても評価される。 As used herein, “positive stimulus” refers to any stimulus that leads the subject (ie, rat or human) to positive emotions or leads the subject (ie, human) to health promotion. The “stimulus” used in the present invention refers to a substance that acts on a living body to cause some phenomenon or reaction, and includes an external stimulus and an internal stimulus. Types of stimuli include physical, chemical, biological, and psychological stimuli. These stimuli are one or more of visual, auditory, gustatory, olfactory, vestibular sensation, somatosensory, visceral sensation, and kinematic sensation. Moreover, the stimulus to be evaluated as to whether or not it is a positive stimulus is evaluated not only for the type but also for the stimulus loading pattern such as intermittent or continuous stimulus.
２－１．ラット陽性刺激評価方法
　本発明に係るラット陽性刺激評価方法は、ラットに評価対象である刺激を負荷する第1のステップと、少なくとも1種のラット陽性感情マーカー遺伝子の発現量を測定する第2のステップと、測定した前記ラット陽性感情マーカー遺伝子の発現量に基づいて、対象刺激がラットに対する陽性刺激であるか否かを判断する第3のステップとを含んでいる。 2-1. Rat positive stimulus evaluation method The rat positive stimulus evaluation method according to the present invention includes a first step of loading a rat with a stimulus to be evaluated, and a second step of measuring an expression level of at least one rat positive emotion marker gene. And a third step of determining whether the target stimulus is a positive stimulus for the rat based on the measured expression level of the rat positive emotion marker gene.
　第1のステップでは、ラットに評価対象である刺激を負荷する。刺激の負荷期間は特に制限されないが、ラット陽性感情マーカー遺伝子として表1に示す遺伝子群を使用する場合には短期間で負荷し、ラット陽性感情マーカー遺伝子として表2に示す遺伝子群を使用する場合には長期間で負荷することが好ましい。なお、本発明において、用語「短期間」は1～数日間（例えば2日、3日、4日又は5日）を、「長期間」は3週間～4週間を指す。 In the first step, the stimulus to be evaluated is loaded on the rat. The stimulation period is not particularly limited, but when the gene group shown in Table 1 is used as a rat positive emotion marker gene, it is loaded in a short period, and the gene group shown in Table 2 is used as a rat positive emotion marker gene. It is preferable to load for a long time. In the present invention, the term “short period” refers to 1 to several days (for example, 2, 3, 4 or 5 days), and “long period” refers to 3 to 4 weeks.
　次いで第2のステップでは、表1、2に示されるラット陽性感情マーカー遺伝子の少なくとも1種の遺伝子の発現量を指定の生体由来試料において測定する。なお、測定対象のラット陽性感情マーカー遺伝子として、表1、2に加えて又はこれに代えて表81～84及び/又は45～59に示す遺伝子の少なくとも1種の発現量を測定してもよいこと、及び表1、2、81～84及び45～59のいずれかに列挙される遺伝子群の発現量を測定してもよいこと、については上記と同様であり、その測定方法についても上記の通りである。 Next, in the second step, the expression level of at least one of the rat positive emotion marker genes shown in Tables 1 and 2 is measured in a designated biological sample. As the rat positive emotion marker gene to be measured, the expression level of at least one of the genes shown in Tables 81 to 84 and / or 45 to 59 may be measured in addition to or instead of Tables 1 and 2. And that the expression level of the gene group listed in any of Tables 1, 2, 81 to 84 and 45 to 59 may be measured, and the measurement method is also described above. Street.
　次いで第3のステップで、第2のステップで測定したラット陽性感情マーカー遺伝子の発現量に基づいて、対象刺激がラット陽性刺激であるか否かを評価する。評価は、上記の通り、第2のステップで測定したラット陽性感情マーカー遺伝子の発現量から算出されるスコアが、当該遺伝子の基準値と異なるかを判定することによって行うことができる。 Then, in the third step, based on the expression level of the rat positive emotion marker gene measured in the second step, it is evaluated whether or not the target stimulus is a rat positive stimulus. As described above, the evaluation can be performed by determining whether the score calculated from the expression level of the rat positive emotion marker gene measured in the second step is different from the reference value of the gene.
　第3のステップにおける対象刺激の評価は、対象刺激の負荷前後のラット陽性感情マーカー遺伝子の発現量を比較することによって行ってもよい。すなわち、対象刺激の負荷前に測定したラット陽性感情マーカー遺伝子の発現量に比較して、刺激負荷後に当該遺伝子の発現量が増加又は減少していれば、当該刺激はラット陽性刺激であると評価することができる。 The evaluation of the target stimulus in the third step may be performed by comparing the expression level of the rat positive emotion marker gene before and after the load of the target stimulus. That is, if the expression level of the gene increases or decreases after the stimulation load compared to the expression level of the rat positive emotion marker gene measured before the target stimulation load, the stimulation is evaluated as a rat positive stimulation. can do.
　したがって、本発明に係るラット陽性刺激評価方法は、ラットに評価の対象である刺激を負荷する第1のステップの前にラット陽性感情マーカー遺伝子の発現量を測定するステップをさらに含むことができる。なおこの場合、生体由来試料として血液、口腔粘膜細胞又は唾液腺を利用する必要があるため、測定対象の陽性感情マーカー遺伝子は、これらの生体由来試料においてラット陽性感情マーカー遺伝子として機能するものを選択する。 Therefore, the rat positive stimulus evaluation method according to the present invention can further include the step of measuring the expression level of the rat positive emotion marker gene before the first step of loading the rat with the stimulus to be evaluated. In this case, since it is necessary to use blood, oral mucosal cells or salivary glands as a biological sample, a positive emotion marker gene to be measured is selected from those that function as a rat positive emotion marker gene in these biological samples. .
　また第3のステップにおける対象刺激の評価は、対照ラットとの比較によって行ってもよい。すなわち、対象刺激を負荷したラットと、対象刺激を負荷しない対照ラットの両者で同一の陽性感情マーカー遺伝子の発現量を測定し、対象刺激を負荷したラットにおいて当該遺伝子の発現量が増加又は減少していれば、対象刺激はラット陽性刺激であると評価することができる。 In addition, the evaluation of the target stimulus in the third step may be performed by comparison with a control rat. That is, the expression level of the same positive emotion marker gene is measured in both the rat loaded with the target stimulus and the control rat not loaded with the target stimulus, and the expression level of the gene increases or decreases in the rat loaded with the target stimulus. If so, the target stimulus can be evaluated as a rat positive stimulus.
２－２．ヒト陽性刺激評価方法
　本発明に係るヒト陽性刺激評価方法は、ヒトに評価対象である刺激を負荷する第1のステップと、少なくとも1種のヒト陽性感情マーカー遺伝子の発現量を測定する第2のステップと、測定した前記ヒト陽性感情マーカー遺伝子の発現量に基づいて、対象刺激がヒトに対する陽性刺激であるか否かを判断する第3のステップとを含んでいる。 2-2. Human positive stimulus evaluation method The human positive stimulus evaluation method according to the present invention includes a first step of loading a stimulus to be evaluated to a human and a second step of measuring an expression level of at least one human positive emotion marker gene. And a third step of determining whether the target stimulus is a positive stimulus for a human based on the measured expression level of the human positive emotion marker gene.
　第1のステップでは、ヒトに評価対象である刺激を負荷する。刺激の負荷期間は特に制限されないが、短期間で負荷することがより好ましい。なお、ヒト陽性刺激評価方法の関連で使用する用語「短期間」は１秒～数分又は数時間を指す。 In the first step, a stimulus to be evaluated is loaded on a human. Although the stimulus loading period is not particularly limited, it is more preferable to load in a short period. The term “short term” used in the context of the human positive stimulus evaluation method refers to 1 second to several minutes or several hours.
　次いで第2のステップでは、遺伝子マーカー1～8に示されるヒト陽性感情マーカー遺伝子の少なくとも1種の遺伝子の発現量を生体由来試料、例えば血液、において測定する。なお、測定対象のヒト陽性感情マーカー遺伝子として、遺伝子マーカー1～8に加えて又はこれに代えてヒト共通遺伝子の少なくとも1種の発現量を測定してもよいこと、及び遺伝子マーカー1～8のいずれかの遺伝子群、又はヒト共通遺伝子の遺伝子群の発現量を網羅的に測定してもよいこと、については上記と同様であり、その測定方法についても上記の通りである。 Next, in the second step, the expression level of at least one of the human positive emotion marker genes shown in gene markers 1 to 8 is measured in a biological sample such as blood. In addition to or instead of gene marker 1-8, the expression level of at least one human common gene may be measured as a human positive emotion marker gene to be measured, and gene markers 1-8 The expression level of any gene group or gene group of human common genes may be comprehensively measured as described above, and the measurement method is also as described above.
　次いで第3のステップで、第2のステップで測定したヒト陽性感情マーカー遺伝子の発現量に基づいて、対象刺激がヒト陽性刺激であるか否かを評価する。評価は、上記の通り、第2のステップで測定したヒト陽性感情マーカー遺伝子の発現量から算出されるスコアが、当該遺伝子の基準値と異なるかを判定することによって行うことができる。 Then, in the third step, whether or not the target stimulus is a human positive stimulus is evaluated based on the expression level of the human positive emotion marker gene measured in the second step. As described above, the evaluation can be performed by determining whether the score calculated from the expression level of the human positive emotion marker gene measured in the second step is different from the reference value of the gene.
　第3のステップにおける対象刺激の評価は、対象刺激の負荷前後のヒト陽性感情マーカー遺伝子の発現量を比較することによって行ってもよい。すなわち、対象刺激の負荷前に測定したヒト陽性感情マーカー遺伝子の発現量に比較して、刺激負荷後に当該遺伝子の発現量が増加又は減少していれば、当該刺激はヒト陽性刺激であると評価することができる。 The evaluation of the target stimulus in the third step may be performed by comparing the expression level of the human positive emotion marker gene before and after the load of the target stimulus. That is, if the expression level of the gene is increased or decreased after the stimulus load compared to the expression level of the human positive emotion marker gene measured before the target stimulus load, the stimulus is evaluated as a human positive stimulus. can do.
　したがって、本発明に係るヒト陽性刺激評価方法は、ヒトに評価の対象である刺激を負荷する第1のステップの前にヒト陽性感情マーカー遺伝子の発現量を測定するステップをさらに含むことができる。 Therefore, the human positive stimulus evaluation method according to the present invention can further include a step of measuring the expression level of the human positive emotion marker gene before the first step of loading a stimulus to be evaluated to a human.
　また第3のステップにおける対象刺激の評価は、対照ヒトとの比較によって行ってもよい。すなわち、対象刺激を負荷したヒトと、対象刺激を負荷しない対照ヒトの両者で同一の陽性感情マーカー遺伝子の発現量を測定し、対象刺激を負荷したヒトにおいて当該遺伝子の発現量が増加又は減少していれば、対象刺激はヒト陽性刺激であると評価することができる。 In addition, the evaluation of the target stimulus in the third step may be performed by comparison with a control human. That is, the expression level of the same positive emotion marker gene is measured in both the human subject loaded with the target stimulus and the control human subject not loaded with the target stimulus, and the expression level of the gene increases or decreases in the human loaded with the target stimulus. If so, the target stimulus can be evaluated as a human positive stimulus.
２－３．ラット共通遺伝子を用いたヒト陽性刺激探索方法
　上記の通り、ラット陽性感情マーカー遺伝子及びヒト陽性感情マーカー遺伝子間で、それぞれTickling刺激及び笑い刺激負荷後に、同様の遺伝子発現挙動を示す40種の共通遺伝子（それぞれラット共通遺伝子、ヒト共通遺伝子と称する）が存在することが見出された。かかるラット共通遺伝子及びヒト共通遺伝子は、ラット及びヒトの陽性感情評価系で特に有用な陽性感情評価マーカーとして利用できると考えられる。さらに、ラットにおいて評価の対象である刺激を負荷し、ラット共通遺伝子の発現量が増加又は減少していれば、対象刺激はラット陽性刺激であることが評価できるとともに、ヒトにおいても同様の効果がある刺激と推測できる。ヒト陽性刺激の探索を行う際、ラット共通遺伝子を用いたラットでの刺激評価は、ヒト陽性刺激の探索ツールとして利用できる。 2-3. Search method for human positive stimuli using common gene of rat As mentioned above, 40 common genes showing similar gene expression behavior after loading Tickling stimulus and laughter stimulus, respectively, between rat positive emotion marker gene and human positive emotion marker gene (Respectively called rat common gene and human common gene) were found to exist. Such rat common gene and human common gene are considered to be useful as positive emotion evaluation markers particularly useful in rat and human positive emotion evaluation systems. Furthermore, if the stimulus to be evaluated is loaded in the rat and the expression level of the common gene of the rat is increased or decreased, it can be evaluated that the target stimulus is a rat positive stimulus, and the same effect is also obtained in humans. It can be inferred as a certain stimulus. When searching for human positive stimuli, stimulation evaluation in rats using a common gene for rats can be used as a search tool for human positive stimuli.
　したがって、本発明のヒトの陽性刺激評価方法は、ラット共通遺伝子を用いて上記ラット陽性刺激評価を行うステップと、陽性刺激であると評価された刺激をヒト陽性刺激であると認定するステップとを含むことができる。 Accordingly, the human positive stimulus evaluation method of the present invention comprises the steps of performing the rat positive stimulus evaluation using a rat common gene, and the step of identifying the stimulus evaluated as a positive stimulus as a human positive stimulus. Can be included.
２－４．ラット陽性感情マーカー遺伝子を用いたヒト陽性感情マーカー遺伝子の探索方法
　ラットのTicking刺激とヒトの笑いについては、共通する生体への生理作用があり、遺伝子発現が類似していることが推測できる。また、Tickling刺激及び笑い刺激負荷後に、同様の遺伝子発現挙動を示す共通遺伝子が存在する。これらのことから、ラット陽性感情マーカー遺伝子のヒトホモログ遺伝子は、負荷した刺激に対してヒトにおいて遺伝子の発現変動が期待できる。そのため、ヒトホモログ遺伝子に注目した陽性感情評価実験を実施することにより、新たなヒト陽性感情マーカーおよびヒト陽性刺激評価方法を構築することができる。したがって、ラット陽性感情マーカーは、新たなヒト陽性感情マーカー遺伝子の探索方法となる。なお、ラット陽性感情マーカーのヒトホモログ遺伝子は、ラット遺伝子のアノテーションの表の「Homology」の欄に記載されている。 2-4. Method for Searching for Human Positive Emotional Marker Gene Using Rat Positive Emotional Marker Gene It can be inferred that rat ticking stimulation and human laughter have a common physiological effect on the living body and gene expression is similar. There are also common genes that show similar gene expression behavior after Tickling stimulation and laughing stimulation loading. From these facts, the human homologous gene of the rat positive emotion marker gene can be expected to change the gene expression in human in response to the applied stimulus. Therefore, a new human positive emotion marker and a human positive stimulus evaluation method can be constructed by conducting a positive emotion evaluation experiment focusing on the human homolog gene. Therefore, the rat positive emotion marker is a search method for a new human positive emotion marker gene. In addition, the human homolog gene of a rat positive emotion marker is described in the column of “Homology” in the table of rat gene annotation.
[実施例1]
発生音声の周波数解析によるラットへの陽性刺激負荷方法の確立
１-１．ラット飼育法の検討実験
　6週令のWistar系雄性ラットの単独飼育群(Single Housing)と集団飼育群(Social Housing、３匹飼い)に対し、陽性刺激であるTickling(下記1-2-1参照)を施行した。単独飼育群は、社会的孤立性を高めるため、ケージ間に衝立を設置した。その結果、単独飼育群ではラット陽性感情の指標である50KHz近傍の高周波音声(physiology & Behavior, 72:167-173, 2001)を確認したのに対し、集団飼育群では当音声は確認できなかった。このことは、単独飼育群では、術者によるTicklingのみが陽性刺激となっているため、Tickling刺激負荷時に陽性感情指標の50KHz近傍の高周波音声が確認できるのに対し、集団飼育群では同居ラット同士でじゃれ合うことによる陽性刺激が術者によるTicklingによる陽性刺激を上回るため、Tickling負荷では50KHz近傍の高周波音声が確認できないと結論付けた。 [Example 1]
Establishment of positive stimulation loading method to rat by frequency analysis of generated voice
1-1. Examination of rat breeding method Tickling (see 1-2-1 below) for 6 week old male Wistar male rats (Single Housing) and group housing group (Social Housing, 3 animals) ) Was enforced. In order to increase social isolation, the single rearing group has a screen between cages. As a result, high-frequency speech near 50 KHz (physiology & Behavior, 72: 167-173, 2001), which is an indicator of rat positive emotion, was confirmed in the single-bred group, but this voice was not confirmed in the group-bred group. . This is because, in the single breeding group, only Tickling by the surgeon is a positive stimulus, so high frequency sound near the positive emotion index of 50 KHz can be confirmed when Tickling stimulation is loaded, whereas in the group fed group, the cohabitation rats It was concluded that high-frequency sound in the vicinity of 50 KHz could not be confirmed with a Tickling load, because the positive stimulation caused by tampering exceeded the positive stimulation caused by the operator.
　以上の結果より、本研究ではラット単独飼育法を採用し、陽性感情指標である50KHz近傍の高周波音声による陽性感情評価を容易にすることにした。 Based on the above results, we decided to use a single rat rearing method in this study to facilitate the evaluation of positive emotions using high-frequency speech near 50 KHz, which is a positive emotion index.
１-２．ラットへのTickling刺激負荷の検討
１-２-１．実験方法（Tickling法、Light Touch法、対照）
1) Tickling刺激負荷法
　Ticklingとは触覚刺激負荷法であり、げっ歯類のRough-and-Tumble Playを模倣した方法である(J. Panksepp: Behavior Genetics 35, 2005)。具体的には、ラットの後から右手で回りこんで、コンピュータのマウスを握るようにラットをつかみ(Dorsal Contact)、ラットの後首筋を指でくすぐる。引き続き直ちにひっくり返し、ラットを床に押し付け(Pinning Behavior)、お腹を全体的に激しくくすぐり(この時、ラットの前足が中に浮くように、腰を床につけるように返すと抵抗がない)、解放する。これらの操作は2秒以内で行ない、15秒間継続する。 1-2. Examination of Tickling stimulation load on rats
1-2-1. Experimental method (Tickling method, Light Touch method, control)
1) Tickling stimulus loading method Tickling is a tactile stimulus loading method, which mimics the rodent Rough-and-Tumble Play (J. Panksepp: Behavior Genetics 35, 2005). Specifically, wrap around with the right hand from behind the rat, grab the rat to hold the computer mouse (Dorsal Contact), and tickle the rat's posterior muscle with your finger. Continue flipping immediately, pushing the rat to the floor (Pinning Behavior), tickling the belly violently (in this case, there is no resistance if the hip is put back on the floor so that the rat's front leg floats inside), release. These operations are done within 2 seconds and continue for 15 seconds.
　本実験では、ラットをTickling用クリーンケージ(内面は黒色フェルトを貼付)に移し、15秒間放置後、15秒のTickling刺激負荷を行なう操作を4回繰り返し、これを１Tickling Sessionとした。１Session終了後1分間の休憩を挟み、さらに２Session目(2分間)を行なった。図1にTickling刺激負荷時程図を示す。 In this experiment, the rat was transferred to a clean cage for Tickling (with black felt on the inner surface), left for 15 seconds, and then subjected to a 15-second Tickling stimulation load, which was repeated 4 times, and this was designated as 1 Tickling IV Session. After the 1st session, a 1-minute break was inserted, and the 2nd session (2 minutes) was performed. Fig. 1 shows the time chart of Tickling stimulation load.
2) Light Touch刺激負荷法
　ラットをTickling用クリーンケージ(上述)に移し15秒間放置の後、Ticklingの変わりに3秒に1回、ラット背部にやさしく触れる。刺激負荷時程は図1の通りとした。 2) Light Touch stimulation method Move the rat to the clean cage for Tickling (described above), leave it for 15 seconds, and then gently touch the back of the rat once every 3 seconds instead of Tickling. The stimulus loading time was as shown in FIG.
3) 対照
　ラットをTickling用クリーンケージ(上述)に移し、5分間(Tickling Session2回＋休憩1分間に相当)放置した。 3) Control Rats were transferred to a Tickling clean cage (described above) and left for 5 minutes (corresponding to 2 Tickling Sessions + 1 minute break).
１-２-２．実験方法（負荷週令、負荷期間）
　３週令(離乳直後)のWistar系雄性ラットを実験スケジュールに合わせ購入、単独飼育(ケージ間には衝立を設置)を開始した。単独飼育4週令目のラットをTickling群4匹、Light Touch群2匹、対照群2匹に、6週令目のラットをTickling群2匹、8週令目のラットをTickling群2匹に割り付けた。Tickling、Light Touchの刺激負荷方法および対照は上述１-２-１記載の方法とし、4週間(月曜～金曜、午後16:30～)継続して行なった。 1-2-2. Experiment method (load age, load period)
Three-week-old (immediately after weaning) Wistar male rats were purchased according to the experimental schedule, and were bred alone (with partitions between cages). Single-bred 4-week-old rats to 4 Tickling groups, 2 Light Touch groups, 2 control groups, 6-week-old rats to 2 Tickling groups, 8-week-old rats to 2 Tickling groups Allocated. The stimulation loading method and control of Tickling and Light Touch were the methods described in the above 1-2-1, and were performed continuously for 4 weeks (Monday to Friday, from 16:30 pm).
１-２-３．実験方法（超広帯域音声の収録）
　Tickling、Light Touchの刺激負荷時と休憩時、および対照実験の相当時点において、ラット陽性刺激の指標として50KHz近傍の音声を解析するため、個体毎に超広帯域音響解析システム(小野測器社製、DS-2100)で音声収録し、周波数解析を行った。高周波マイクロホン(小野測器社製、MI-3140)でラット音声を収録し、超広帯域音響解析システム(小野測器社製、DS-2100)でその時の時間波形をフーリエ変換の後、各周波数成分のピーク値をグラフ化して解析に供した。 1-2-3. Experimental method (recording of ultra-wideband audio)
Ultra-wideband acoustic analysis system (by Ono Sokki Co., Ltd.) for each individual in order to analyze sounds near 50 KHz as an index of rat positive stimulation at the time of Tickling, Light Touch stimulation and at rest, and at the equivalent time of the control experiment Audio was recorded with DS-2100) and frequency analysis was performed. Rat sound is recorded with a high-frequency microphone (Ono Sokki Co., Ltd., MI-3140), and the time waveform at that time is Fourier-transformed with an ultra-wideband acoustic analysis system (Ono Sokki Co., Ltd., DS-2100). The peak value of was graphed and used for analysis.
１-２-４．実験方法（接近待機時間の測定）
　Tickling、Light Touch刺激負荷の2Session終了後(対照は5分間放置後)、ラットをTickling用ケージ(上述)のコーナーに置き、対角線上(距離50cm)にある術者の手に接近接触するまでの時間を測定した。各刺激が快であれば、接近待機時間は短縮される。30秒以上経過しても接近接触しない場合は「30秒」とした。 1-2-4. Experimental method (measurement of approach waiting time)
After 2 sessions of Tickling and Light Touch stimulation load (control is left for 5 minutes), place the rat in the corner of the Tickling cage (described above) until it touches the operator's hand diagonally (distance 50 cm) Time was measured. If each stimulus is pleasant, the approach waiting time is shortened. If there is no close contact even after 30 seconds, “30 seconds” was set.
１-２-５．結果
1)超広帯域音声の解析
　超広帯域音響解析システムでフーリエ変換した各周波数成分のピーク値を下表４の通り示す。
Figure JPOXMLDOC01-appb-T000003
 1-2-5. result
1) Analysis of ultra-wideband speech The peak value of each frequency component that is Fourier transformed by the ultra-wideband acoustic analysis system is shown in Table 4 below.
Figure JPOXMLDOC01-appb-T000003
　４週令ラット及び６週令ラットでは、Tickling刺激初日から50KHz近傍(本実験では、FFT解析装置を使用したため、先行研究より10KHz程度高周波側にシフト)の音声を発したが、８週令ラットでは、全期間を通じ該音声を発しなかった。また、4週令ラットと６週令ラットでは、刺激開始２週目でTickling休憩時(15秒)にも陽性感情の表す該音声を発するようになった。Light Touchあるいは対照ラットでは、全期間を通じ発生音声は認められなかった。なお、本報告書には全データを記載できないが、刺激開始２週目が音声発生のピークであった。 In the 4-week-old rats and the 6-week-old rats, a voice of around 50 KHz from the first day of Tickling stimulation (in this experiment, the FFT analyzer was used, so it shifted to the high frequency side by about 10 KHz from the previous study). Then, the voice was not emitted throughout the whole period. In addition, the 4-week-old rat and the 6-week-old rat started to emit the voice representing positive emotion at the time of Tickling break (15 seconds) at the second week after the start of stimulation. In Light Touch or control rats, no utterances were observed throughout the period. In addition, although all data cannot be described in this report, the peak of voice generation was in the second week after the start of stimulation.
2)接近待機時間の測定
　刺激開始直後、刺激開始翌日、刺激開始２週目、刺激開始４週目における４週令ラットへの各処置直後の接近待機時間をそれぞれ図42、図43、図44、図45に示す。Tickling群の接近待機時間は、Light Touch群、対照群と比較すると有意に短縮(2週目のLight Touch群に対してはP=0.4)していた。刺激開始4週目になると３群間で差を認めなくなった。 2) Measurement of waiting time for approach Immediately after starting stimulation, the next day after starting stimulation, the second week of starting stimulation, and the fourth week of starting stimulation, the approaching waiting time immediately after each treatment to a 4-week-old rat is shown in FIGS. 42, 43, and 44, respectively. As shown in FIG. The approach waiting time of the Tickling group was significantly shortened compared to the Light Touch group and the control group (P = 0.4 for the Light Touch group at 2 weeks). At 4 weeks after the start of stimulation, there was no difference between the 3 groups.
１-２-６．考察・結論
　Tickling刺激は、離乳直後(３週令)から６週目までの施行が有効であり、８週目まで個別飼育するとTickling刺激に反応して発する50KHz近傍の音声を確認できなかった。これは、ラットの成長に伴う心身の変化や個別飼育に伴う過剰な慢性ストレスによるものと考えられた。また、Light Touch群では該音声を発しないことから、Tickling刺激は弁別刺激となっている。刺激開始２週目が最も反応する時期であり、Tickling刺激の休憩中(15秒間)でも陽性感情指標である該音声を発するようになり、接近待機時間も短縮されている。しかし、2週目になるとLight Touch群、対照群でも接近待機時間が短縮し、４週目で３群間に差を認めなくなるのは術者にラットが馴れてくるためであると推測された。 1-2-6. Discussion / Conclusion Tickling stimulation was effective from the first weaning (3 weeks old) to the 6th week, and when it was individually reared until the 8th week, it was not possible to confirm the voice near 50 KHz that was generated in response to the Tickling stimulation. This was thought to be due to changes in mind and body associated with the growth of rats and excessive chronic stress associated with individual breeding. In addition, since the sound is not emitted in the Light Touch group, the Tickling stimulus is a discrimination stimulus. The second week of the stimulus start is the most responsive time, and the voice, which is a positive emotion index, is emitted even during the tickling stimulus break (15 seconds), and the approach waiting time is also shortened. However, it was speculated that the approach waiting time was shortened in the Light Touch group and the control group at the second week, and that the difference between the three groups was not recognized at the fourth week because the rats became accustomed to the surgeon. .
　これらの結果より、Tickling刺激はラットにとって陽性感情をもたらす刺激であると結論付けた。 From these results, it was concluded that the Tickling stimulus is a stimulus that brings positive emotion to the rat.

[実施例２]
遺伝子発現解析実験
　上記１-２-２の４週令(離乳直後[３週令]から個別飼育)からTickling刺激を負荷したラット、Light Touch刺激を負荷したラット、対照ラットについて遺伝子発現解析を行なった。
[Example 2]
Gene expression analysis experiment Gene expression analysis was performed on rats loaded with Tickling stimulus, rats loaded with Light Touch stimulus, and control rats from 4 weeks of 1-2-2 above (individual breeding immediately after weaning [3 weeks of age]). It was.
２－１．長期間（４週間）のTickling刺激
２-１-１．実験方法（解析対象臓器の摘出）
　臓器摘出の順番は、開胸→心採血→生食灌流(腎動脈瀉血)→断頭→開頭→脳摘出→唾液腺摘出の順とした。 2-1. Long-term (4 weeks) Tickling stimulation
2-1-1. Experimental method (extraction of analysis target organ)
The order of organ removal was as follows: thoracotomy-> heart blood sampling-> saline perfusion (renal artery hemoptysis)->decapitation->craniotomy-> brain extraction-> salivary gland extraction.
1) 心採血・生食灌流
　エーテル麻酔下に開胸し心採血(約1mLをPAXgene専用採血管に採取)を行ない、留置針にて生食(約20mL)灌流／腎動脈瀉血を行なった。 1) Cardiac blood sampling and perfusion of the heart The heart was opened under ether anesthesia, and cardiac blood sampling (about 1 mL was collected in a PAXgene dedicated blood collection tube) was performed.
2) 脳摘出
　頭頂部から開頭、脳を摘出し、Glowinski(J Neurochem, 1966)の脳7分割法に従い、氷冷したアルミ板上で線条体、海馬、視床下部、皮質を分けた。脳下垂体はトルコ鞍より直接摘出した。摘出組織は約3mm角にトリミングし、RNA安定化試薬RNAlater(QIAGEN社製)に浸漬、-80℃で保存した。 2) Brain extraction The craniotomy was performed from the top of the head, the brain was extracted, and the striatum, hippocampus, hypothalamus, and cortex were separated on an ice-cooled aluminum plate according to the brain division method of Glowinski (J Neurochem, 1966). The pituitary gland was removed directly from the Turkish anther. The extracted tissue was trimmed to about 3 mm square, immersed in RNA stabilizing reagent RNAlater (manufactured by QIAGEN) and stored at −80 ° C.
3) 唾液腺摘出
　大唾液腺２部位(顎下腺、耳下腺)を摘出、約3mm角にトリミングし、RNA安定化試薬RNAlater(QIAGEN社製)に浸漬、-80℃で保存した。 3) Salivary gland excision Two major salivary glands (submandibular gland, parotid gland) were excised, trimmed to about 3 mm square, immersed in RNA stabilizing reagent RNAlater (manufactured by QIAGEN), and stored at -80 ° C.
２-１-２．実験方法（ラット組織からのRNA調製・検定）
1) 血液試料からの全RNA調製
　PAXgene Blood RNA Kit (QIAGEN社製)を用いて全RNA標品を調製した。 2-1-2. Experimental method (preparation and assay of RNA from rat tissue)
1) Preparation of total RNA from blood sample Total RNA preparation was prepared using PAXgene Blood RNA Kit (manufactured by QIAGEN).
2) 組織からの全RNA調製
　RNAlater浸漬組織(遺伝子発現解析対象である視床下部、線条体、２唾液腺のみ)を、RNeasy Buffer RLT中でホモジナイズ〔TissueLyzer(QIAGEN社製)使用〕し、以下Kitプロトコールに従い全RNA標品を調製した。各群(Tickling、Light Touch、対照)の試料は部位毎に、全RNAを等量混合した。 2) Preparation of total RNA from tissue RNAlater immersion tissue (hypothalamic, striatum, and salivary gland only for gene expression analysis) was homogenized (using TissueLyzer (QIAGEN)) in RNeasy Buffer RLT, and the following Kit A total RNA preparation was prepared according to the protocol. Samples of each group (Tickling, Light Touch, control) were mixed with equal amounts of total RNA for each site.
3) 全RNA純度と無傷度の検定
　個体毎(全RNA標品混合前)に、全RNA標品の濃度とA260/280比、28S/18S rRNA比を求めた。28S/18S rRNA比は、マイクロチップ型電気泳動システム(日立コスモアイ、SV1210)により検定・算出した。 3) Total RNA purity and intactness test For each individual (before mixing the total RNA sample), the concentration of the total RNA sample, the A260 / 280 ratio, and the 28S / 18S rRNA ratio were determined. The 28S / 18S rRNA ratio was assayed and calculated using a microchip electrophoresis system (Hitachi Cosmo Eye, SV1210).
２-１-３．実験方法（DNAチップ解析）
　上記(２-１-２)の全RNA標品を出発材料として、T7RNA Polymerase Promoter配列を付加したOligo(dT)24をプライマーとしたcDNA合成を行った。引き続き、これを鋳型としてアミノアリル-dUTP 存在下にT7RNA PolymeraseによるmRNA増幅を行って、cRNAにアミノ基を導入し、Cyanine 3[Cy3と略](Light Touch群、対照群)あるいはCyanine 5[Cy5と略] (Tickling群)のサクシニミド誘導体とカップリング反応させることにより蛍光標識を行いDNAチップ解析に供した。 2-1-3. Experimental method (DNA chip analysis)
Using the total RNA preparation of (2-1-2) as a starting material, cDNA synthesis was performed using Oligo (dT) 24 with a T7 RNA Polymerase Promoter sequence added as a primer. Subsequently, mRNA was amplified with T7RNA Polymerase in the presence of aminoallyl-dUTP using this as a template, amino groups were introduced into cRNA, and Cyanine 3 [abbreviated as Cy3] (Light Touch group, control group) or Cyanine 5 [Cy5 and (Omitted) Fluorescent labeling was performed by a coupling reaction with a succinimide derivative of (Tickling group) and subjected to DNA chip analysis.
　DNAチップ解析は以下の通り行った。各Cyanine色素で蛍光標識したRNA標品を等量混合し、DNAチップ〔Agilent社製(約41,012オリゴDNA搭載)、Whole Rat Genome DNA Microarray〕上で65℃、17時間ハイブリダイゼーション反応を行い(未反応分子はハイブリダイゼーション反応後洗浄除去)、共焦点レーザースキャナー(Agilent社製、G2565)で各色素に由来する蛍光強度を測定した。バックグランド補正とグローバル正規化の後、信号強度50未満の遺伝子(トランスクリプトを含む)を除外し、Cy5/Cy3比を求めた。 DNA chip analysis was performed as follows. Equal amounts of RNA preparations fluorescently labeled with each cyanine dye are mixed, and a hybridization reaction is carried out at 65 ° C. for 17 hours on a DNA chip (Agilent (approx. 41,012 oligo DNA), WholeWGenome DNA Microarray). The reaction molecules were washed and removed after the hybridization reaction), and the fluorescence intensity derived from each dye was measured with a confocal laser scanner (Agilent, G2565). After background correction and global normalization, genes with a signal intensity of less than 50 (including transcripts) were excluded and the Cy5 / Cy3 ratio was determined.
２-１-３．結果
　本結果で用いる「プローブ」と「遺伝子(トランスクリプトを含む)」の用語は同義であり、図表で用いる凡例は以下の通りである。 2-1-3. Results The terms “probe” and “gene (including transcript)” used in this result are synonymous, and the legend used in the chart is as follows.
T：Tickling
L：Light Touch
C：対照
Bl：血液
Hy：視床下部
St：線条体
Sg：唾液腺
２-１-４-１．信号強度分布
　Tickling／対照間、Tickling／Light Touch間の遺伝子発現の差をDNAチップ法にて解析した際の、DNAチップ上の各プローブの信号強度の分布を図46に示す。縦軸は信号強度、横軸はその信号強度を示す遺伝子数を示すが、組織試料は相互に類似の信号強度分布を示したが、血液試料では、高信号強度を示す遺伝子数が少なかった。これは、網状赤血球由来のグロビン遺伝子の影響を受けているためである。 T: Tickling
L: Light Touch
C: Control
Bl: Blood
Hy: hypothalamus
St: Striatum
Sg: Salivary gland
2-1-4-1. Signal intensity distribution FIG. 46 shows the signal intensity distribution of each probe on the DNA chip when the difference in gene expression between Tickling / control and Tickling / Light Touch is analyzed by the DNA chip method. The vertical axis represents the signal intensity, and the horizontal axis represents the number of genes indicating the signal intensity. The tissue samples showed similar signal intensity distributions to each other, but the blood sample had a small number of genes indicating high signal intensity. This is because it is influenced by the globin gene derived from reticulocytes.
２-１-４-２．発現変化を認めた遺伝子
　対照群と比較してTickling群で1.5倍超の発現を認めた末梢血液(Bl)の遺伝子の「Target Accession」及び「UniGene」を下記表4に示す。
Figure JPOXMLDOC01-appb-T000004
 2-1-4-2. Table 4 below shows the “Target Accession” and “UniGene” of the peripheral blood (Bl) gene in which expression was more than 1.5 times in the Tickling group compared to the gene control group in which expression change was observed .
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-I000010
Figure JPOXMLDOC01-appb-I000010
Figure JPOXMLDOC01-appb-I000011
Figure JPOXMLDOC01-appb-I000011
Figure JPOXMLDOC01-appb-I000012
Figure JPOXMLDOC01-appb-I000012
　対照群と比較してTickling群で1/1.5倍未満の発現を認めた末梢血液(Bl)の遺伝子の「Target Accession」及び「UniGene」を下記表5に示す。
Figure JPOXMLDOC01-appb-T000005
 Table 5 below shows the “Target Accession” and “UniGene” of the peripheral blood (Bl) gene that was found to be expressed in the Tickling group by less than 1 / 1.5-fold compared to the control group.
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-I000013
Figure JPOXMLDOC01-appb-I000013
Figure JPOXMLDOC01-appb-I000014
Figure JPOXMLDOC01-appb-I000014
Figure JPOXMLDOC01-appb-I000015
Figure JPOXMLDOC01-appb-I000015
Figure JPOXMLDOC01-appb-I000016
Figure JPOXMLDOC01-appb-I000016
　Light Touch群と比較してTickling群で1.5倍超の発現を認めた末梢血液(Bl)の遺伝子の「Target Accession」及び「UniGene」を下記表6に示す。
Figure JPOXMLDOC01-appb-T000006
 Table 6 below shows the “Target Accession” and “UniGene” of peripheral blood (Bl) genes that have been expressed more than 1.5 times in the Tickling group compared to the Light Touch group.
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-I000017
Figure JPOXMLDOC01-appb-I000017
Figure JPOXMLDOC01-appb-I000018
Figure JPOXMLDOC01-appb-I000018
　Light Touch群と比較してTickling群で1/1.5倍未満の発現を認めた末梢血液(Bl)の遺伝子の「Target Accession」及び「UniGene」を下記表7に示す。
Figure JPOXMLDOC01-appb-T000007
 Table 7 below shows the “Target Accession” and “UniGene” of peripheral blood (Bl) genes that were found to be expressed in the Tickling group by less than 1 / 1.5-fold compared to the Light Touch group.
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-I000019
Figure JPOXMLDOC01-appb-I000019
Figure JPOXMLDOC01-appb-I000020
Figure JPOXMLDOC01-appb-I000020
Figure JPOXMLDOC01-appb-I000021
Figure JPOXMLDOC01-appb-I000021
　対照群と比較してTickling群で1.5倍超の発現を認めた視床下部(Hy)の遺伝子の「Target Accession」及び「UniGene」を下記表8に示す。
Figure JPOXMLDOC01-appb-T000008
 Table 8 below shows the “Target Accession” and “UniGene” of the hypothalamic (Hy) gene that was found to express more than 1.5 times in the Tickling group compared to the control group.
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-I000022
Figure JPOXMLDOC01-appb-I000022
　対照群と比較してTickling群で1/1.5倍未満の発現を認めた視床下部(Hy)の遺伝子の「Target Accession」及び「UniGene」を下記表9に示す。
Figure JPOXMLDOC01-appb-T000009
 Table 9 below shows the “Target Accession” and “UniGene” of the hypothalamic (Hy) gene that was found to be expressed in the Tickling group by less than 1 / 1.5-fold compared to the control group.
Figure JPOXMLDOC01-appb-T000009
　Light Touch群と比較してTickling群で1.5倍超の発現を認めた視床下部(Hy)の遺伝子の「Target Accession」及び「UniGene」を下記表10に示す。
Table 10 below shows the “Target Accession” and “UniGene” of hypothalamic genes (Hy) that have been expressed more than 1.5 times in the Tickling group compared to the Light Touch group.
Figure JPOXMLDOC01-appb-I000023
Figure JPOXMLDOC01-appb-I000023
　Light Touch群と比較してTickling群で1/1.5倍未満の発現を認めた視床下部(Hy)の遺伝子の「Target Accession」及び「UniGene」を下記表11に示す。
Figure JPOXMLDOC01-appb-T000011
 Table 11 below shows “Target Accession” and “UniGene” of hypothalamic (Hy) genes in which expression in the Tickling group was found to be less than 1 / 1.5 fold compared to the Light Touch group.
Figure JPOXMLDOC01-appb-T000011
Figure JPOXMLDOC01-appb-I000024
Figure JPOXMLDOC01-appb-I000024
　対照群と比較してTickling群で1.5倍超の発現を認めた線条体(St)の遺伝子の「Target Accession」及び「UniGene」を下記表12に示す。
Figure JPOXMLDOC01-appb-T000012
 Table 12 below shows the “Target Accession” and “UniGene” of the striatum (St) gene that was expressed more than 1.5 times in the Tickling group compared to the control group.
Figure JPOXMLDOC01-appb-T000012
Figure JPOXMLDOC01-appb-I000025
Figure JPOXMLDOC01-appb-I000025
Figure JPOXMLDOC01-appb-I000026
Figure JPOXMLDOC01-appb-I000026
　対照群と比較してTickling群で1/1.5倍未満の発現を認めた線条体(St)の遺伝子の「Target Accession」及び「UniGene」を下記表13に示す。
Figure JPOXMLDOC01-appb-T000013
 Table 13 below shows the “Target Accession” and “UniGene” of the striatum (St) gene that was found to be expressed in the Tickling group by less than 1 / 1.5-fold compared to the control group.
Figure JPOXMLDOC01-appb-T000013
Figure JPOXMLDOC01-appb-I000027
Figure JPOXMLDOC01-appb-I000027
Figure JPOXMLDOC01-appb-I000028
Figure JPOXMLDOC01-appb-I000028
　Light Touch群と比較してTickling群で1.5倍超の発現を認めた線条体(St)の遺伝子の「Target Accession」及び「UniGene」を下記表14に示す。
Figure JPOXMLDOC01-appb-T000014
 Table 14 below shows the “Target Accession” and “UniGene” of the striatum (St) gene that was expressed more than 1.5 times in the Tickling group as compared to the Light Touch group.
Figure JPOXMLDOC01-appb-T000014
　Light Touch群と比較してTickling群で1/1.5倍未満の発現を認めた線条体(St)の遺伝子の「Target Accession」及び「UniGene」を下記表15に示す。
Figure JPOXMLDOC01-appb-T000015
 Table 15 below shows the “Target Accession” and “UniGene” of the striatum (St) gene in which expression of less than 1 / 1.5 times in the Tickling group compared to the Light Touch group was observed.
Figure JPOXMLDOC01-appb-T000015
　対照群と比較してTickling群で1.5倍超の発現を認めた唾液腺(Sg)の遺伝子の「Target Accession」及び「UniGene」を下記表16に示す。
Figure JPOXMLDOC01-appb-T000016
 Table 16 below shows the “Target Accession” and “UniGene” of salivary gland (Sg) genes that were found to express more than 1.5 times in the Tickling group compared to the control group.
Figure JPOXMLDOC01-appb-T000016
Figure JPOXMLDOC01-appb-I000029
Figure JPOXMLDOC01-appb-I000029
Figure JPOXMLDOC01-appb-I000030
Figure JPOXMLDOC01-appb-I000030
Figure JPOXMLDOC01-appb-I000031
Figure JPOXMLDOC01-appb-I000031
Figure JPOXMLDOC01-appb-I000032
Figure JPOXMLDOC01-appb-I000032
　対照群と比較してTickling群で1/1.5倍未満の発現を認めた唾液腺(Sg)の遺伝子の「Target Accession」及び「UniGene」を下記表17に示す。
Figure JPOXMLDOC01-appb-T000017
 Table 17 below shows the “Target Accession” and “UniGene” of salivary gland (Sg) genes that were found to be expressed in the Tickling group by less than 1 / 1.5-fold compared to the control group.
Figure JPOXMLDOC01-appb-T000017
Figure JPOXMLDOC01-appb-I000033
Figure JPOXMLDOC01-appb-I000033
Figure JPOXMLDOC01-appb-I000034
Figure JPOXMLDOC01-appb-I000034
Figure JPOXMLDOC01-appb-I000035
Figure JPOXMLDOC01-appb-I000035
Figure JPOXMLDOC01-appb-I000036
Figure JPOXMLDOC01-appb-I000036
　Light Touch群と比較してTickling群で1.5倍超の発現を認めた唾液腺(Sg)の遺伝子の「Target Accession」及び「UniGene」を下記表18に示す。
Figure JPOXMLDOC01-appb-T000018
 Table 18 below shows the “Target Accession” and “UniGene” of salivary gland (Sg) genes that were expressed more than 1.5 times in the Tickling group compared to the Light Touch group.
Figure JPOXMLDOC01-appb-T000018
Figure JPOXMLDOC01-appb-I000037
Figure JPOXMLDOC01-appb-I000037
　Light Touch群と比較してTickling群で1/1.5倍未満の発現を認めた唾液腺(Sg)の遺伝子の「Target Accession」及び「UniGene」を下記表19に示す。
Figure JPOXMLDOC01-appb-T000019
 Table 19 below shows the “Target Accession” and “UniGene” of salivary gland (Sg) genes that were found to be expressed in the Tickling group by less than 1 / 1.5-fold compared to the Light Touch group.
Figure JPOXMLDOC01-appb-T000019
Figure JPOXMLDOC01-appb-I000038
Figure JPOXMLDOC01-appb-I000038
　対照群と比較してTickling群で2.0倍超の発現を認めた末梢血液(Bl)の遺伝子のアノテーションを下記表20に示す。
Figure JPOXMLDOC01-appb-T000020
 Table 20 below shows the annotations of peripheral blood (Bl) genes that were expressed more than 2.0 times in the Tickling group compared to the control group.
Figure JPOXMLDOC01-appb-T000020
Figure JPOXMLDOC01-appb-I000039
Figure JPOXMLDOC01-appb-I000039
Figure JPOXMLDOC01-appb-I000040
Figure JPOXMLDOC01-appb-I000040
Figure JPOXMLDOC01-appb-I000041
Figure JPOXMLDOC01-appb-I000041
　対照群と比較してTickling群で0.5倍未満の発現を認めた末梢血液(Bl)の遺伝子のアノテーションを下記表21に示す。
Figure JPOXMLDOC01-appb-T000021
 Table 21 below shows the annotations of peripheral blood (Bl) genes that showed less than 0.5-fold expression in the Tickling group compared to the control group.
Figure JPOXMLDOC01-appb-T000021
　Light Touch群と比較してTickling群で2.0倍超の発現を認めた末梢血液(Bl)の遺伝子のアノテーションを下記表22に示す。
Figure JPOXMLDOC01-appb-T000022
 Table 22 below shows gene annotations of peripheral blood (Bl) in which expression was more than 2.0 times in the Tickling group compared to the Light Touch group.
Figure JPOXMLDOC01-appb-T000022
Figure JPOXMLDOC01-appb-I000042
Figure JPOXMLDOC01-appb-I000042
Figure JPOXMLDOC01-appb-I000043
Figure JPOXMLDOC01-appb-I000043
Figure JPOXMLDOC01-appb-I000044
Figure JPOXMLDOC01-appb-I000044
Figure JPOXMLDOC01-appb-I000045
Figure JPOXMLDOC01-appb-I000045
Figure JPOXMLDOC01-appb-I000046
Figure JPOXMLDOC01-appb-I000046
Figure JPOXMLDOC01-appb-I000047
Figure JPOXMLDOC01-appb-I000047
Figure JPOXMLDOC01-appb-I000048
Figure JPOXMLDOC01-appb-I000048
Figure JPOXMLDOC01-appb-I000049
Figure JPOXMLDOC01-appb-I000049
　Light Touch群と比較してTickling群で0.5倍未満の発現を認めた末梢血液(Bl)の遺伝子のアノテーションを下記表23に示す。
Figure JPOXMLDOC01-appb-T000023
 Table 23 below shows annotations of genes in peripheral blood (Bl) that showed expression of 0.5 times less in the Tickling group than in the Light Touch group.
Figure JPOXMLDOC01-appb-T000023
Figure JPOXMLDOC01-appb-I000050
Figure JPOXMLDOC01-appb-I000050
Figure JPOXMLDOC01-appb-I000051
Figure JPOXMLDOC01-appb-I000051
Figure JPOXMLDOC01-appb-I000052
Figure JPOXMLDOC01-appb-I000052
Figure JPOXMLDOC01-appb-I000053
Figure JPOXMLDOC01-appb-I000053
Figure JPOXMLDOC01-appb-I000054
Figure JPOXMLDOC01-appb-I000054
　対照群と比較してTickling群で2.0倍超の発現を認めた視床下部(Hy)の遺伝子のアノテーションを下記表24に示す。
Figure JPOXMLDOC01-appb-T000024
 Table 24 below shows the annotation of the hypothalamic gene (Hy) in which the expression was more than 2.0 times in the Tickling group compared to the control group.
Figure JPOXMLDOC01-appb-T000024
Figure JPOXMLDOC01-appb-I000055
Figure JPOXMLDOC01-appb-I000055
Figure JPOXMLDOC01-appb-I000056
Figure JPOXMLDOC01-appb-I000056
　対照群と比較してTickling群で0.5倍未満の発現を認めた視床下部(Hy)の遺伝子のアノテーションを下記表25に示す。
Figure JPOXMLDOC01-appb-T000025
 Table 25 below shows the annotation of the hypothalamic gene (Hy) in which the expression was less than 0.5-fold in the Tickling group compared to the control group.
Figure JPOXMLDOC01-appb-T000025
Figure JPOXMLDOC01-appb-I000057
Figure JPOXMLDOC01-appb-I000057
Figure JPOXMLDOC01-appb-I000058
Figure JPOXMLDOC01-appb-I000058
Figure JPOXMLDOC01-appb-I000059
Figure JPOXMLDOC01-appb-I000059
Figure JPOXMLDOC01-appb-I000060
Figure JPOXMLDOC01-appb-I000060
　Light Touch群と比較してTickling群で2.0倍超の発現を認めた視床下部(Hy)の遺伝子のアノテーションを下記表26に示す。
Figure JPOXMLDOC01-appb-T000026
 Table 26 below shows the hypothalamic (Hy) gene annotations that have been expressed more than 2.0 times in the Tickling group compared to the Light Touch group.
Figure JPOXMLDOC01-appb-T000026
Figure JPOXMLDOC01-appb-I000061
Figure JPOXMLDOC01-appb-I000061
　Light Touch群と比較してTickling群で0.5倍未満の発現を認めた視床下部(Hy)の遺伝子のアノテーションを下記表27に示す。
Figure JPOXMLDOC01-appb-T000027
 Table 27 below shows hypothalamic (Hy) gene annotations that were found to be less than 0.5-fold more expressed in the Tickling group than in the Light Touch group.
Figure JPOXMLDOC01-appb-T000027
Figure JPOXMLDOC01-appb-I000062
Figure JPOXMLDOC01-appb-I000062
Figure JPOXMLDOC01-appb-I000063
Figure JPOXMLDOC01-appb-I000063
Figure JPOXMLDOC01-appb-I000064
Figure JPOXMLDOC01-appb-I000064
Figure JPOXMLDOC01-appb-I000065
Figure JPOXMLDOC01-appb-I000065
Figure JPOXMLDOC01-appb-I000066
Figure JPOXMLDOC01-appb-I000066
　対照群と比較してTickling群で2.0倍超の発現を認めた線条体(St)の遺伝子のアノテーションを下記表28に示す。
Figure JPOXMLDOC01-appb-T000028
 Table 28 below shows the annotation of the striatum (St) gene that was found to express more than 2.0 times in the Tickling group compared to the control group.
Figure JPOXMLDOC01-appb-T000028
Figure JPOXMLDOC01-appb-I000067
Figure JPOXMLDOC01-appb-I000067
Figure JPOXMLDOC01-appb-I000068
Figure JPOXMLDOC01-appb-I000068
　対照群と比較してTickling群で0.5倍未満の発現を認めた線条体(St)の遺伝子のアノテーションを下記表29に示す。
Figure JPOXMLDOC01-appb-T000029
 Table 29 below shows the annotation of the striatum (St) gene that was found to be less than 0.5-fold expressed in the Tickling group compared to the control group.
Figure JPOXMLDOC01-appb-T000029
Figure JPOXMLDOC01-appb-I000069
Figure JPOXMLDOC01-appb-I000069
Figure JPOXMLDOC01-appb-I000070
Figure JPOXMLDOC01-appb-I000070
Figure JPOXMLDOC01-appb-I000071
Figure JPOXMLDOC01-appb-I000071
　Light Touch群と比較してTickling群で2.0倍超の発現を認めた線条体(St)の遺伝子のアノテーションを下記表30に示す。
Figure JPOXMLDOC01-appb-T000030
 Table 30 below shows the annotations of the striatum (St) genes that were found to express more than 2.0 times in the Tickling group compared to the Light Touch group.
Figure JPOXMLDOC01-appb-T000030
　Light Touch群と比較してTickling群で0.5倍未満の発現を認めた線条体(St)の遺伝子のアノテーションを下記表31に示す。
Figure JPOXMLDOC01-appb-T000031
 Table 31 below shows the annotation of the striatum (St) gene that was found to be less than 0.5-fold expressed in the Tickling group compared to the Light Touch group.
Figure JPOXMLDOC01-appb-T000031
Figure JPOXMLDOC01-appb-I000072
Figure JPOXMLDOC01-appb-I000072
Figure JPOXMLDOC01-appb-I000073
Figure JPOXMLDOC01-appb-I000073
Figure JPOXMLDOC01-appb-I000074
Figure JPOXMLDOC01-appb-I000074
　対照群と比較してTickling群で2.0倍超の発現を認めた唾液腺(Sg)の遺伝子のアノテーションを下記表32に示す。
Figure JPOXMLDOC01-appb-T000032
 Table 32 below shows annotations of salivary gland (Sg) genes that were expressed more than 2.0 times in the Tickling group compared to the control group.
Figure JPOXMLDOC01-appb-T000032
Figure JPOXMLDOC01-appb-I000075
Figure JPOXMLDOC01-appb-I000075
Figure JPOXMLDOC01-appb-I000076
Figure JPOXMLDOC01-appb-I000076
Figure JPOXMLDOC01-appb-I000077
Figure JPOXMLDOC01-appb-I000077
Figure JPOXMLDOC01-appb-I000078
Figure JPOXMLDOC01-appb-I000078
Figure JPOXMLDOC01-appb-I000079
Figure JPOXMLDOC01-appb-I000079
Figure JPOXMLDOC01-appb-I000080
Figure JPOXMLDOC01-appb-I000080
Figure JPOXMLDOC01-appb-I000081
Figure JPOXMLDOC01-appb-I000081
Figure JPOXMLDOC01-appb-I000082
Figure JPOXMLDOC01-appb-I000082
Figure JPOXMLDOC01-appb-I000083
Figure JPOXMLDOC01-appb-I000083
Figure JPOXMLDOC01-appb-I000084
Figure JPOXMLDOC01-appb-I000084
Figure JPOXMLDOC01-appb-I000085
Figure JPOXMLDOC01-appb-I000085
Figure JPOXMLDOC01-appb-I000086
Figure JPOXMLDOC01-appb-I000086
Figure JPOXMLDOC01-appb-I000087
Figure JPOXMLDOC01-appb-I000087
Figure JPOXMLDOC01-appb-I000088
Figure JPOXMLDOC01-appb-I000088
Figure JPOXMLDOC01-appb-I000089
Figure JPOXMLDOC01-appb-I000089
Figure JPOXMLDOC01-appb-I000090
Figure JPOXMLDOC01-appb-I000090
Figure JPOXMLDOC01-appb-I000091
Figure JPOXMLDOC01-appb-I000091
Figure JPOXMLDOC01-appb-I000092
Figure JPOXMLDOC01-appb-I000092
Figure JPOXMLDOC01-appb-I000093
Figure JPOXMLDOC01-appb-I000093
Figure JPOXMLDOC01-appb-I000094
Figure JPOXMLDOC01-appb-I000094
Figure JPOXMLDOC01-appb-I000095
Figure JPOXMLDOC01-appb-I000095
Figure JPOXMLDOC01-appb-I000096
Figure JPOXMLDOC01-appb-I000096
Figure JPOXMLDOC01-appb-I000097
Figure JPOXMLDOC01-appb-I000097
Figure JPOXMLDOC01-appb-I000098
Figure JPOXMLDOC01-appb-I000098
Figure JPOXMLDOC01-appb-I000099
Figure JPOXMLDOC01-appb-I000099
Figure JPOXMLDOC01-appb-I000100
Figure JPOXMLDOC01-appb-I000100
Figure JPOXMLDOC01-appb-I000101
Figure JPOXMLDOC01-appb-I000101
Figure JPOXMLDOC01-appb-I000102
Figure JPOXMLDOC01-appb-I000102
　対照群と比較してTickling群で0.5倍未満の発現を認めた唾液腺(Sg)の遺伝子のアノテーションを下記表33に示す。
Figure JPOXMLDOC01-appb-T000033
 Table 33 below shows annotations of salivary gland (Sg) genes that showed expression less than 0.5-fold in the Tickling group compared to the control group.
Figure JPOXMLDOC01-appb-T000033
Figure JPOXMLDOC01-appb-I000103
Figure JPOXMLDOC01-appb-I000103
Figure JPOXMLDOC01-appb-I000104
Figure JPOXMLDOC01-appb-I000104
Figure JPOXMLDOC01-appb-I000105
Figure JPOXMLDOC01-appb-I000105
Figure JPOXMLDOC01-appb-I000106
Figure JPOXMLDOC01-appb-I000106
Figure JPOXMLDOC01-appb-I000107
Figure JPOXMLDOC01-appb-I000107
Figure JPOXMLDOC01-appb-I000108
Figure JPOXMLDOC01-appb-I000108
Figure JPOXMLDOC01-appb-I000109
Figure JPOXMLDOC01-appb-I000109
Figure JPOXMLDOC01-appb-I000110
Figure JPOXMLDOC01-appb-I000110
Figure JPOXMLDOC01-appb-I000111
Figure JPOXMLDOC01-appb-I000111
Figure JPOXMLDOC01-appb-I000112
Figure JPOXMLDOC01-appb-I000112
Figure JPOXMLDOC01-appb-I000113
Figure JPOXMLDOC01-appb-I000113
Figure JPOXMLDOC01-appb-I000114
Figure JPOXMLDOC01-appb-I000114
Figure JPOXMLDOC01-appb-I000115
Figure JPOXMLDOC01-appb-I000115
Figure JPOXMLDOC01-appb-I000116
Figure JPOXMLDOC01-appb-I000116
Figure JPOXMLDOC01-appb-I000117
Figure JPOXMLDOC01-appb-I000117
Figure JPOXMLDOC01-appb-I000118
Figure JPOXMLDOC01-appb-I000118
Figure JPOXMLDOC01-appb-I000119
Figure JPOXMLDOC01-appb-I000119
Figure JPOXMLDOC01-appb-I000120
Figure JPOXMLDOC01-appb-I000120
Figure JPOXMLDOC01-appb-I000121
Figure JPOXMLDOC01-appb-I000121
Figure JPOXMLDOC01-appb-I000122
Figure JPOXMLDOC01-appb-I000122
Figure JPOXMLDOC01-appb-I000123
Figure JPOXMLDOC01-appb-I000123
Figure JPOXMLDOC01-appb-I000124
Figure JPOXMLDOC01-appb-I000124
Figure JPOXMLDOC01-appb-I000125
Figure JPOXMLDOC01-appb-I000125
Figure JPOXMLDOC01-appb-I000126
Figure JPOXMLDOC01-appb-I000126
Figure JPOXMLDOC01-appb-I000127
Figure JPOXMLDOC01-appb-I000127
Figure JPOXMLDOC01-appb-I000128
Figure JPOXMLDOC01-appb-I000128
Figure JPOXMLDOC01-appb-I000129
Figure JPOXMLDOC01-appb-I000129
　Light Touch群と比較してTickling群で2.0倍超の発現を認めた唾液腺(Sg)の遺伝子のアノテーションを下記表34に示す。
Figure JPOXMLDOC01-appb-T000034
 Table 34 below shows the annotation of the salivary gland (Sg) gene that was expressed more than 2.0 times in the Tickling group compared to the Light Touch group.
Figure JPOXMLDOC01-appb-T000034
Figure JPOXMLDOC01-appb-I000130
Figure JPOXMLDOC01-appb-I000130
Figure JPOXMLDOC01-appb-I000131
Figure JPOXMLDOC01-appb-I000131
　Light Touch群と比較してTickling群で0.5倍未満の発現を認めた唾液腺(Sg)の遺伝子のアノテーションを下記表35に示す。
Figure JPOXMLDOC01-appb-T000035
 Table 35 below shows the annotation of salivary gland (Sg) genes that showed expression of less than 0.5-fold in the Tickling group compared to the Light Touch group.
Figure JPOXMLDOC01-appb-T000035
Figure JPOXMLDOC01-appb-I000132
Figure JPOXMLDOC01-appb-I000132
Figure JPOXMLDOC01-appb-I000133
Figure JPOXMLDOC01-appb-I000133
Figure JPOXMLDOC01-appb-I000134
Figure JPOXMLDOC01-appb-I000134
　なお、表20～35中の「No.」は、上記表2中の「No.」に対応している。 Note that “No.” in Tables 20 to 35 corresponds to “No.” in Table 2 above.
　Tickling群において末梢血液で２倍超の発現を認める遺伝子は、Light Touch群と比較した場合は41遺伝子存在し、そのうち未知遺伝子が12、機能推定あるいは構造類似している遺伝子は10、部分配列である遺伝子は５つであった。一方、対照群と比較した場合は10遺伝子存在し、そのうち１遺伝子が未知遺伝子、２遺伝子が機能推定あるいは構造類似している遺伝子であった。末梢血液で0.5倍未満の発現を認める遺伝子は、Light Touch群と比較した場合は30遺伝子、対照群と比較した場合は２遺伝子存在した。同様に発現差を認める遺伝子をカウントすると、視床下部で２倍超の発現を認める遺伝子は、Light Touch群と比較した場合は５遺伝子、対照群と比較した場合は13遺伝子存在し、0.5倍未満の発現を認める遺伝子は、Light Touch群と比較した場合は30遺伝子、対照群と比較した場合は11遺伝子存在した。また、線条体では２倍超の発現を認める遺伝子は、Light Touch群と比較した場合は３遺伝子、対照群と比較した場合は８遺伝子存在し、0.5倍未満の発現を認める遺伝子は、Light Touch群と比較した場合は８遺伝子、対照群と比較した場合は12遺伝子存在した。唾液腺では、対照群と比較した場合に発現差を認める遺伝子が多く、２倍超の発現を認める遺伝子は161、0.5倍未満の発現を認める遺伝子は143存在した。Light Touch群と比較した場合は、それぞれ21遺伝子、22遺伝子であった。 In the Tickling group, there are 41 genes whose expression in the peripheral blood is more than doubled when compared with the Light 、 Touch group, of which 12 are unknown genes, 10 are genes whose function is estimated or structurally similar, and partial sequences. There were 5 genes. On the other hand, when compared with the control group, there were 10 genes, of which 1 gene was an unknown gene, and 2 genes were genes whose function was estimated or structurally similar. There were 30 genes whose expression was less than 0.5-fold in peripheral blood when compared with the Light Touch group and 2 genes when compared with the control group. Similarly, when the number of genes with differential expression is counted, there are 5 genes in the hypothalamus that are more than 2-fold expressed compared to the Light 床 Touch group, 13 genes compared to the control group, and less than 0.5-fold. 30 genes were found to be expressed in the light-touch group and 11 genes were compared to the control group. In addition, there are 3 genes in the striatum that are expressed more than 2 times compared to the Light Touch group, 8 genes that are compared to the control group, and genes that are expressed less than 0.5 times are compared to the Light group. There were 8 genes when compared with the Touch group and 12 genes when compared with the control group. In the salivary glands, there were many genes that showed a difference in expression when compared with the control group, 161 genes that showed more than 2-fold expression, and 143 genes that showed less than 0.5-fold expression. When compared with the Light Touch group, there were 21 genes and 22 genes, respectively.
　注目すべき遺伝子としては、Ticklingにより視床下部でUp-regulateされたGalp、Slc6a3、Down-regulateされたChrna3、Slc5a7、Slc18a3、Avp、線条体でDown-regulateされたCck、唾液腺でUp-regulateされたAmy1、Klks3、Ton、Klk12、Ngfg、Down-regulateされたFetub、Serpina1等がある。 Notable genes include Galp, Slc6a3, Down-regulated Chrna3, Slc5a7, Slc18a3, Avp up-regulated in the hypothalamus by Tickling, Cck down-regulated in the striatum, Up-regulate in salivary glands Amy1, Klks3, Ton, Klk12, Ngfg, down-regulated Fetub, Serpina1, etc.
２-１-４-３．クラスタリング解析
　いずれか２組の解析におけるCy3およびCy5の信号強度が50以上あったプローブは26,075遺伝子(トランスクリプトを含む)存在し、これらをクラスタリングした像を図47に示し、少なくとも２組の解析において２倍超あるいは0.5倍未満の発現差を認めたプローブのクラスタリング像を図48に示した。発現差の程度は、TicklingによりUp-regulateされた遺伝子(トランスクリプトを含む)は赤色、Down-regulateされた遺伝子(トランスクリプトを含む)は緑色の濃さの程度で表した(カラーバーは図中右側に表示)。また、信号強度が低く(50未満)、発現差を表示できない遺伝子(トランスクリプトを含む)は灰色で表示した。 2-1-4-3. Clustering analysis There are 26,075 genes (including transcripts) that have a Cy3 and Cy5 signal intensity of 50 or more in any two sets of analysis. Figure 47 shows an image of clustering these probes. FIG. 48 shows a clustering image of probes in which an expression difference of more than 2 times or less than 0.5 times was recognized. The degree of expression difference is expressed in red for genes up-regulated by Tickling (including transcripts) and green for down-regulated genes (including transcripts). (Displayed on the middle right). In addition, genes with low signal intensity (less than 50) that cannot display differential expression (including transcripts) are shown in gray.
　対照群あるいはLight Touch群と比較してTickling群の唾液腺組織でそれぞれ1.5倍超の発現差を認めた593プローブと171プローブのクラスタリング像を図49と図50に示した。また、両比較において発現差を認めた遺伝子(トランスクリプトを含む)の数を図51のベン図に示したが、共通して発現差を認める遺伝子は48存在した。一方、両比較でそれぞれ1/1.5倍未満の発現差を認めた525プローブと166プローブのクラスタリング像を図52と図53に示し、図54のベン図には両比較において発現差を認めた遺伝子(トランスクリプトを含む)数を示したが、共通して発現差を認めた遺伝子は56存在した。 FIG. 49 and FIG. 50 show clustering images of the 593 probe and the 171 probe in which the expression difference of more than 1.5 times was observed in the salivary gland tissue of the Tickling group as compared with the control group or the Light Touch group. In addition, the number of genes (including transcripts) in which expression differences were observed in both comparisons is shown in the Venn diagram of FIG. 51, but there were 48 genes in which expression differences were commonly recognized. On the other hand, clustering images of the 525 probe and the 166 probe that showed an expression difference of less than 1 / 1.5 times in both comparisons are shown in FIGS. 52 and 53, and the Venn diagram in FIG. 56 genes (including transcripts) showed common expression differences.
　対照群あるいはLight Touch群と比較してTickling群の視床下部組織でそれぞれ1.5倍超の発現差を認めた157プローブと136プローブのクラスタリング像を図55と図56に示した。また、両比較において発現差を認めた遺伝子(トランスクリプトを含む)の数を図57のベン図に示したが、共通して発現差を認める遺伝子は39存在した。一方、両比較でそれぞれ1/1.5倍未満の発現差を認めた57プローブと185プローブのクラスタリング像を図58と図59に示し、図60のベン図には両比較において発現差を認めた遺伝子(トランスクリプトを含む)数を示したが、共通して発現差を認めた遺伝子は11存在した。 FIG. 55 and FIG. 56 show clustered images of the 157 probe and the 136 probe in which the expression difference of 1.5 times or more was recognized in the hypothalamic tissue of the Tickling group as compared with the control group or the Light Touch group. Further, the number of genes (including transcripts) in which expression differences were observed in both comparisons is shown in the Venn diagram of FIG. 57, but 39 genes in which expression differences were commonly recognized existed. On the other hand, the clustered images of 57 probes and 185 probes that each showed an expression difference of less than 1 / 1.5 times in both comparisons are shown in FIGS. 58 and 59, and the Venn diagram in FIG. There were 11 genes that showed differential expression in common.
２-１-４-４．オントロジー解析
　当解析は、対照群あるいはLight Touch群と比較してTickling群で1.5倍超あるいは1/1.5倍未満の発現差を認める遺伝子を対象とし、該条件にて抽出される視床下部あるいは線条体の遺伝子と共通する末梢血液の遺伝子と、唾液腺、視床下部の遺伝子に関し行なった。抽出されたプローブ数と遺伝子数は下記表36に示す。
Figure JPOXMLDOC01-appb-T000036
 2-1-4-4. Ontology analysis This analysis targets genes that show an expression difference of more than 1.5 times or less than 1 / 1.5 times in the Tickling group compared to the control group or the Light Touch group, and the hypothalamus or striatum extracted under these conditions Peripheral blood genes in common with body genes, salivary glands, and hypothalamic genes. The number of extracted probes and the number of genes are shown in Table 36 below.
Figure JPOXMLDOC01-appb-T000036
　DNAチップ上のプローブと遺伝子(GeneID)との対応付けは、Agilent社の提供する情報とNCBI Entrez(http://www.ncbi.nlm.nih.gov/)の情報に基づいて行なった。GeneIDとGOの対応付けはBiNGO (http://www.psb.ugent.be.cbd/papers/BiNGO/)を用いて行い、GOの階層図はCytoscape (http://www.cytoscape.org/)を用いて描画した。各GO Termについて、Agilent社製Whole Rat Genome Microarray上の全遺伝子中での出現頻度に比べて抽出遺伝子群中での出現頻度が高くなっているかHypergeometric testを用いて有意水準FDR(False Discovery rate)<0.1で検定した。該検定で有意と判定されたGO Termを抽出遺伝子群に特徴的なGOとした。表37から表44に、各試料で抽出された遺伝子(表36参照)のGO解析結果を示す。
Figure JPOXMLDOC01-appb-T000037
Figure JPOXMLDOC01-appb-T000038
Figure JPOXMLDOC01-appb-T000039
Figure JPOXMLDOC01-appb-T000040
Figure JPOXMLDOC01-appb-T000041
Figure JPOXMLDOC01-appb-T000042
 The association between the probe on the DNA chip and the gene (GeneID) was performed based on information provided by Agilent and NCBI Entrez (http://www.ncbi.nlm.nih.gov/). GeneID and GO are mapped using BiNGO (http: //www.psb.ugent.be.cbd/papers/BiNGO/), and the hierarchy of GO is Cytoscape (http://www.cytoscape.org/ ). For each GO Term, the frequency of occurrence in the extracted gene group is higher than the frequency of occurrence in all genes on the Agilent Whole Rat Genome Microarray. Tested at <0.1. GO Term determined to be significant by the test was defined as GO characteristic of the extracted gene group. Tables 37 to 44 show the GO analysis results of the genes (see Table 36) extracted from each sample.
Figure JPOXMLDOC01-appb-T000037
Figure JPOXMLDOC01-appb-T000038
Figure JPOXMLDOC01-appb-T000039
Figure JPOXMLDOC01-appb-T000040
Figure JPOXMLDOC01-appb-T000041
Figure JPOXMLDOC01-appb-T000042
Figure JPOXMLDOC01-appb-I000135
Figure JPOXMLDOC01-appb-T000043
Figure JPOXMLDOC01-appb-I000135
Figure JPOXMLDOC01-appb-T000043
Figure JPOXMLDOC01-appb-I000136
Figure JPOXMLDOC01-appb-I000136
Figure JPOXMLDOC01-appb-I000137
Figure JPOXMLDOC01-appb-T000044
Figure JPOXMLDOC01-appb-I000137
Figure JPOXMLDOC01-appb-T000044
　また、それぞれについてCytoscapeを用いて作図したGO階層図は、図62から図72に示した。なお、図61には色の濃淡とP値の関係を表した。色が濃いほどそのGO Termに割り当てられる有意性が高く、円の大きさが大きいほど割り当てられる遺伝子数が多いこと示す。 Also, the GO hierarchy diagram drawn using Cytoscape for each is shown in FIG. 62 to FIG. FIG. 61 shows the relationship between color shading and P value. The darker the color, the higher the significance assigned to that GO Term, and the larger the circle, the more genes assigned.
　対照群と比較してTickling群の唾液腺で相対的に発現変動している遺伝子、およびLight Touch群と比較してTickling群の視床下部で相対的に発現変動している遺伝子のGO解析では、それぞれ新規ストレス指標探索、健康増進遺伝子探索のための遺伝子を特定することができた。 In the GO analysis of genes that are relatively expressed in the salivary glands of the Tickling group compared to the control group and genes that are relatively expressed in the hypothalamus of the Tickling group compared to the Light Touch group, We were able to identify genes for new stress index search and health promotion gene search.
　これらのGO解析で注目される唾液腺でUp-regulateされる遺伝子としては、機能上GO ID: 17017のMAPキナーゼ脱リン酸化酵素に割り当てられる４遺伝子(表45)、組織カリクレイン活性(GO ID: 4293)に割り当てられる５遺伝子(表46)、細胞外成分(GO ID: 5576, 44421, 5615)として割り当てられる67遺伝子(表47)、生物学経路として脂質代謝(GO ID: 6629, 44255)に割り当てられる９遺伝子(表48)、ドーパミン合成(GO ID: 42416)に割り当てられる３遺伝子(表49)が抽出された。Down-regulateされる遺伝子としては、機能上、アクチン結合(GO ID; 3779)に割り当てられる13遺伝子(表50)、細胞外に局在する成分(GO ID: 5576, 44421, 5615)として割り当てられる66遺伝子(表51)、生物学経路として核酸構成糖の代謝(GO ID: 9225)に割り当てられる２遺伝子(表52)が抽出された。 The genes that are up-regulated in the salivary glands noted in these GO analyzes include 4 genes that are functionally assigned to MAP kinase dephosphorylating enzyme of GO ID: 17017 (Table 45), tissue kallikrein activity (GO ID: 4293) ) Assigned to 5 genes (Table 46), 67 genes (Table 47) assigned as extracellular components (GO ID: 5576, 44421, 5615), and lipid metabolism (GO ID: 6629, 44255) as biological pathways 9 genes (Table 48) and 3 genes (Table 49) assigned to dopamine synthesis (GO ID: 42416) were extracted. Down-regulated genes are functionally assigned as 13 genes assigned to actin binding (GO ID; 3779) (Table 50) and components localized outside the cell (GO ID: 5576, 44421, 5615) 66 genes (Table 51) and 2 genes (Table 52) assigned to metabolism of nucleic acid constituent sugars (GO ID: 9225) as biological pathways were extracted.
　視床下部でUp-regulateされる遺伝子としては、機能上、ホルモン活性(GO ID: 5184, 5179)に割り当てられる２遺伝子(表53)、モノアミントランスポーター(GO ID: 8504)に割り当てられる３遺伝子(表54)、受容体結合(GO ID: 5102)に割り当てられる４遺伝子(表55)、生物学的経路として食行動(GO ID: 7631)に割り当てられる６遺伝子(表56)、生体アミン合成(GO ID: 42401, 42426, 42416, 9309)に割り当てられる３遺伝子(表57)、生体アミン代謝(GO ID: 9308, 6584, 6576)に割り当てられる１遺伝子(表58)が抽出された。Down-regulateされる遺伝子としては、機能上、陽イオン結合(GO ID; 5509, 43169)に割り当てられる21遺伝子(表59)が抽出された。
Figure JPOXMLDOC01-appb-T000045
Figure JPOXMLDOC01-appb-T000046
 The genes that are up-regulated in the hypothalamus are functionally 2 genes (Table 53) assigned to hormone activity (GO ID: 5184, 5179) and 3 genes assigned to monoamine transporter (GO ID: 8504) ( Table 54), 4 genes assigned to receptor binding (GO ID: 5102) (Table 55), 6 genes assigned to eating behavior (GO ID: 7631) as biological pathways (Table 56), biogenic amine synthesis (Table 56) Three genes (Table 57) assigned to GO ID: 42401, 42426, 42416, 9309) and 1 gene (Table 58) assigned to biogenic amine metabolism (GO ID: 9308, 6584, 6576) were extracted. As genes to be down-regulated, 21 genes (Table 59) that were functionally assigned to cation binding (GO ID; 5509, 43169) were extracted.
Figure JPOXMLDOC01-appb-T000045
Figure JPOXMLDOC01-appb-T000046
Figure JPOXMLDOC01-appb-I000138
Figure JPOXMLDOC01-appb-T000047
Figure JPOXMLDOC01-appb-I000138
Figure JPOXMLDOC01-appb-T000047
Figure JPOXMLDOC01-appb-I000139
Figure JPOXMLDOC01-appb-I000139
Figure JPOXMLDOC01-appb-I000140
Figure JPOXMLDOC01-appb-I000140
Figure JPOXMLDOC01-appb-I000141
Figure JPOXMLDOC01-appb-I000141
Figure JPOXMLDOC01-appb-I000142
Figure JPOXMLDOC01-appb-I000142
Figure JPOXMLDOC01-appb-I000143
Figure JPOXMLDOC01-appb-I000143
Figure JPOXMLDOC01-appb-I000144
Figure JPOXMLDOC01-appb-I000144
Figure JPOXMLDOC01-appb-I000145
Figure JPOXMLDOC01-appb-I000145
Figure JPOXMLDOC01-appb-I000146
Figure JPOXMLDOC01-appb-I000146
Figure JPOXMLDOC01-appb-I000147
Figure JPOXMLDOC01-appb-I000147
Figure JPOXMLDOC01-appb-I000148
Figure JPOXMLDOC01-appb-I000148
Figure JPOXMLDOC01-appb-I000149
Figure JPOXMLDOC01-appb-I000149
Figure JPOXMLDOC01-appb-I000150
Figure JPOXMLDOC01-appb-I000150
Figure JPOXMLDOC01-appb-I000151
Figure JPOXMLDOC01-appb-I000151
Figure JPOXMLDOC01-appb-I000152
Figure JPOXMLDOC01-appb-I000152
Figure JPOXMLDOC01-appb-I000153
Figure JPOXMLDOC01-appb-I000153
Figure JPOXMLDOC01-appb-I000154
Figure JPOXMLDOC01-appb-I000154
Figure JPOXMLDOC01-appb-I000155
Figure JPOXMLDOC01-appb-T000048
Figure JPOXMLDOC01-appb-I000155
Figure JPOXMLDOC01-appb-T000048
Figure JPOXMLDOC01-appb-I000156
Figure JPOXMLDOC01-appb-I000156
Figure JPOXMLDOC01-appb-I000157
Figure JPOXMLDOC01-appb-T000049
Figure JPOXMLDOC01-appb-T000050
Figure JPOXMLDOC01-appb-I000157
Figure JPOXMLDOC01-appb-T000049
Figure JPOXMLDOC01-appb-T000050
Figure JPOXMLDOC01-appb-I000158
Figure JPOXMLDOC01-appb-I000158
Figure JPOXMLDOC01-appb-I000159
Figure JPOXMLDOC01-appb-I000159
Figure JPOXMLDOC01-appb-I000160
Figure JPOXMLDOC01-appb-T000051
Figure JPOXMLDOC01-appb-I000160
Figure JPOXMLDOC01-appb-T000051
Figure JPOXMLDOC01-appb-I000161
Figure JPOXMLDOC01-appb-I000161
Figure JPOXMLDOC01-appb-I000163
Figure JPOXMLDOC01-appb-I000163
Figure JPOXMLDOC01-appb-I000164
Figure JPOXMLDOC01-appb-I000164
Figure JPOXMLDOC01-appb-I000165
Figure JPOXMLDOC01-appb-I000165
Figure JPOXMLDOC01-appb-I000166
Figure JPOXMLDOC01-appb-I000166
Figure JPOXMLDOC01-appb-I000167
Figure JPOXMLDOC01-appb-I000167
Figure JPOXMLDOC01-appb-I000168
Figure JPOXMLDOC01-appb-I000168
Figure JPOXMLDOC01-appb-I000169
Figure JPOXMLDOC01-appb-I000169
Figure JPOXMLDOC01-appb-I000170
Figure JPOXMLDOC01-appb-I000170
Figure JPOXMLDOC01-appb-I000171
Figure JPOXMLDOC01-appb-I000171
Figure JPOXMLDOC01-appb-I000172
Figure JPOXMLDOC01-appb-I000172
Figure JPOXMLDOC01-appb-I000173
Figure JPOXMLDOC01-appb-I000173
Figure JPOXMLDOC01-appb-I000174
Figure JPOXMLDOC01-appb-T000052
Figure JPOXMLDOC01-appb-T000053
Figure JPOXMLDOC01-appb-T000054
Figure JPOXMLDOC01-appb-T000055
Figure JPOXMLDOC01-appb-T000056
Figure JPOXMLDOC01-appb-I000174
Figure JPOXMLDOC01-appb-T000052
Figure JPOXMLDOC01-appb-T000053
Figure JPOXMLDOC01-appb-T000054
Figure JPOXMLDOC01-appb-T000055
Figure JPOXMLDOC01-appb-T000056
Figure JPOXMLDOC01-appb-I000175
Figure JPOXMLDOC01-appb-T000057
Figure JPOXMLDOC01-appb-T000058
Figure JPOXMLDOC01-appb-T000059
Figure JPOXMLDOC01-appb-I000175
Figure JPOXMLDOC01-appb-T000057
Figure JPOXMLDOC01-appb-T000058
Figure JPOXMLDOC01-appb-T000059
Figure JPOXMLDOC01-appb-I000176
Figure JPOXMLDOC01-appb-I000176
Figure JPOXMLDOC01-appb-I000177
Figure JPOXMLDOC01-appb-I000177
Figure JPOXMLDOC01-appb-I000178
Figure JPOXMLDOC01-appb-I000178
Figure JPOXMLDOC01-appb-I000179
Figure JPOXMLDOC01-appb-I000179
Figure JPOXMLDOC01-appb-I000180
Figure JPOXMLDOC01-appb-I000180
Figure JPOXMLDOC01-appb-I000181
Figure JPOXMLDOC01-appb-I000181
Figure JPOXMLDOC01-appb-I000182
Figure JPOXMLDOC01-appb-I000182
Figure JPOXMLDOC01-appb-I000183
Figure JPOXMLDOC01-appb-I000183
Figure JPOXMLDOC01-appb-I000184
Figure JPOXMLDOC01-appb-I000184
２-１-５．考察・結論
　今回の実験ではラットへのTickling刺激を４週間の長期に渡り施しているため、刺激による即時反応の結果というより体質の変化に伴う各組織の遺伝子の発現差を主に観察していると推測される。すなわち、外界から一時的な刺激に対して、生体は恒常性維持のため神経－内分泌－免疫系を介して各組織が協調反応するので遺伝子の発現レベルでは連動することが予測できたが、長期の刺激は各組織の本来の機能に対して作用するため、その結果は体質の変化として表出するものと考えられ、組織間で連動して変化する遺伝子は少なかった。 2-1-5. Discussion / Conclusion In this experiment, Tickling stimulation was applied to rats over a long period of 4 weeks, so we mainly observed differences in gene expression in each tissue associated with changes in constitution rather than immediate response to stimulation. It is estimated that In other words, in response to temporary stimuli from the outside world, it was predicted that each tissue cooperates through the neuro-endocrine-immune system in order to maintain homeostasis, so that the expression level of the gene is linked. Since the stimuli act on the original functions of each tissue, the results are thought to be expressed as changes in the constitution, and few genes change in conjunction with each other.
　クラスタリング解析において、視床下部における対照群/Tickling群、Light Touch群/Tickling群で観察される発現差が一部の遺伝子クラスターで逆のパターンを描くことが明らかになった(図55)。線条体および唾液腺(軽度な差異は図50で認めるが)では、これら２比較間で顕著な差は観察されなかった。すなわち、視床下部では対照とLight Touchの差がTickling刺激のベースとして他組織より大きく反映されるものと考えられた。 Clustering analysis revealed that the expression differences observed in the control group / Tickling group, Light Touch group / Tickling group in the hypothalamus draw a reverse pattern in some gene clusters (FIG. 55). No significant differences were observed between these two comparisons in the striatum and salivary glands (although slight differences are seen in FIG. 50). In other words, in the hypothalamus, the difference between the control and the Light と し て Touch was considered to be more greatly reflected than the other tissues as the base of Tickling stimulation.
　視床下部は生命維持の中枢であるため、GO解析において他組織と比較して多くのGO Termに抽出遺伝子が割り当てられる結果となった。興味ある結果としては、対照群/Tickling群間の解析ではTicklingでDown-regulateされる遺伝子が広範囲のTermに割り当てられるのに対し、Up-regulateされる遺伝子は限局したTermに割り当てられたことである。一方、Light Touch群/Tickling群間の解析では、TicklingでUp-regulateされる遺伝子が広範囲のTermに割り当てられるのに対し、Down-regulateされる遺伝子は狭い範囲のTermに割り当てられたことである。すなわち、一方向に対しては、非特異的なTermに割り当てられる遺伝子により生体反応がマクロに反応し、それとは逆方向のregulationを受ける特異的なTermに割り当てられた遺伝子により微調節を受けていることである。例えば、Light Touch群と比較してTickling群でDown-regulateされる遺伝子群は陰イオン結合に関するTermに属し、Ticklingによる視床下部のCaイオンを介した細胞内刺激伝達、コリン作動性神経による異常興奮を非特異的に制御しているものと考えられる。逆に、Up-regulateされる遺伝子群は食行動や生体アミン合成・代謝に関与する限局した作用を有するTermに属し、特異的に働いていることが特徴的である。特に、食行動に関与する具体的遺伝子は、ガラニン様ペプチド(Galp)、オピオメラノコルチン前躯体(Pomc)、メラニン濃縮ホルモン(Pmch)、Agouti関連蛋白(Agrp)、ハイポクレチン(Hcrt)、ニューロペプチドY(Npy)が抽出されたが、いずれも食行動を活性化する作用を有している。また、対照群/Tickling群間の比較において、Tickling群でストレスホルモン(Avp)が抽出されたことは、本実験において対照として採用したラットの個別飼育は、負のストレス刺激になっていることを検証する結果となった。 Since the hypothalamus is the center of life support, compared to other tissues in GO analysis, extracted genes were assigned to more GO Term. An interesting result is that in the analysis between the control group and the Tickling group, genes that are down-regulated by Tickling are assigned to a wide range of terms, whereas genes that are up-regulated are assigned to limited terms. is there. On the other hand, in the analysis between Light Touch group / Tickling group, genes that are up-regulated by Tickling are assigned to a wide range of terms, whereas genes that are down-regulated are assigned to a narrow range of terms. . That is, for one direction, the biological reaction reacts macroscopically with a gene assigned to a non-specific Term, and finely regulated by a gene assigned to a specific Term that undergoes the reverse regulation. It is that you are. For example, the group of genes down-regulated in the Tickling group compared to the Light Touch group belongs to the term related to anion binding, intracellular stimulation transmission via Ca ions in the hypothalamus by Tickling, abnormal excitation by cholinergic nerves Is thought to be non-specifically controlled. On the other hand, the genes that are up-regulated belong to a term having a limited action involved in eating behavior and biogenic amine synthesis / metabolism, and are characterized by working specifically. In particular, specific genes involved in eating behavior are galanin-like peptide (Galp), opiomelanocortin precursor (Pomc), melanin-concentrating hormone (Pmch), Agouti-related protein (Agrp), hypocretin (Hcrt), neuropeptide Y (Npy) was extracted, but all have the effect of activating eating behavior. In addition, in the comparison between the control group and the Tickling group, the stress hormone (Avp) was extracted in the Tickling group, which means that the individual rearing of the rats used as controls in this experiment was a negative stress stimulus. It became the result to verify.
　唾液腺の解析では、多くの遺伝子がTicklingにより発現変動しており、この結果から演繹される唾液成分が新規なストレス指標となる可能性を示唆している。例えば、対照群と比較してTickling群でUp-regulateされたトニン(Ton)、顎下腺カリクレイン(Klks2)、腺性カリクイレイン(Klk12)、神経成長因子(Ngfg)や、Down-regulateされるセリンプロテアーゼインヒビター(Serpina1)、フェツイン(Fetub)はその第一候補となりえる。その他、多くの構造蛋白をコードする遺伝子がUp-regulateされており、これらの成分もストレス指標として利用できる可能性を示唆している。 In the analysis of salivary glands, the expression of many genes fluctuates due to Tickling, and it is suggested that saliva components deduced from these results may be a novel stress indicator. For example, Tonin (Ton), submandibular gland kallikrein (Klks2), glandular caliculein (Klk12), nerve growth factor (Ngfg), and serine down-regulated compared to the control group Protease inhibitor (Serpina1) and fetubin can be the first candidates. In addition, genes encoding many structural proteins have been up-regulated, suggesting the possibility that these components can also be used as stress indicators.
２-１-５-１．健康Responsible遺伝子候補
　生命維持の中枢としての視床下部の役割を考えると、Light Touch群と比較してTickling群で発現差を認める遺伝子が健康Responsible遺伝子の第１候補として位置づけられる。非特異的なGO Termに割り当てられるDown-regulateされた遺伝子よりも、特異的なGO Termに割り当てられるUp-regulateされた遺伝子に注目すると、食行動に関与する上記６遺伝子とドーパミントランスポータ(Slc6a3)、チロシン水酸化酵素(Th)がその候補であるが、今後の課題として、短期的なTickling、末梢細胞での発現変化との関連性解析が待たれる。 2-1-5-1. Considering the role of the hypothalamus as the center of life support for healthy Responsible gene candidates, genes that show differential expression in the Tickling group compared to the Light Touch group are positioned as the first candidate for the healthy Responsible gene. Focusing on the up-regulated gene assigned to the specific GO Term rather than the down-regulated gene assigned to the non-specific GO Term, the above 6 genes involved in eating behavior and the dopamine transporter (Slc6a3 ), Tyrosine hydroxylase (Th) is a candidate for this, but as future issues, it is awaited to analyze the relationship between short-term Tickling and expression changes in peripheral cells.
２-１-５-２．新規ストレス指標の推定
　今後の体外診断用医薬品には非侵襲・非拘束であることが要求されていることを鑑みると、唾液成分を測定することの臨床的意義が拡大することは容易に推測できる。本研究で実施したTicklingによる唾液腺の遺伝子発現解析から新規ストレス指標を提案するためには、ラットにおける陽性感情と陰性感情の隔たりが大きい対照群/Tickling群間で発現差を認める唾液腺遺伝子から演繹される唾液成分がその候補として適切であると判断する。この選択基準に合致する具体的唾液成分としては、前項記載のトニン(Ton)、顎下腺カリクレイン(Klks2)、腺性カリクイレイン(Klk12)、神経成長因子(Ngfg)、セリンプロテアーゼインヒビター(Serpina1)、フェツイン(Fetub)をはじめとして、唾液中の多くの分泌蛋白が候補として挙げられる。刺激負荷後のこれら遺伝子発現の時間変化を予め調べておけば、或る時点での複数の唾液腺遺伝子由来の唾液成分濃度により生体がストレス状態に向かうのか回復期にあるのか判断することが可能になる。また、本実験により特定した複数の唾液成分濃度をパターン解析することによりストレスの種類を判定することも可能になり応用範囲は広い。 2-1-5-2. Estimating new stress indicators Given the fact that future in vitro diagnostics are required to be non-invasive and non-constrained, it can be easily guessed that the clinical significance of measuring saliva components will expand . In order to propose a new stress index from the salivary gland gene expression analysis by Tickling conducted in this study, it was deduced from the salivary gland genes that showed a difference in expression between the control group / Tickling group with a large gap between positive and negative emotions in rats. It is determined that the saliva component is appropriate as the candidate. Specific saliva components that meet this selection criteria include Tonin (Ton), submandibular gland kallikrein (Klks2), glandular kallikrein (Klk12), nerve growth factor (Ngfg), serine protease inhibitor (Serpina1), Many secreted proteins in saliva, including fetub, are candidates. If the time changes in the expression of these genes after a stress load are examined in advance, it is possible to determine whether the living body is in a stress state or in the recovery phase based on the saliva component concentrations derived from multiple salivary gland genes at a certain point in time. Become. In addition, it is possible to determine the type of stress by pattern analysis of a plurality of saliva component concentrations specified by this experiment, and the application range is wide.
２-１-６．総括
　Ticklingがラットに対して陽性感情を表出する刺激となることを、50KHz近傍の発生音声の解析により検証し、Tickling(4週間)により発現変化する遺伝子群を血液、脳組織(視床下部、線条体)、唾液腺で解析した。 2-1-6. General Tickling is a stimulus that expresses positive emotions to rats by verifying the analysis of generated speech near 50 KHz, and the genes that change expression by Tickling (4 weeks) are expressed in blood, brain tissue (hypothalamic, hypothalamus, Striatum) and salivary glands.
　その結果、長期(4週間)に渡るTicklingによる遺伝子の発現変化は中枢/末梢血液では連動せず、また線条体での発現変化も少ないことが明らかとなった。これは、Ticklingの効果が体質の変化として現れるためであると考察した。Ticklingの長期効果がより多く認められた組織は視床下部と唾液腺であり、前者における遺伝子発現解析より食行動とドーパミン代謝に関わる健康Responsible遺伝子、後者より新規ストレス指標の候補となる唾液成分をコード化する遺伝子を特定することができた。 As a result, it was clarified that the gene expression change by Tickling over a long period (4 weeks) is not linked in the central / peripheral blood, and the expression change in the striatum is small. This was considered because the effect of Tickling appeared as a change in constitution. The tissues with more long-term effects of Tickling are the hypothalamus and salivary gland, which encodes healthy responsible genes related to eating behavior and dopamine metabolism from the former gene expression analysis, and saliva components that are candidates for new stress indicators from the latter I was able to identify the gene to be.
２－２．短期間（２日間）のTickling刺激
　６週令(離乳直後[３週令]から個別飼育)になってから２日間、Tickling刺激を負荷したラット、Light Touch刺激(対照)を負荷したラットついて遺伝子発現解析を行なった。Tickling刺激は上記１-２-２記載の方法に従い、ラットをTickling用クリーンケージ(内面は黒色フェルトを貼付)に移し、15秒間放置後、15秒のTickling刺激負荷を行なう操作を4回繰り返して１Tickling Sessionとし、１Session終了後1分間の休憩を挟み、さらに２Session目(2分間)のTicklingを行なった。Tickling刺激負荷時程図は図１に示す通りである。 2-2. Genes for rats loaded with Tickling stimulation and rats loaded with Light Touch stimulation (control) for 2 days after short-term (2 days) Tickling stimulation 6 weeks of age (individually reared immediately after weaning [3 weeks of age]) Expression analysis was performed. For tickling stimulation, follow the method described in 1-2-2 above. Transfer the rat to a clean cage for Tickling (with black felt on the inner surface), leave it for 15 seconds, and repeat the operation of tickling stimulation load for 15 seconds 4 times. A 1-tickling session was set, and a 1-minute break was placed after the end of the 1-session, and a second Tickling (2 minutes) was performed. Tickling stimulus loading time chart is as shown in FIG.
　一方、Light Touch刺激は、ラットをTickling用クリーンケージ(上述)に移し15秒間放置の後、Ticklingの変わりに3秒に1回、ラット背部にやさしく触れることにより負荷した。刺激負荷時程はTickling刺激負荷と同様(図1)とした。 On the other hand, the Light Touch stimulation was applied by gently touching the rat back once every 3 seconds instead of Tickling after moving the rat to the Tickling clean cage (described above) and leaving it for 15 seconds. The stimulus load time was the same as the Tickling stimulus load (FIG. 1).
　Tickling負荷２日目及びLight Touch負荷２日目のラット発生音声の周波数成分解析図をそれぞれ図73と図74に示す。Tickling刺激時には50KHz近傍(60KHz)の音声を確認することができたが、対照であるLight Touch刺激では当音声は確認できず、１-２-５．の結果を再現した。 Fig. 73 and Fig. 74 show the frequency component analysis charts of rat-generated speech on the second day of Tickling load and the second day of Light Touch load, respectively. Although the voice in the vicinity of 50KHz (60KHz) was able to be confirmed at the time of Tickling stimulation, this voice was not able to be confirmed with the Light Touch stimulation as a control, 1-2-5. The result of was reproduced.
２-２-１．実験方法（解析対象臓器の摘出）
　臓器摘出の順番は、開胸→心採血→生食灌流(腎動脈瀉血)→口腔内粘膜細胞擦過→断頭→開頭→脳摘出の順とした。 2-2-1. Experimental method (extraction of analysis target organ)
The order of organ removal was as follows: thoracotomy-> heart blood sampling-> saline perfusion (renal artery hemoptysis)-> oral mucosal cell abrasion->decapitation->craniotomy-> brain removal.
1) 心採血・生食灌流
　エーテル麻酔下に開胸し心採血(約1mLをPAXgene専用採血管に採取)を行ない、留置針にて生食(約20mL) 灌流／腎動脈瀉血を行なった。 1) Cardiac blood sampling and saline perfusion The heart was opened under ether anesthesia, blood sampling was performed (approximately 1 mL was collected in a dedicated blood collection tube for PAXgene), and saline (approximately 20 mL) was perfused / perfused with renal artery.
2) 口腔内粘膜細胞擦過
　生食にて口腔内を洗浄、キムワイプにて口腔内残液を吸収後、マイクロスパーテルで口腔内粘膜細胞を擦過採取し、RNA安定化試薬RNAlater(QIAGEN社製)に浸漬、-80℃で保存した。 2) Oral mucosal cell rubbing After washing the oral cavity with a raw diet, absorbing the oral residual liquid with Kimwipe, scraping the oral mucosal cells with a micro spatula and immersing them in the RNA stabilizing reagent RNAlater (QIAGEN) And stored at -80 ° C.
3) 脳摘出
　頭頂部から開頭、脳を摘出し、Glowinski(J Neurochem, 1966)の脳7分割法に従い、氷冷したアルミ板上で線条体、海馬、視床下部、皮質を分けた。脳下垂体はトルコ鞍より直接摘出した。摘出組織は約3mm角にトリミングし、RNA安定化試薬RNAlater(QIAGEN社製)に浸漬、-80℃で保存した。 3) Brain extraction The craniotomy was performed from the top of the head, the brain was extracted, and the striatum, hippocampus, hypothalamus, and cortex were separated on an ice-cooled aluminum plate according to the brain division method of Glowinski (J Neurochem, 1966). The pituitary gland was removed directly from the Turkish anther. The extracted tissue was trimmed to about 3 mm square, immersed in RNA stabilizing reagent RNAlater (manufactured by QIAGEN) and stored at −80 ° C.
２-２-２．実験方法（ラット組織からのRNA調製・検定）
1) 血液試料からの全RNA調製
　PAXgene Blood RNA Kit (QIAGEN社製)を用いて全RNA標品を調製した。 2-2-2. Experimental method (preparation and assay of RNA from rat tissue)
1) Preparation of total RNA from blood sample Total RNA preparation was prepared using PAXgene Blood RNA Kit (manufactured by QIAGEN).
2) 口腔内粘膜擦過細胞および脳組織からの全RNA調製
　RNAlater浸漬組織(遺伝子発現解析対象である擦過細胞、視床下部、線条体のみ)を、RNeasy Buffer RLT中でホモジナイズ〔TissueLyzer(QIAGEN社製)使用〕し、以下Kitプロトコールに従い全RNA標品を調製した。各群(Tickling、Light Touch)の口腔粘膜擦過細胞は全RNAを全量混合、脳組織は等量混合した。 2) Total RNA preparation from oral mucosal scraping cells and brain tissue RNAlater-immersed tissue (scratched cells, hypothalamus, and striatum only for gene expression analysis) were homogenized in RNeasy Buffer RLT (TissueLyzer (QIAGEN) The total RNA preparation was prepared according to the following Kit protocol. Oral mucosal scratched cells in each group (Tickling, Light Touch) were mixed with total RNA, and brain tissue was mixed with equal volume.
3) 全RNA純度と無傷度の検定
　個体毎(全RNA標品混合前)に、全RNA標品の濃度とA260/280比、28S/18S rRNA比を求めた。28S/18S rRNA比は、マイクロチップ型電気泳動システム(日立コスモアイ、SV1210)により検定・算出した。 3) Total RNA purity and intactness test For each individual (before mixing the total RNA sample), the concentration of the total RNA sample, the A260 / 280 ratio, and the 28S / 18S rRNA ratio were determined. The 28S / 18S rRNA ratio was assayed and calculated using a microchip electrophoresis system (Hitachi Cosmo Eye, SV1210).
２-２-３．実験方法（DNAチップ解析）
　上記２-２-２の全RNA標品を出発材料として、T7RNA Polymerase Promoter配列を付加したOligo(dT)24をプライマーとしたcDNA合成を行った。引き続き、これを鋳型としてアミノアリル-dUTP 存在下にT7RNA PolymeraseによるmRNA増幅を行って、cRNAにアミノ基を導入し、Cyanine 3[Cy3と略](Light Touch群)あるいはCyanine 5[Cy5と略] (Tickling群)のサクシニミド誘導体とカップリング反応させることにより蛍光標識を行いDNAチップ解析に供した。 2-2-3. Experimental method (DNA chip analysis)
Using the above 2-2-2 total RNA preparation as a starting material, cDNA synthesis was performed using Oligo (dT) 24 to which a T7 RNA Polymerase Promoter sequence was added as a primer. Subsequently, using this as a template, mRNA amplification was performed with T7RNA Polymerase in the presence of aminoallyl-dUTP to introduce amino groups into cRNA, and Cyanine 3 [abbreviated as Cy3] (Light Touch group) or Cyanine 5 [abbreviated as Cy5] ( Tickling group) was fluorinated with a coupling reaction with a succinimide derivative and subjected to DNA chip analysis.
　DNAチップ解析は以下の通り行った。各Cyanine色素で蛍光標識したRNA標品を等量混合し、DNAチップ〔Agilent社製(約41,012オリゴDNA搭載)、Whole Rat Genome DNA Microarray〕上で65℃、17時間ハイブリダイゼーション反応を行い(未反応分子はハイブリダイゼーション反応後洗浄除去)、共焦点レーザースキャナー(Agilent社製、G2565)で各色素に由来する蛍光強度を測定した。バックグランド補正とグローバル正規化の後、信号強度50未満の遺伝子(トランスクリプトを含む)を除外し、Cy5/Cy3比を求めた。 DNA chip analysis was performed as follows. Equal amounts of RNA preparations fluorescently labeled with each cyanine dye are mixed, and a hybridization reaction is carried out at 65 ° C. for 17 hours on a DNA chip (Agilent (approx. 41,012 oligo DNA), WholeWGenome DNA Microarray). The reaction molecules were washed and removed after the hybridization reaction), and the fluorescence intensity derived from each dye was measured with a confocal laser scanner (Agilent, G2565). After background correction and global normalization, genes with a signal intensity of less than 50 (including transcripts) were excluded and the Cy5 / Cy3 ratio was determined.
２-２-４．結果
　本結果で用いる「プローブ」と「遺伝子(トランスクリプトを含む)」の用語は同義であり、図表で用いる凡例は以下の通りである。 2-2-4. Results The terms “probe” and “gene (including transcript)” used in this result are synonymous, and the legend used in the chart is as follows.
T：Tickling
L：Light Touch
Bl：血液
Bc：口腔内粘膜擦過細胞
Hy：視床下部
St：線条体
２-２-４-１．信号強度分布
　Tickling／Light Touch間の遺伝子発現の差をDNAチップ法にて解析した際の、DNAチップ上の各プローブの信号強度の分布を図75に示す。縦軸は信号強度、横軸はその信号強度を示す遺伝子数を示すが、脳組織試料は相互に類似の信号強度分布を示したが、血液試料及び口腔粘膜擦過細胞試料では、高信号強度を示す遺伝子数が少なかった。 T: Tickling
L: Light Touch
Bl: Blood
Bc: Oral mucosal scraping cells
Hy: hypothalamus
St: Striatum
2-2-4-1. When analyzing the differences in gene expression between the signal intensity distribution Tickling / Light Touch by DNA chip method, the distribution of the signal intensities for each probe on the DNA chip shown in FIG. 75. The vertical axis shows the signal intensity, and the horizontal axis shows the number of genes indicating the signal intensity, but the brain tissue samples showed similar signal intensity distributions, but the blood sample and the oral mucosal scratched cell sample showed a high signal intensity. Fewer genes were shown.
２-２-４-２．発現変化を認めた遺伝子
　Light Touch群と比較してTickling群で1.5倍超の発現を認めた末梢血液(Bl)の遺伝子の「Target Accession」及び「UniGene」を下記表60に示す。
Figure JPOXMLDOC01-appb-T000060
 2-2-4-2. Table 60 below shows “Target Accession” and “UniGene” of peripheral blood (Bl) genes whose expression was observed more than 1.5 times in the Tickling group as compared to the gene Light Touch group in which expression change was observed .
Figure JPOXMLDOC01-appb-T000060
　Light Touch群と比較してTickling群で1/1.5倍未満の発現を認めた末梢血液(Bl)の遺伝子の「Target Accession」及び「UniGene」を下記表61に示す。
Figure JPOXMLDOC01-appb-T000061
 Table 61 below shows the “Target Accession” and “UniGene” of peripheral blood (Bl) genes whose expression was found to be less than 1 / 1.5-fold in the Tickling group compared to the Light Touch group.
Figure JPOXMLDOC01-appb-T000061
　Light Touch群と比較してTickling群で1.5倍超の発現を認めた視床下部(Hy)の遺伝子の「Target Accession」及び「UniGene」を下記表62に示す。
Figure JPOXMLDOC01-appb-T000062
 Table 62 below shows the “Target Accession” and “UniGene” of hypothalamic genes (Hy) in which expression of 1.5 times more was observed in the Tickling group than in the Light Touch group.
Figure JPOXMLDOC01-appb-T000062
　Light Touch群と比較してTickling群で1/1.5倍未満の発現を認めた視床下部(Hy)の遺伝子の「Target Accession」及び「UniGene」を下記表63に示す。
Figure JPOXMLDOC01-appb-T000063
 Table 63 below shows the “Target Accession” and “UniGene” of hypothalamic (Hy) genes that were found to be expressed in the Tickling group by less than 1 / 1.5-fold compared to the Light Touch group.
Figure JPOXMLDOC01-appb-T000063
　Light Touch群と比較してTickling群で1.5倍超の発現を認めた線条体(St)の遺伝子の「Target Accession」及び「UniGene」を下記表64に示す。
Figure JPOXMLDOC01-appb-T000064
 Table 64 below shows the “Target Accession” and “UniGene” of the striatum (St) gene that was expressed more than 1.5 times in the Tickling group compared to the Light Touch group.
Figure JPOXMLDOC01-appb-T000064
Figure JPOXMLDOC01-appb-I000185
Figure JPOXMLDOC01-appb-I000185
　Light Touch群と比較してTickling群で1/1.5倍未満の発現を認めた線条体(St)の遺伝子の「Target Accession」及び「UniGene」を下記表65に示す。
Figure JPOXMLDOC01-appb-T000065
 Table 65 below shows the “Target Accession” and “UniGene” of the striatum (St) gene that was found to be expressed in the Tickling group by less than 1 / 1.5-fold compared to the Light Touch group.
Figure JPOXMLDOC01-appb-T000065
　Light Touch群と比較してTickling群で1.5倍超の発現を認めた口腔内細胞(Bc)の遺伝子の「Target Accession」及び「UniGene」を下記表66に示す。
Figure JPOXMLDOC01-appb-T000066
 Table 66 below shows the “Target Accession” and “UniGene” of the genes of oral cells (Bc) that have been expressed more than 1.5 times in the Tickling group as compared to the Light Touch group.
Figure JPOXMLDOC01-appb-T000066
Figure JPOXMLDOC01-appb-I000186
Figure JPOXMLDOC01-appb-I000186
Figure JPOXMLDOC01-appb-I000187
Figure JPOXMLDOC01-appb-I000187
　Light Touch群と比較してTickling群で1/1.5倍未満の発現を認めた口腔内細胞(Bc)の遺伝子の「Target Accession」及び「UniGene」を下記表67に示す。
Figure JPOXMLDOC01-appb-T000067
 Table 67 below shows the “Target Accession” and “UniGene” of genes of oral cells (Bc) that were found to be expressed in the Tickling group in an expression of less than 1 / 1.5-fold compared to the Light Touch group.
Figure JPOXMLDOC01-appb-T000067
Figure JPOXMLDOC01-appb-I000188
Figure JPOXMLDOC01-appb-I000188
　Light Touch群と比較してTickling群で2.0倍超の発現を認めた末梢血液(Bl)の遺伝子のアノテーションを下記表68に示す。
Figure JPOXMLDOC01-appb-T000068
 Table 68 below shows annotations of genes in peripheral blood (Bl) that were expressed more than 2.0 times in the Tickling group compared to the Light Touch group.
Figure JPOXMLDOC01-appb-T000068
　Light Touch群と比較してTickling群で0.5倍未満の発現を認めた末梢血液(Bl)の遺伝子のアノテーションを下記表69に示す。
Figure JPOXMLDOC01-appb-T000069
 Table 69 below shows gene annotations of peripheral blood (Bl) in which expression of less than 0.5-fold was observed in the Tickling group compared to the Light Touch group.
Figure JPOXMLDOC01-appb-T000069
　Light Touch群と比較してTickling群で2.0倍超の発現を認めた視床下部(Hy)の遺伝子のアノテーションを下記表70に示す。
Figure JPOXMLDOC01-appb-T000070
 Table 70 below shows annotations of hypothalamic genes (Hy) that have been expressed more than 2.0 times in the Tickling group compared to the Light Touch group.
Figure JPOXMLDOC01-appb-T000070
　Light Touch群と比較してTickling群で0.5倍未満の発現を認めた視床下部(Hy)の遺伝子のアノテーションを下記表71に示す。
Figure JPOXMLDOC01-appb-T000071
 Table 71 below shows hypothalamic (Hy) gene annotations in which expression of less than 0.5-fold was observed in the Tickling group compared to the Light Touch group.
Figure JPOXMLDOC01-appb-T000071
　Light Touch群と比較してTickling群で2.0倍超の発現を認めた線条体(St)の遺伝子のアノテーションを下記表72に示す。
Figure JPOXMLDOC01-appb-T000072
 Table 72 below shows the annotation of the striatum (St) gene that was found to express more than 2.0 times in the Tickling group compared to the Light Touch group.
Figure JPOXMLDOC01-appb-T000072
Figure JPOXMLDOC01-appb-I000189
Figure JPOXMLDOC01-appb-I000189
Figure JPOXMLDOC01-appb-I000190
Figure JPOXMLDOC01-appb-I000190
Figure JPOXMLDOC01-appb-I000191
Figure JPOXMLDOC01-appb-I000191
Figure JPOXMLDOC01-appb-I000193
Figure JPOXMLDOC01-appb-I000193
　Light Touch群と比較してTickling群で0.5倍未満の発現を認めた線条体(St)の遺伝子のアノテーションを下記表73に示す。
Figure JPOXMLDOC01-appb-T000073
 Table 73 below shows the annotation of the striatum (St) gene in which the expression was less than 0.5-fold in the Tickling group compared to the Light Touch group.
Figure JPOXMLDOC01-appb-T000073
　Light Touch群と比較してTickling群で2.0倍超の発現を認めた口腔内細胞(Bc)の遺伝子のアノテーションを下記表74に示す。
Figure JPOXMLDOC01-appb-T000074
 Table 74 below shows annotations of genes of oral cells (Bc) that have been expressed more than 2.0 times in the Tickling group compared to the Light Touch group.
Figure JPOXMLDOC01-appb-T000074
Figure JPOXMLDOC01-appb-I000194
Figure JPOXMLDOC01-appb-I000194
Figure JPOXMLDOC01-appb-I000195
Figure JPOXMLDOC01-appb-I000195
　Light Touch群と比較してTickling群で0.5倍未満の発現を認めた口腔内細胞(Bc)の遺伝子のアノテーションを下記表75に示す。
Figure JPOXMLDOC01-appb-T000075
 Table 75 below shows gene annotations of oral cells (Bc) in which expression in the Tickling group was less than 0.5 times that of the Light Touch group.
Figure JPOXMLDOC01-appb-T000075
Figure JPOXMLDOC01-appb-I000196
Figure JPOXMLDOC01-appb-I000196
　なお、表68～75中の「No.」は、上記表1中の「No.」に対応している。 “No.” in Tables 68 to 75 corresponds to “No.” in Table 1 above.
　Tickling群の末梢血液で２倍以上の発現を認める遺伝子は３つ、0.5倍以下の発現を認める遺伝子は１つしか存在しなかった。同様に発現差を認める遺伝子をカウントすると、口腔粘膜擦過細胞、視床下部、線条体で２倍以上の発現を認める遺伝子はそれぞれ12、３、25遺伝子存在し、0.5倍以下の発現を認める遺伝子はそれぞれ13、２、２遺伝子存在した。 In the Tickling group, there were 3 genes that showed more than 2-fold expression in the peripheral blood and only 1 gene that showed 0.5-fold or less expression. Similarly, when genes that show differential expression are counted, there are 12, 3 and 25 genes that are expressed more than twice in oral mucosal scratched cells, hypothalamus, and striatum, and genes that are expressed less than 0.5 times. There were 13, 2, and 2 genes, respectively.
　注目すべき遺伝子としては、Ticklingにより口腔粘膜細胞でUp-regulateされたGsta5、Aldh1a1、Yc2、Gsta2、Cdig1l、Selenbp1、Down-regulateされたCdkn1c、Gda、視床下部でUp-regulateされたSlc6a3、線条体でUp-regulateされたAvp、Neurod2、Vip、Slc17a7、Cck、Sstr1、Cort、Nrn1、Prss、Down-regulateされたTtrがある。 Notable genes include Gsta5, Aldh1a1, Yc2, Gsta2, Cdig1l, Selenbp1, Down-regulated Cdkn1c, Gda, Slc6a3 up-regulated in the hypothalamus, lines There are Avp, Neurod2, Vip, Slc17a7, Cck, Sstr1, Cort, Nrn1, Prss, and Down-regulated Ttr that are up-regulated in the striatum.
２-２-４-３．クラスタリング解析
　いずれかの解析においてCy3およびCy5の信号強度が50以上あったプローブは24,076遺伝子(トランスクリプトを含む)存在し、これらをクラスタリングした像を図76に示し、また、いずれかの解析において1.5倍以上あるいは1/1.5倍以下の発現差を認めた868プローブのクラスタリング像を図77に、２倍以上あるいは0.5倍以下の発現差を認めた88プローブのクラスタリング像を図78に示した。発現差の程度は、TicklingによりUp-regulateされた遺伝子(トランスクリプトを含む)は赤色、Down-regulateされた遺伝子(トランスクリプトを含む)は緑色の濃さの程度で表した(カラーバーは図中右側に表示)。また、信号強度が低く(50未満)、発現差を表示できない遺伝子(トランスクリプトを含む)は灰色で表示した。血液と口腔粘膜擦過細胞において、双方向(Up-regulationあるいはDown-regulation)に発現変動する遺伝子が顕著であり、特に後者では変動幅が大きい遺伝子(２倍以上あるいは0.5倍以下)を多く含んでいた。視床下部での発現変動は双方向共に鈍いのに対し、線条体ではUp-regulationした遺伝子が多く存在し、その変動幅も２倍以上のものが多かった。 2-2-4-3. Clustering analysis There were 24,076 genes (including transcripts) in which the signal intensity of Cy3 and Cy5 was 50 or more in any analysis, and an image obtained by clustering them is shown in Fig. 76. FIG. 77 shows a clustering image of 868 probes in which an expression difference of more than double or 1 / 1.5 times or less was observed, and FIG. 78 shows a clustering image of 88 probes in which an expression difference of 2 times or more or 0.5 times or less was recognized. The degree of expression difference is expressed in red for genes up-regulated by Tickling (including transcripts) and green for down-regulated genes (including transcripts). (Displayed on the middle right). In addition, genes with low signal intensity (less than 50) that cannot display differential expression (including transcripts) are shown in gray. In blood and oral mucosal scraping cells, genes that change in both directions (Up-regulation or Down-regulation) are prominent, especially in the latter, which contain many genes with a large fluctuation range (more than 2 times or less than 0.5 times). It was. The expression variation in the hypothalamus was slow in both directions, whereas in the striatum, there were many up-regulated genes, and the variation range was more than twice as many.
　また、上記のTickling長期（4週間）刺激時のデータも解析対象とし、いずれかの解析において1.5倍以上あるいは1/1.5倍以下の発現差を認めた4,397プローブのクラスタリング像を図79(プローブの並びは長期刺激を基準)と図80(プローブの並びは短期刺激を基準)に、２倍以上あるいは0.5倍以下の発現差を認めた865プローブのクラスタリング像を図81(プローブの並びは長期刺激を基準)と図82(プローブの並びは短期刺激を基準)に示した。長期Tickling刺激負荷を行ったラット血液において双方向(Up-regulationあるいはDown-regulation)に発現変動する遺伝子が多く、その変動幅も２倍以上のものが多かった。長期Tickling負荷を行ったラット脳組織(視床下部、線条体)でも、同様に双方向に発現変動する遺伝子群が存在したが、これらは血液で発現変動する遺伝子とは異なっていた。さらに、血液と線条体では、長期Tickling刺激で発現変動する遺伝子の変化方向と短期Tickling刺激で発現変動する遺伝子の変化方向が逆であることが特徴的であった。 The above Tickling long-term (4 weeks) stimulation data was also analyzed, and clustering images of 4,397 probes that showed an expression difference of 1.5 times or more or 1 / 1.5 times or less in any analysis were shown in Fig. 79 (probe of probe). Fig. 81 (probe alignment is long-term stimulation) and Fig. 80 (probe alignment is based on short-term stimulation) and Fig. 81 (probe alignment is long-term stimulation). ) And FIG. 82 (probe alignment is based on short-term stimulation). In the blood of rats subjected to long-term tickling stimulation, there were many genes that fluctuated in both directions (Up-regulation or Down-regulation), and the fluctuation range was more than twice as many. In rat brain tissues (hypothalamus, striatum) subjected to long-term tickling load, there were genes that fluctuated in both directions, but these were different from genes that fluctuated in blood. Furthermore, in blood and striatum, it was characteristic that the direction of change of the gene whose expression was changed by long-term Tickling stimulation was opposite to the direction of change of the gene whose expression was changed by short-term Tickling stimulation.
２-２-４-４．オントロジー解析
　当解析は、Light Touch群と比較してTickling群で1.5倍以上あるいは1/1.5倍以下の発現差を認める遺伝子を対象とし、該条件にて抽出される視床下部、線条体の遺伝子に関し行なった。抽出されたプローブ数と遺伝子数は下記表76に示す。
Figure JPOXMLDOC01-appb-T000076
 2-2-4-4. Ontology analysis This analysis targets genes that have an expression difference of 1.5 times or more or 1 / 1.5 times or less in the Tickling group compared to the Light Touch group, and the hypothalamus and striatum genes extracted under these conditions It performed about. The number of extracted probes and the number of genes are shown in Table 76 below.
Figure JPOXMLDOC01-appb-T000076
　DNAチップ上のプローブと遺伝子(GeneID)との対応付けは、Agilent社の提供する情報とNCBI Entrez(http://www.ncbi.nlm.nih.gov/)の情報に基づいて行なった。GeneIDとGOの対応付けはBiNGO (http://www.psb.ugent.be/cbd/papers/BiNGO/)を用いて行い、GOの階層図はCytoscape (http://www.cytoscape.org/)を用いて描画した。各GO Termについて、Agilent社製Whole Rat Genome Microarray上の全遺伝子中での出現頻度に比べて抽出遺伝子群中での出現頻度が高くなっているかHypergeometric testを用いて有意水準FDR(False Discovery rate)<0.1で検定した。該検定で有意と判定されたGO Termを抽出遺伝子群に特徴的なGOとした。表77から80に、各試料で抽出された遺伝子(表76参照)のGO解析結果を示した。また、有意と判定されたGO Termが存在する解析のそれぞれについてCytoscapeを用いて作図したGO階層図は、図84から図88に示した。尚、図83には色の濃淡とP値の関係を表した。色が濃いほどそのGO Termに割り当てられる有意性が高く、円の大きさが大きいほど割り当てられる遺伝子数が多いこと示す。
Figure JPOXMLDOC01-appb-T000077
 The association between the probe on the DNA chip and the gene (GeneID) was performed based on information provided by Agilent and NCBI Entrez (http://www.ncbi.nlm.nih.gov/). GeneID and GO are mapped using BiNGO (http://www.psb.ugent.be/cbd/papers/BiNGO/), and the hierarchy of GO is Cytoscape (http://www.cytoscape.org/ ). For each GO Term, the frequency of occurrence in the extracted gene group is higher than the frequency of occurrence in all genes on the Agilent Whole Rat Genome Microarray. Tested at <0.1. GO Term determined to be significant by the test was defined as GO characteristic of the extracted gene group. Tables 77 to 80 show the GO analysis results of the genes (see Table 76) extracted from each sample. In addition, FIGS. 84 to 88 show GO hierarchy diagrams drawn using Cytoscape for each analysis in which a GO Term determined to be significant exists. FIG. 83 shows the relationship between color shading and P value. The darker the color, the higher the significance assigned to that GO Term, and the larger the circle, the more genes assigned.
Figure JPOXMLDOC01-appb-T000077
Figure JPOXMLDOC01-appb-I000197
Figure JPOXMLDOC01-appb-I000197
Figure JPOXMLDOC01-appb-I000198
Figure JPOXMLDOC01-appb-T000078
Figure JPOXMLDOC01-appb-I000198
Figure JPOXMLDOC01-appb-T000078
Figure JPOXMLDOC01-appb-I000199
Figure JPOXMLDOC01-appb-T000079
Figure JPOXMLDOC01-appb-I000199
Figure JPOXMLDOC01-appb-T000079
Figure JPOXMLDOC01-appb-I000200
Figure JPOXMLDOC01-appb-I000200
Figure JPOXMLDOC01-appb-I000201
Figure JPOXMLDOC01-appb-I000201
Figure JPOXMLDOC01-appb-I000202
Figure JPOXMLDOC01-appb-I000202
Figure JPOXMLDOC01-appb-I000203
Figure JPOXMLDOC01-appb-T000080
Figure JPOXMLDOC01-appb-I000203
Figure JPOXMLDOC01-appb-T000080
Figure JPOXMLDOC01-appb-I000204
Figure JPOXMLDOC01-appb-I000204
Figure JPOXMLDOC01-appb-I000205
Figure JPOXMLDOC01-appb-I000205
　線条体でUp-regulateされる遺伝子のGO解析では、健康増進遺伝子探索のための候補遺伝子を特定できた。機能上GOID:5184に割り当てられる神経ペプチドホルモン活性に関する遺伝子(表81)、生物学的経路としてGOID：50880、42310、8015、1978に割り当てられる血圧調節に関する遺伝子(表82)、GOID:7631に割り当てられる食行動に関する遺伝子(表83)、GOID：7623、48511に割り当てられる生体リズムに関する遺伝子(表84)は注目に値する。
Figure JPOXMLDOC01-appb-T000081
 The GO analysis of genes up-regulated in the striatum was able to identify candidate genes for the search for health promoting genes. Genes related to neuropeptide hormone activity functionally assigned to GOID: 5184 (Table 81), genes related to blood pressure regulation assigned to GOID: 50880, 42310, 8015, 1978 as biological pathways (Table 82), assigned to GOID: 7631 Notable are genes related to eating behavior (Table 83) and genes related to biological rhythms (Table 84) assigned to GOID: 7623, 48511.
Figure JPOXMLDOC01-appb-T000081
Figure JPOXMLDOC01-appb-I000206
Figure JPOXMLDOC01-appb-T000082
Figure JPOXMLDOC01-appb-I000206
Figure JPOXMLDOC01-appb-T000082
Figure JPOXMLDOC01-appb-I000207
Figure JPOXMLDOC01-appb-I000207
Figure JPOXMLDOC01-appb-I000208
Figure JPOXMLDOC01-appb-T000083
Figure JPOXMLDOC01-appb-T000084
Figure JPOXMLDOC01-appb-I000208
Figure JPOXMLDOC01-appb-T000083
Figure JPOXMLDOC01-appb-T000084
２-２-４-５．長期Tickling刺激時と短期Tickling刺激時とで共通して発現変化した遺伝子
　長期Tickling刺激時と短期Tickling刺激時に血液、視床下部、線条体において1.5倍以上あるいは1/1.5倍以下に発現量が変化した遺伝子に関し、共通性を解析した結果を表85に示す。
Figure JPOXMLDOC01-appb-T000085
 2-2-4-5. Genes that have changed in expression during long-term Tickling stimulation and during short-term Tickling stimulation Expression changes in blood, hypothalamus, and striatum at 1.5 times or less or 1 / 1.5 times or less during long-term Tickling stimulation and short-term Tickling stimulation Table 85 shows the result of analyzing the commonality of the obtained genes.
Figure JPOXMLDOC01-appb-T000085
　血液と視床下部では発現増加した遺伝子、発現減少した遺伝子は共に長期Tickling刺激時に多く、発現変化(増加あるいは減少)した遺伝子はそれぞれ641、321存在し、短期Tickling刺激時の148、32遺伝子を大きく上回った。一方、線条体では短期Tickling刺激時に発現増加した遺伝子が多かった(140遺伝子)のが特徴的であった。しかしながら、長短期間で順方向に発現変化する共通遺伝子はいずれの部位においても1～３程度であったのに対し、血液と線条体では逆方向に発現変化する遺伝子が多く、短期Tickling刺激ではUp-regulateされるが長期Tickling刺激ではDown-regulateされる遺伝子がそれぞれ32、28存在した。それぞれの遺伝子名については、表86と表87に示す。
Figure JPOXMLDOC01-appb-T000086
Figure JPOXMLDOC01-appb-T000087
 In the blood and hypothalamus, genes with increased expression and genes with decreased expression are both present during long-term Tickling stimulation, and there are 641 and 321 genes whose expression changes (increase or decrease), respectively, and 148 and 32 genes during short-term Tickling stimulation are large. Exceeded. On the other hand, the striatum was characterized in that there were many genes (140 genes) whose expression increased upon short-term Tickling stimulation. However, while there are about 1 to 3 common genes whose expression changes in the forward direction over a short period of time, there are many genes whose expression changes in the reverse direction in blood and striatum, and in short-term Tickling stimulation There were 32 and 28 genes that were up-regulated but were down-regulated by long-term tickling stimulation. Each gene name is shown in Table 86 and Table 87.
Figure JPOXMLDOC01-appb-T000086
Figure JPOXMLDOC01-appb-T000087
２-２-５．考察・結論
　血液と視床下部ではTicklingによる長期の繰り返し刺激がラットの体質を変化させ、これに伴う遺伝子の発現変化が顕著であるが、これらの組織でのTicklingの短期効果は遺伝子の発現変化としては現れづらかった。一方、線条体では逆に短期刺激で発現増加する遺伝子が多く、外界からの刺激入路がこの部位を経由し、刺激への対応遺伝子を発現させるものと考えられた。長短期Tickling刺激間で共通して発現変化した遺伝子に注目すると、順方向に発現変動する共通遺伝子は極めて少なく、機能面から見ても視床下部での「陽性」刺激の伝達に関わるドーパミントランスポータが長短期を問わず発現増加していることと、血液肥満細胞における過剰免疫への関与が示唆されるキマーゼ遺伝子が長短共に発現減少していることのみがTicklingの陽性刺激の効果であった。一方、逆方向に発現変動する共通遺伝子は、血液と線条体で認められるが、血液における共通遺伝子は推定遺伝子が多く注目に値する遺伝子は存在しなかった。線条体では後述の注目遺伝子を相当数含んでいた。 2-2-5. Discussion / Conclusion In the blood and hypothalamus, long-term repeated stimulation by Tickling changes the constitution of the rat, and the accompanying changes in gene expression are remarkable, but the short-term effect of Tickling in these tissues is a change in gene expression Was hard to appear. On the other hand, in the striatum, on the other hand, there are many genes whose expression increases by short-term stimulation, and it is considered that the stimulus entry from the outside world passes through this site and expresses the corresponding gene for stimulation. Focusing on genes that are expressed and changed in common between long- and short-term Tickling stimuli, there are very few common genes whose expression changes in the forward direction, and from a functional perspective, dopamine transporters involved in the transmission of "positive" stimuli in the hypothalamus The effect of Tickling's positive stimulation was only that expression increased in both short and long periods, and that the chymase gene, which was suggested to be involved in hyperimmunity in blood mast cells, decreased in both long and short expression. On the other hand, common genes whose expression varies in the opposite direction are found in blood and striatum, but there are many genes that are notable for common genes in blood. The striatum contained a considerable number of genes of interest described below.
　個々の組織において発現変化した遺伝子で、健康増進遺伝子候補として位置づけられる可能性を有している注目遺伝子としては、口腔粘膜擦過細胞で発現増加したGsta5、Aldh1a1、Yc2、Gsta2、Cdig1l、Selenbp1があり、前５者は口腔内の解毒に関わる酵素等をコードしており、Selenbp1は味覚に関与するセレン担体をコード化する遺伝子である。一方、発現減少した遺伝子としては、外界からの刺激による細胞ダメージに反応して細胞周期を調節するCDK阻害因子をコードするCdkn1c、アミノ酸代謝を調節する酵素をコードするHpd、肝障害指標であるグアニンデアミナーゼをコードするGdaがあるが、その発現変化が何を意味しているかは現時点では不明である。 Among the genes of which expression has changed in individual tissues, the genes of interest that have the potential to be positioned as health promotion gene candidates include Gsta5, Aldh1a1, Yc2, Gsta2, Cdig1l, and Selenbp1 that have increased expression in oral mucosal scraping cells. The former 5 encode enzymes and the like involved in oral detoxification, and Selenbp1 is a gene encoding a selenium carrier involved in taste. On the other hand, genes with decreased expression include Cdkn1c, which encodes a CDK inhibitor that regulates the cell cycle in response to cell damage caused by external stimuli, Hpd, which encodes an enzyme that regulates amino acid metabolism, and guanine, an indicator of liver damage. There is Gda that encodes deaminase, but it is unclear at this time what its expression change means.
　視床下部では、「陽性」刺激の伝達に関与するドーパミントランスポータをコードするSlc6a3が長期Tickling刺激時と同じく発現増加しており、Tickling刺激の極初期から継続して発現レベルが高くなっていると考えられた。その他Cga遺伝子の発現が増えているが、この遺伝子が雌性に関わるホルモン産生を調整していることを鑑みると、雄性ラットで増える意義についてはマクロでの考察を要する。 In the hypothalamus, Slc6a3, which encodes a dopamine transporter involved in the transmission of “positive” stimuli, is up-regulated in the same way as long-term Tickling stimulation, and the expression level continues to increase from the very beginning of Tickling stimulation. it was thought. In addition, the expression of the Cga gene is increasing, but considering that this gene regulates hormone production related to females, the significance of increasing in male rats requires macroscopic consideration.
　線条体では、発現増加した遺伝子が最も多く、血圧調節に関与するAvp、Vip、神経成長や分化に関与するNeurod2、Nrn１、神経伝達に関与するSlc17a7、摂食調節に関与するCck、アルツハイマー病に関係するβアミロイド蛋白のレベルを下げ記憶力の改善に関与するSstr1、Cort、精神薄弱の原因遺伝子ともいわれ学習と記憶に関与するPrssの各遺伝子が健康増進遺伝子の候補としてリストアップできた。一方、βアミロイド蛋白の繊維形成を阻害するTtrが線条体でなぜ発現低下するかは不明であるが、Ttr本体の機能(プレアルブミンとしての担体機能)との関係があるのかも知れない。 In the striatum, the most commonly expressed genes are Avp and Vip involved in blood pressure regulation, Neurod2 and Nrn1 involved in nerve growth and differentiation, Slc17a7 involved in neurotransmission, Cck involved in feeding regulation, Alzheimer's disease Sstr1, Cort, which is involved in the improvement of memory ability by lowering the level of β-amyloid protein related to, and the Prss gene involved in learning and memory were listed as candidates for health promoting genes. On the other hand, it is unclear why the expression of Ttr, which inhibits fiber formation of β-amyloid protein, is decreased in the striatum, but it may be related to the function of Ttr main body (carrier function as prealbumin).
　GO解析結果からは、線条体での発現増加した遺伝子群での解析以外は各GO Termに割り当てられる遺伝子が少なく有効な情報が得られなかった。線条体でUp-regulateされた遺伝子が有意に割り当てられたGO Termとしては、シグナル伝達関連、神経ペプチドホルモン活性、血圧調節関連、食行動関連、生体リズム関連のTermがあるが、健康増進遺伝子候補として注目すべきは、後４者のTermに割り当てられる遺伝子である。神経ペプチドホルモン活性に割り当てられる遺伝子としては、上記遺伝子の他にGrp、Adcyap1、Nppcがあるが、Nppcは血圧調節関連のTermに割り当てられるNppa、Avp、Vipと共に血液循環機能を調節する健康増進遺伝子候補となり得る。また、血圧調節関連のTermにはアドレナリン受容体遺伝子も割り当てられている。食行動に関連するTermには長期Tickling刺激実験でのGO解析で抽出されたCck、Pmch以外にBdnfが抽出されたことはストレスとの関連で注目に値する。その他、生体リズム関連のGO Termに有意に遺伝子が割り当てられたことも、Ticklingの短期刺激効果として注目すべきであり、特に睡眠との関係が示唆されているPtgds遺伝子は、動物の「やる気」に関する行動を規定するものとして、健康増進に繋がる可能性が秘められている。 From the GO analysis results, there were few genes assigned to each GO Term except for the analysis with the gene group whose expression increased in the striatum, and effective information could not be obtained. The GO Term to which genes up-regulated in the striatum are significantly assigned include signal transmission-related, neuropeptide hormone activity, blood pressure regulation-related, dietary behavior-related, biological rhythm-related Term, but health promoting genes What should be noted as a candidate is a gene assigned to the latter four terms. In addition to the above genes, genes assigned to neuropeptide hormone activity include Grp, Adcyap1, and Nppc. Nppc, along with Nppa, Avp, and Vip assigned to blood pressure regulation-related terms, regulates blood circulation functions. Can be a candidate. An adrenergic receptor gene is also assigned to a term related to blood pressure regulation. It is worth noting that Bdnf was extracted in Term related to eating behavior in addition to Cck and Pmch extracted by GO analysis in the long-term Tickling stimulation experiment. In addition, the significant assignment of genes to biological rhythm-related GO Term is also notable as a short-term stimulation effect of Tickling, and the Ptgds gene, which is particularly suggested to be related to sleep, is an animal's `` motivation '' There is a possibility that it may lead to health promotion as a prescription for behavior.
２-２-６．総括
　ラットを用いて陽性刺激に応じて発現変化する中枢神経系ほかの遺伝子（群）を探索するモデル実験系を作製したが、その刺激期間としてまず４週間を採用し、刺激効果の確実化を図った。その結果、視床下部や唾液腺等において発現変動する遺伝子を特定できたが、長期の繰り返し刺激はラットの体質変化を伴った。４週齢のラットにTickling刺激を与えると、ラットは刺激初日から50 KHz近傍の音声を発し、その音声のピークが２週間であったことから、刺激の受容過程の把握には短期の方が有利と考えられた。短期間（2日間）刺激では、予想した通り、線条体における遺伝子の発現変化が主に観察された。陽性刺激の受容がこの部位を経由するという仮説を支持するものである。健康増進が外界からの刺激に対する心身の適応能力の向上との立場に立てば、短～長期にわたり持続的に活性化される遺伝子は、健康増進遺伝子の候補の一つに挙げることができる。 2-2-6. A model experimental system was developed to search the central nervous system and other genes (groups) whose expression changes in response to positive stimulation using generalized rats. The stimulation period is initially set to 4 weeks to ensure the stimulation effect. planned. As a result, genes whose expression changes in the hypothalamus and salivary glands could be identified, but long-term repeated stimulation was accompanied by changes in the constitution of the rat. When Tickling stimulation was given to a 4-week-old rat, the rat uttered a voice in the vicinity of 50 KHz from the first day of stimulation, and the peak of the voice was 2 weeks. It was considered advantageous. In short-term (2 days) stimulation, as expected, gene expression changes in the striatum were mainly observed. It supports the hypothesis that the reception of positive stimuli goes through this site. From the standpoint that health promotion is an improvement in the ability of the body and mind to adapt to stimuli from the outside world, genes that are continuously activated over a short to long term can be listed as candidates for health promotion genes.

[実施例３]
DNAチップデータの精度の検証
　上記DNAチップを用いた実験は、一般的には「一次スクリーニングのための手法」、すなわち「DNAチップ上の遺伝子の中から更なる機能解析に用いる遺伝子を選び出す手法」と考えられている。そのため、次のステップとして、得られたDNAチップデータが遺伝子発現状態を正しく反映しているか否かを、定量性の高い方法で確認する必要がある。そこで、DNAチップに比べ1000倍程度感度が良いリアルタイムPCR解析方法を用いて、選抜した遺伝子の発現定量解析を行い、DNAチップデータの検証を行った。
[Example 3]
Verification of the accuracy of DNA chip data Experiments using the above DNA chip are generally “primary screening methods”, that is, “methods for selecting genes for further functional analysis from genes on DNA chips”. It is believed that. Therefore, as the next step, it is necessary to confirm whether or not the obtained DNA chip data correctly reflects the gene expression state by a highly quantitative method. Therefore, using a real-time PCR analysis method that is about 1000 times more sensitive than the DNA chip, we performed quantitative expression analysis of the selected genes and verified the DNA chip data.
　本実施例では、陽性刺激を4週間負荷したラットで発現変動することが認められた遺伝子群(上記実施例2参照)の中から、視床下部の食行動に関与する6遺伝子、ドーパミントランスポータ(Slc6a3)、及びコレシストキニン（Cck）遺伝子を選別し、遺伝子発現定量解析を実施した。 In this example, 6 genes involved in hypothalamic eating behavior, dopamine transporter (from the gene group (see Example 2 above)) that was found to change expression in rats loaded with positive stimulation for 4 weeks, Slc6a3) and cholecystokinin (Cck) genes were selected, and gene expression quantitative analysis was performed.
3-1．方法
　本実施例における遺伝子発現解析実験の流れは図89に示される。 3-1. Method The flow of the gene expression analysis experiment in this example is shown in FIG.
3-1-1．供試材料
　本実施例では、4週令(離乳直後[3週令]から個別飼育)から陽性刺激を4週間負荷したラット(Tickling 4個体、Light Touch 2個体)の視床下部から、上記(2-2-2)と同様にして調製したTotal RNAを用いた。 3-1-1. The test materials present embodiment, the hypothalamus 4 weeks old (weaning immediately [3 weeks old from individual rearing) Load 4 weeks positive stimulus from rat (Tickling 4 individuals, Light Touch 2 individuals), the (2 Total RNA prepared in the same manner as in -2-2) was used.
3-1-2.Total RNA純度と無傷度の検定
　個体毎(Total RNA標品混合前)に、Total RNAの濃度とA260/280比を求めた。 3-1-2. Total RNA purity and intactness assay The total RNA concentration and A260 / 280 ratio were determined for each individual (before mixing the total RNA preparation).
3-1-3．逆転写反応
　DNAチップ解析では、Tickling群とLight Touch群の2群での比較を行う。そのため、Tickling 4個体、Light Touch 2個体については、各群の個体のTotal RNAを等量混合し、Tickling群とLight Touch群の2群を逆転写に用いるRNAサンプルとした。 3-1-3. In reverse transcription reaction DNA chip analysis, two groups, Tickling group and Light Touch group, are compared. Therefore, for Tickling 4 individuals and Light Touch 2 individuals, equal amounts of total RNA of individuals in each group were mixed, and two groups of Tickling group and Light Touch group were used as RNA samples for reverse transcription.
　逆転写反応には、High Capacity cDNA Reverse Transcription Kit with RNase IH(アプライドバイオシステムズ社製)を用いて行った。反応液組成については、Total RNA量を5μg、その他の反応液については説明書に記載の量を加え、トータルボリュームを100μlとした。逆転写反応は、製品説明書に記載の時間と温度で実施した。 The reverse transcription reaction was performed using High® Capacity® cDNA® Reverse® Transcription® Kit with with RNase® IH (Applied Biosystems). Regarding the reaction solution composition, the total amount of RNA was 5 μg, and for other reaction solutions, the amount described in the instructions was added to make the total volume 100 μl. The reverse transcription reaction was performed at the time and temperature described in the product instructions.
3-1-4．リアルタイムPCRによる遺伝子の発現定量解析
3-1-4-1．リアルタイムPCR
　リアルタイムPCRには、TaqMan アッセイを選択し、7300リアルタイムPCRシステム(アプライドバイオシステムズ社製)を用いて行った。反応は、TaqMan Gene Expression Assaysを用いて、製品説明書に記載の方法に従った。定量方法にはΔΔCt法を用い、相対定量実験を行った。定量結果が得られた後、ΔΔCt法の解析方法により、Light Touchに対するTicklingでの遺伝子発現量の相対値を算出した。 3-1-4. Quantitative analysis of gene expression by real-time PCR
3-1-4-1. Real-time PCR
For real-time PCR, TaqMan assay was selected and performed using a 7300 real-time PCR system (Applied Biosystems). The reaction was performed according to the method described in the product manual using TaqMan Gene Expression Assays. The ΔΔCt method was used as the quantitative method, and a relative quantitative experiment was conducted. After obtaining the quantification results, the relative value of the gene expression level in Tickling with respect to Light Touch was calculated by the analysis method of ΔΔCt method.
3-1-4-2．解析対象遺伝子
　下記表88の8遺伝子について、発現定量を行った。また、初期RNA量の補正のための内在性コントロール遺伝子には、actin, beta遺伝子、およびribosomal protein, large P2遺伝子を使用した（表89）。
Figure JPOXMLDOC01-appb-T000088
Figure JPOXMLDOC01-appb-T000089
 3-1-4-2. Genes to be analyzed Expression quantification was performed for 8 genes in Table 88 below. In addition, actin, beta gene, ribosomal protein, and large P2 gene were used as endogenous control genes for correction of the initial RNA amount (Table 89).
Figure JPOXMLDOC01-appb-T000088
Figure JPOXMLDOC01-appb-T000089
3-1-4-3．Unknown Sample
　3-1-3で、Total RNAを等量混合したTickling群、Light Touch群の2群について、Total RNAから逆転写反応によって合成したcDNAをUnknown Sampleとした。 3-1-4-3. Unknown Sample
For the two groups of Tickling group and Light Touch group mixed with equal amount of total RNA in 3-1-3, cDNA synthesized from total RNA by reverse transcription reaction was used as Unknown Sample.
3-2．結果
3-2-1．全RNA純度と無傷度の検定
　Tickling群、Light Touch群の各個体のTotal RNA濃度、およびA260/280比を表90に示す。
Figure JPOXMLDOC01-appb-T000090
 3-2. result
3-2-1. Test of total RNA purity and intactness Table 90 shows the total RNA concentration of each individual in the Tickling group and Light Touch group, and the A260 / 280 ratio.
Figure JPOXMLDOC01-appb-T000090
　上記の結果、調製した各個体毎のTotal RNAは、純度、無傷度について問題のないレベルの品質であると判断した。 As a result of the above, it was determined that the prepared total RNA for each individual was of a quality with no problem with respect to purity and intactness.
3-2-2．リアルタイムPCRによる遺伝子の発現定量解析
　3-1-4のリアルタイムPCRによる遺伝子の発現定量解析を実施した。定量結果をもとに、ΔΔCt法の解析方法により算出した相対値を表91に示す。
Figure JPOXMLDOC01-appb-T000091
 3-2-2. Gene expression quantitative analysis by real-time PCR The gene expression quantitative analysis by real-time PCR of 3-1-4 was performed. Table 91 shows the relative values calculated by the ΔΔCt analysis method based on the quantification results.
Figure JPOXMLDOC01-appb-T000091
3-2-3．DNAチップ解析結果と、リアルタイムPCR解析結果との比較
　3-1-1の供試材料のDNAチップ解析における発現データを表92に示す。
Figure JPOXMLDOC01-appb-T000092
 3-2-3. Comparison between DNA chip analysis result and real-time PCR analysis result Table 92 shows the expression data in DNA chip analysis of the test material of 3-1-1.
Figure JPOXMLDOC01-appb-T000092
　解析対象8遺伝子の相対発現量について、リアルタイムPCR解析結果と比較するため、DNAチップ解析の発現データについて、内在性コントロール遺伝子を用いて補正を行い、算出された相対値を表93に示す。
Figure JPOXMLDOC01-appb-T000093
 In order to compare the relative expression levels of the 8 genes to be analyzed with the results of real-time PCR analysis, the expression data of DNA chip analysis was corrected using endogenous control genes, and the calculated relative values are shown in Table 93.
Figure JPOXMLDOC01-appb-T000093
　リアルタイムPCR解析とDNAチップ解析とを比較したところ、ともにNo.1～No.7の遺伝子については1.5～2倍以上、No.8の遺伝子については約0.5倍の相対値であり、両者の遺伝子発現の定量値は、ほぼ同程度の値を示した。 When comparing real-time PCR analysis and DNA chip analysis, the relative values of both No.1 to No.7 genes are 1.5 to 2 times or more, and No.8 gene is about 0.5 times. The quantitative value of the expression was almost the same value.
3-3．結論
　8つの特定遺伝子において、DNAチップ解析およびリアルタイムPCR解析で同程度の遺伝子発現量という結果となり、DNAチップデータが遺伝子発現状態を正しく反映していることを検証できた。 3-3. Conclusion In 8 specific genes, DNA chip analysis and real-time PCR analysis resulted in the same level of gene expression, and it was verified that the DNA chip data correctly reflected the gene expression status.

[実施例４]
　本実施例では、ラットを用いたモデル実験の結果を踏まえて、ヒトに陽性刺激（笑い）を負荷し、血液で発現変動する遺伝子を解析することにより、健康に関与する遺伝子の探索と同定に関する研究を行った。
[Example 4]
In this example, based on the results of model experiments using rats, positive stimulation (laughing) is loaded on humans, and genes that vary in expression in blood are analyzed. I did research.
4-1．ヒト末梢血白血球細胞において、陽性刺激としての笑いにより発現変動する健康増進遺伝子候補（群）の特定
　研究イベント「笑いと健康Part4：歯周病を合併した糖尿病患者における陽性ストレス負荷後の遺伝子発現の変化に関する研究」として実施した。本実験はヒト・ゲノム遺伝子解析研究に関する倫理指針に準じ、筑波大学医の倫理委員会の承認を得た後、被験者の同意を得て行った。 4-1. Specific research event for candidate group (s) of health-enhancing genes that change in expression due to laughter as positive stimulus in human peripheral blood leukocytes "Laughter and health Part4: Gene expression after positive stress in diabetic patients with periodontal disease Conducted as a study on change. This experiment was carried out with the consent of the subject after obtaining approval from the University of Tsukuba Medical Ethics Committee in accordance with the ethical guidelines for human genome analysis.
4-1-1．被験者
　糖尿病患者はFair ControlがHbA1c値6.5～7.9％領域の患者を対象とした。対象被験者の募集は、糖尿病患者は糖尿病専門医に依頼し行い、健常被験者は新聞等の公募により行った。 4-1-1. Subjects Diabetes patients were those whose Fair Control HbA1c value ranged from 6.5 to 7.9%. Recruitment of target subjects was requested by diabetics for diabetic patients, and healthy subjects were recruited through public recruitment of newspapers.
4-1-1-1．歯周病指標
　1)歯周病の指標として、WHO基準のCommunity Periodontal Index of Treatment Needs (CPITN)法を用いた（表94）。所定数の歯を測定し、その中で最大の値を示すCPITN値を被験者ごとのCPITN値とした。CPITN値が3以上を示した被験者群を歯周病を有する群(歯周病罹病者群)、2以下を示した被験者群を歯周病を有しない群(歯周病非罹病者群)とした。
Figure JPOXMLDOC01-appb-T000094
 4-1-1-1. Periodontal disease index 1) As an index of periodontal disease, WHO-standard Community Periodontal Index of Treatment Needs (CPITN) method was used (Table 94). A predetermined number of teeth were measured, and the CPITN value showing the maximum value among them was used as the CPITN value for each subject. The group of subjects with CPITN values of 3 or more had a periodontal disease (periodontal disease affected group), the group of subjects with a CPITN value of 2 or less had no periodontal disease (periodontal disease non-affected group) It was.
Figure JPOXMLDOC01-appb-T000094
　2)各被験者の歯周病の自覚症状の程度は歯科医師作成の問診表を使用し、「はい」の数を数値化して表した。 2) The degree of subjective symptoms of periodontal disease in each subject was expressed by using a questionnaire prepared by a dentist and quantifying the number of “yes”.
4-1-1-2．糖尿病指標
　糖尿病患者の血糖コントロール指標としてHbA1C値を用いた。測定には、下記(4-1-2)の時程図に示すC1時にサンプリングした血液を用いた。 4-1-1-2. Diabetes index HbA1C value was used as a blood glucose control index of diabetic patients. For the measurement, blood sampled at C1 shown in the timing diagram of (4-1-2) below was used.
4-1-1-3．被験者プロフィール
　被験者プロフィールを表95に示した。糖尿病患者群のHbA1C値とCPITN値は、それぞれ7.2 ± 0.6%、3.4 ± 0.7、健常者群は5.1 ± 0.3%、2.7 ± 0.6であり、糖尿病患者群のCPITN値は健常者群に比較して有意(p<0.05)に高かった。このことは、糖尿病患者は歯周病疾患に罹りやすいという臨床上の傾向を検証する結果となった。
Figure JPOXMLDOC01-appb-T000095
 4-1-1-3. Subject profile Subject profiles are shown in Table 95. The HbA1C and CPITN values in the diabetic patient group are 7.2 ± 0.6% and 3.4 ± 0.7, respectively, and the healthy group is 5.1 ± 0.3% and 2.7 ± 0.6. The CPITN value in the diabetic patient group is compared with that in the healthy group. Significantly higher (p <0.05). This resulted in the verification of the clinical trend that diabetics are more susceptible to periodontal disease.
Figure JPOXMLDOC01-appb-T000095
4-1-2．実験方法（実験デザイン）
　実験は2日間に渡って行った。被験者に両日とも500Kcalの昼食をとって頂き、昼食後1日目(対照)は単調な講義を45分間聴講してもらった。2日目は、コントを生ステージ鑑賞（45分間）して笑いを体験してもらった。鑑賞（あるいは聴講）前後において、心理テスト、唾液の採取と採血、口腔内観察を行った。口腔内観察は歯科医師(茨城県歯科医師会に協力を得た）が行った。図90に実験時程図を示す。 4-1-2. Experimental method (experimental design)
The experiment was conducted over 2 days. The subjects had a lunch of 500 Kcal on both days, and on the first day after lunch (control), they had a 45-minute monotonous lecture. On the second day, they enjoyed laughing by watching the live stage of Conte (45 minutes). Before and after appreciation (or audition), psychological tests, saliva collection and blood sampling, and intraoral observation were performed. Intraoral observation was performed by a dentist (with cooperation from the Ibaraki Dental Association). FIG. 90 shows the experimental schedule.
4-1-3．心理・生理・生化学指標の解析
4-1-3-1．実験方法（心理・生理・生化学指標の測定）
　上記4-1-2の時程図C1、C2、T1、T2時に心理・生理・生化学指標を測定した。測定項目は次の通りである：
心理テスト；POMS、STAI-S
血液；血糖値、白血球数、白血球分画(好中球比、リンパ球比)、NK細胞活性、コルチゾール、C反応性蛋白、HbA1C値（C1のみ）
唾液；量、緩衝能、酸化還元電位、アミラーゼ活性、コルチゾール、s-IgA
統計解析；解析ソフトはエクセルのアドインソフトの「エクセルStatcelQC (S_QC)」を使用した。P値はウィルコクソン符号付順位和検定を用い、危険率5％でP値(両側確率)を算出した。 4-1-3. Analysis of psychological, physiological and biochemical indicators
4-1-3-1. Experimental method (measurement of psychological, physiological and biochemical indicators)
Psychological, physiological and biochemical indices were measured at time charts C1, C2, T1 and T2 in 4-1-2 above. The measurement items are as follows:
Psychological test: POMS, STAI-S
Blood; blood glucose level, white blood cell count, white blood cell fraction (neutrophil ratio, lymphocyte ratio), NK cell activity, cortisol, C-reactive protein, HbA1C level (C1 only)
Saliva; volume, buffer capacity, redox potential, amylase activity, cortisol, s-IgA
Statistical analysis: Excel's add-in software “Excel StatcelQC (S_QC)” was used as the analysis software. P-value was calculated by using Wilcoxon signed rank sum test, and P-value (two-sided probability) was calculated with a risk rate of 5%.
4-1-3-2．結果・考察
4-1-3-2-1．糖尿病非罹病かつ歯周病非罹病者群(以下、A群と称する)の解析
　解析結果を表96に示す。食後2時間血糖値上昇は講義でもコントでも共に有意差が見られなかった。s-IgA(唾液中の分泌型免疫グロブリンA)は講義後に減少したが、コント後では増加した。感染や炎症時に増加するC反応性蛋白は、講義前後で有意差が見られなかったが、コント後では減少した。
Figure JPOXMLDOC01-appb-T000096
 4-1-3-2. Results and discussion
4-1-3-2-1. Table 96 shows the analysis results of the non-diabetic and periodontal disease non-affected group (hereinafter referred to as group A) . There was no significant difference in blood glucose elevation for 2 hours after meals in both lectures and contes. s-IgA (secretory immunoglobulin A in saliva) decreased after the lecture, but increased after contest. C-reactive protein, which increases during infection and inflammation, was not significantly different between before and after the lecture, but decreased after contest.
Figure JPOXMLDOC01-appb-T000096
　これらの結果より、A群では、コント鑑賞により、s-IgAの増加から免疫機能が活性化され、またC反応性蛋白の減少から感染・炎症が鎮静しているときの状態になったといえ、コント鑑賞による健康維持・増進が認められた。 From these results, it can be said that in group A, the immune function was activated from the increase in s-IgA, and the infection and inflammation were sedated from the decrease in C-reactive protein. Health maintenance / promotion by appreciation of comte was recognized.
4-1-3-2-2．糖尿病非罹病かつ歯周病罹病者群(以下、B群と称する）の解析
　解析結果を表97に示す。食後2時間血糖値上昇は講義でもコントでも共に有意差が見られなかった。TMD値（総合感情障害度）は講義聴講前後では差を示さなかったが、コント鑑賞後有意(p<0.05)に減少した。s-IgAは講義聴講前後では有意差を示さなかったが、コント後では増加した。また、白血球数はコント後に有意(p<0.05)に増加した。
Figure JPOXMLDOC01-appb-T000097
 4-1-3-2-2. Table 97 shows the analysis results of a group of non-diabetic and periodontal disease patients (hereinafter referred to as group B) . There was no significant difference in blood glucose elevation for 2 hours after meals in both lectures and contes. The TMD value (total emotional disorder) did not show any difference before and after the lecture, but decreased significantly (p <0.05) after watching the contest. s-IgA did not show a significant difference before and after the lecture, but increased after the contest. In addition, the white blood cell count increased significantly (p <0.05) after control.
Figure JPOXMLDOC01-appb-T000097
　これらの結果より、B群では、コント鑑賞により、s-IgAの増加から免疫機能が活性化されたと推測でき、コント鑑賞による健康維持・増進が認められた。 From these results, it can be inferred from the increase in s-IgA that the immune function was activated by appreciation in group B, and health maintenance and enhancement by appreciation were observed.
4-1-3-2-3．糖尿病罹病かつ歯周病非罹病者群(以下、C群と称する）の解析
　解析結果を表98に示す。食後2時間血糖値は講義では145.5 ±12.0mg/dL、コントでは94.0 ±1.4 mg/dLと上昇し、コント鑑賞により上昇が51.5mg/dLと著しく抑制された。ストレスの指標となる唾液中アミラーゼ活性は、講義後に減少し、コント後では著しく減少した。白血球数は、講義聴講前後では差を示さなかったが、コント後に増加した。免疫指標であるリンパ球比は、講義後に減少したが、コント後に増加した。ストレス指標である好中球比は、講義聴講前後では差を示さなかったが、コント後に減少した。感染や炎症時に増加するC反応性蛋白は、講義前後で差が見られなかったが、コント後では減少した。
Figure JPOXMLDOC01-appb-T000098
 4-1-3-2-3. Table 98 shows the analysis analysis results of the group with diabetes and non-periodontal disease (hereinafter referred to as group C) . The blood glucose level for 2 hours after meal increased to 145.5 ± 12.0 mg / dL in the lecture and 94.0 ± 1.4 mg / dL in the conte, and the increase was remarkably suppressed to 51.5 mg / dL by the comte appreciation. The amylase activity in saliva, which is an index of stress, decreased after the lecture and decreased significantly after the contest. The white blood cell count did not show any difference before and after the lecture, but it increased after the contest. The lymphocyte ratio, an immune index, decreased after the lecture, but increased after the contest. The neutrophil ratio, which is a stress index, did not show a difference before and after the lecture, but decreased after contesting. The C-reactive protein that increased during infection and inflammation did not show any difference between before and after the lecture, but decreased after contest.
Figure JPOXMLDOC01-appb-T000098
　これらの結果をまとめると、C群では、コント鑑賞により、食後2時間血糖値上昇が著しく抑制され、ストレス指標である唾液中アミラーゼ活性、好中球比が減少し、免疫指標であるリンパ球比は増加し、C反応性蛋白が減少した。コント鑑賞により、ストレスが減り、免疫機能が活性化し、食後2時間血糖値上昇の抑制効果が認められた。 Summarizing these results, in Group C, the increase in blood glucose level for 2 hours after meals was markedly suppressed by conte appreciation, amylase activity in saliva and neutrophil ratio as stress indicators decreased, and lymphocyte ratio as immune index Increased and C-reactive protein decreased. Conte appreciation reduced stress, activated immune function, and suppressed blood glucose elevation for 2 hours after meals.
4-1-3-2-4．糖尿病罹病かつ歯周病罹病者群(以下、D群と称する）の解析
　解析結果を表99に示す。食後2時間血糖値は講義では147.0 ±55.3mg/dL、コントでは135.9 ±58.0 mg/dLと上昇し、コント鑑賞により上昇が11.1mg/dL抑制された。TMD値（総合感情障害度）は講義聴講前後では差を示さなかったが、コント鑑賞後有意(p<0.05)に減少した。s-IgA(唾液中の分泌型免疫グロブリンA)は講義後、コント後とも増加した。感染や炎症時に増加するC反応性蛋白は、講義後、コント後とも減少した。
Figure JPOXMLDOC01-appb-T000099
 4-1-3-2-4. Table 99 shows the analysis results of the group with diabetes and periodontal disease (hereinafter referred to as group D) . The blood glucose level for 2 hours after meal increased to 147.0 ± 55.3 mg / dL in the lecture and 135.9 ± 58.0 mg / dL in the conte, and the increase was suppressed by 11.1 mg / dL by the comte appreciation. The TMD value (total emotional disorder) did not show any difference before and after the lecture, but decreased significantly (p <0.05) after watching the contest. s-IgA (secretory immunoglobulin A in saliva) increased both after the lecture and after the contest. The C-reactive protein that increases during infection and inflammation decreased both after the lecture and after the contest.
Figure JPOXMLDOC01-appb-T000099
　これらの結果をまとめると、糖尿病罹病かつ歯周病罹病者群では、コント鑑賞により、食後2時間血糖値上昇が抑制され、TMD値（総合感情障害度）が有意に減少した。コント鑑賞により、感情障害が改善され、食後2時間血糖値上昇の抑制効果が認められた。 Summarizing these results, in the group with diabetes and periodontal disease, the increase in blood glucose level for 2 hours after meal was suppressed and the TMD value (total affective disorder) was significantly decreased by the appreciation. Contest appreciation improved emotional disorder and showed a 2-hour postprandial blood glucose level suppression effect.
4-1-3-3-1．糖尿病罹病者群／糖尿病非罹病者群(C＋D群/A＋B群)間の解析
　採取した心理・生理・生化学データを糖尿病罹病者群／糖尿病非罹病者群間で比較解析した。講義前後、コント前後の心理・生理・生化学データをプロットした図を図91～106に示し、各群の解析結果を表100、101に示す。
Figure JPOXMLDOC01-appb-T000100
Figure JPOXMLDOC01-appb-T000101
 4-1-3-3-1. Analysis between diabetic group / non-diabetic group (C + D group / A + B group) The collected psychological / physiological / biochemical data were compared between diabetic group / non-diabetic group. Plots of psychological, physiological, and biochemical data before and after the lecture and before and after the contest are shown in FIGS. 91 to 106, and the analysis results for each group are shown in Tables 100 and 101.
Figure JPOXMLDOC01-appb-T000100
Figure JPOXMLDOC01-appb-T000101
　糖尿病罹病者群においては、食後2時間値血糖値は講義では146.8 ±51.8mg/dL、コントでは130.9 ±56.0 mg/dLと上昇し、コント鑑賞により上昇が15.9mg/dL抑制された。POMSの6感情指標より算出したTMD値（総合感情障害度）は講義聴講前後では有意差を示さなかったが、コント鑑賞後有意(p<0.05)に減少した。唾液中の酸化還元電位は講義後もコント後も減少した。白血球数は講義後もコント後も有意(p<0.05)に増加した。 In the group suffering from diabetes, the 2-hour postprandial blood glucose level increased to 146.85 ± 51.8mg / dL in lectures and 130.9 ± 56.0 / mg / dL in comtes, and the increase was suppressed by 15.9mg / dL by comte appreciation. The TMD values (total emotional disorder) calculated from the POMS 6 emotional index did not show a significant difference before and after the lecture, but decreased to a significant (p <0.05) after watching the contest. The redox potential in saliva decreased after the lecture and after the contest. The leukocyte count increased significantly (p <0.05) after the lecture and after the contest.
　これらの結果をまとめると、糖尿病罹病者群では、コント鑑賞により、食後2時間血糖値上昇が抑制され、TMD値（総合感情障害度）が有意に減少した。コント鑑賞により、感情障害が改善され、食後2時間血糖値上昇の抑制効果が認められた。 Summarizing these results, in the group of people with diabetes, the increase in blood glucose level for 2 hours after meal was suppressed and the TMD level (total affective disorder) was significantly decreased by watching the contest. Contest appreciation improved emotional disorder and showed a 2-hour postprandial blood glucose level suppression effect.
　糖尿病非罹病者群においては、食後2時間血糖値上昇は講義でもコントでも共に有意差が見られなかった。TMD値は講義聴講前後では有意差を示さなかったが、コント鑑賞後有意(p<0.05)に減少した。白血球数は講義後もコント後も有意(p<0.05)に増加した。s-IgAは講義前後では有意差がなかったが、コント後有意(p<0.05)に増加した。唾液量は、コント後に増加した。唾液中の酸化還元電位は講義後もコント後も有意(p<0.05)に減少した。C反応性蛋白は講義前後でもコント前後でも共に有意(p<0.05)に減少した。 In the non-diabetic group, there was no significant difference in the blood glucose level for 2 hours after meals in either the lecture or the conte. The TMD value did not show a significant difference before and after the lecture attendance, but decreased significantly (p <0.05) after watching the contest. The leukocyte count increased significantly (p <0.05) after the lecture and after the contest. Although s-IgA was not significantly different before and after the lecture, it increased significantly (p <0.05) after contest. The amount of saliva increased after the contest. The redox potential in saliva decreased significantly (p <0.05) after lecture and after contest. C-reactive protein decreased significantly (p <0.05) both before and after the lecture.
　これらの結果より、糖尿病非罹病者群では、コント鑑賞により、TMD値（総合感情障害度）が有意に減少し、唾液量が増加し、s-IgAの増加から免疫機能が活性化され、コント鑑賞による健康維持・増進が認められた。 From these results, in the non-diabetic group, TMD levels (total emotional disorder) decreased significantly, saliva increased, and immune function was activated by increasing s-IgA. Health maintenance and enhancement by appreciation was recognized.
4-1-3-3-2．歯周病罹病者群／歯周病非罹病者群(B＋D群/A＋C群)間の解析
　それぞれの群の講義聴講前後、コント鑑賞前後で心理・生理・生化学データの比較解析を行った。歯周病罹病者群に健常被験者が含まれる割合と、歯周病非罹病者群に糖尿病被験者が含まれる割合は同様であった。講義前後、コント前後の各心理・生理・生化学データをプロットした図を図107～122に示し、各群の解析結果を表102、103に示す。
Figure JPOXMLDOC01-appb-T000102
Figure JPOXMLDOC01-appb-T000103
 4-1-3-3-2. Analysis between periodontal disease affected group / periodontal disease non-affected group (B + D group / A + C group) Comparative analysis of psychological / physiological / biochemical data before and after listening to lectures and before / after watching each group. The proportion of healthy subjects in the periodontal disease affected group was similar to the proportion of diabetic subjects in the periodontal disease unaffected group. Figures 107-122 show plots of psychological, physiological, and biochemical data before and after the lecture and before and after the contest, and Tables 102 and 103 show the analysis results for each group.
Figure JPOXMLDOC01-appb-T000102
Figure JPOXMLDOC01-appb-T000103
　歯周病罹病者群では、TMD値は講義前後では有意差を示さなかったが、コント後有意(p<0.05)に減少した。唾液中の酸化還元電位は講義後もコント後も共に有意(p<0.05)に減少した。白血球数は講義後もコント後も共に有意(p<0.05)に増加した。s-IgAは講義後もコント後も増加した。NK細胞活性は講義後もコント後も増加した。C反応性蛋白は講義後もコント後も減少した。 In the group suffering from periodontal disease, TMD values did not show a significant difference before and after the lecture, but decreased to a significant (p <0.05) after control. The redox potential in saliva decreased significantly (p <0.05) both after the lecture and after the contest. The leukocyte count increased significantly (p <0.05) both after the lecture and after the contest. s-IgA increased after the lecture and after the contest. NK cell activity increased after lecture and after contest. C-reactive protein decreased after lecture and after contest.
　歯周病非罹患者群では、TMD値は講義前後では有意差を示さなかったが、コント後に減少した。s-IgAは講義後は減少したが、コント後に増加した。唾液アミラーゼ活性は、コント後に減少した。唾液中の酸化還元電位は講義後もコント後も減少した。白血球数は講義前後では有意差がなかったが、コント後有意(p<0.05)に増加した。C反応性蛋白は講義前後では有意差がなかったが、コント後有意(p<0.05)に減少した。 In the non-periodontal disease group, TMD values did not show a significant difference before and after the lecture, but decreased after contest. s-IgA decreased after the lecture, but increased after the contest. Salivary amylase activity decreased after control. The redox potential in saliva decreased after the lecture and after the contest. The white blood cell count was not significantly different between before and after the lecture, but increased significantly (p <0.05) after contest. C-reactive protein was not significantly different between before and after the lecture, but decreased significantly after control (p <0.05).
　これらの結果より、歯周病非罹患者群では、コント鑑賞により、TMD値（総合感情障害度）が減少し、ストレス指標である唾液中アミラーゼ活性が減少し、s-IgAの増加から免疫機能が活性化され、またC反応性蛋白の減少から感染・炎症が鎮静しているときの状態になったといえ、コント鑑賞による健康維持・増進が認められた。 From these results, in the group without periodontal disease, TMD levels (total affective disorder) decreased, salivary amylase activity as a stress index decreased, and s-IgA increased, resulting in immune function. Was activated, and the decrease in C-reactive protein resulted in a state of calming infection / inflammation.
4-1-4．遺伝子発現解析実験
　糖尿病罹病者群（C＋D群）と糖尿病非罹病者群（A＋B群）で、笑い体験、講義聴講による遺伝子発現変化をDNAチップ法により解析した。糖尿病非罹病かつ歯周病非罹病者群(A群)、糖尿病非罹病かつ歯周病罹病者群(B群)、糖尿病罹病かつ歯周病非罹病者群（C群）、糖尿病罹病かつ歯周病罹病者群（D群）、歯周病非罹病者群（A＋C群）、歯周病罹病者群（B＋D群）の組合せでは、笑い体験時のみの遺伝子発現変化解析を行なった。DNAチップ解析の組合せを表104に示す。
Figure JPOXMLDOC01-appb-T000104
 4-1-4. Gene expression analysis experiment In the group with diabetes (C + D) and non-diabetes (A + B), changes in gene expression due to laughter and lectures were analyzed by the DNA chip method. Non-diabetic and non-periodontal disease group (Group A), Non-diabetes and periodontal disease group (Group B), Diabetes and non-periodontal disease group (Group C), Diabetes and dental In the combination of the group suffering from periodontal disease (group D), the group not suffering from periodontal disease (group A + C), and the group suffering from periodontal disease (group B + D), gene expression change analysis was performed only during laughter experience. Table 104 shows combinations of DNA chip analysis.
Figure JPOXMLDOC01-appb-T000104
4-1-4-1．実験方法
4-1-4-1-1．解析対象サンプルの採取
血液の採取
　PAXgene専用採血管に採取を行ない、室温で4時間、4℃で1晩放置後、-30℃で保存した。 4-1-4-1. experimental method
4-1-4-1-1. Collection of sample to be analyzed
Collection of blood Samples were collected in a PAXgene blood collection tube, left at room temperature for 4 hours and at 4 ° C overnight, and then stored at -30 ° C.
4-1-4-1-2．解析対象サンプルからのRNA調製・検定
1) 血液試料からの全RNA調製
　PAXgene Blood RNA Kit (QIAGEN社製)を用いて全RNA標品を調製した。 4-1-4-1-2. RNA preparation and assay from analysis sample
1) Preparation of total RNA from blood sample Total RNA preparation was prepared using PAXgene Blood RNA Kit (manufactured by QIAGEN).
2) 全RNA純度と無傷度の検定
　被験者試料毎(全RNA標品混合前)に、全RNA標品の濃度とA260/280比、A230/260比、28S/18S rRNA比を求めた。28S/18S rRNA比は、マイクロチップ型電気泳動システム(日立コスモアイ、SV1210)により検定・算出した。 2) Total RNA purity and intactness test For each subject sample (before mixing the total RNA sample), the concentration of the total RNA sample and the A260 / 280 ratio, A230 / 260 ratio, and 28S / 18S rRNA ratio were determined. The 28S / 18S rRNA ratio was assayed and calculated using a microchip electrophoresis system (Hitachi Cosmo Eye, SV1210).
4-1-4-1-3．DNAチップ解析法
　上記(4-1-4-1-1)の血液試料から得られた全RNA標品を出発材料として、T7RNA Polymerase Promoter配列を付加したOligo(dT)24をプライマーとしてcDNA合成し、さらにT7 RNA ポリメラーゼによりmRNAの増幅を行った。この時の増幅反応の基質として、Cyanine [Cy]3-CTP（講義前あるいはコント前）、Cy5-CTP（講義後あるいはコント後）を用いることにより、蛍光標識したcRNAを得、DNAチップ解析に供した。DNAチップ解析は以下の通り行った。各Cyanine色素で蛍光標識したRNA標品を等量混合し、DNAチップ〔Agilent社製(約41,000オリゴDNA搭載)、Whole Human Genome DNA Microarray 4×44K〕上で65℃、17時間ハイブリダイゼーション反応を行い(未反応分子はハイブリダイゼーション反応後洗浄除去)、共焦点レーザースキャナー(Agilent社製、G2565)で各色素に由来する蛍光強度を測定した。バックグランド補正とグローバル正規化の後、Cy5/Cy3比を遺伝子の発現変化として求めた。 4-1-4-1-3. DNA chip analysis method Using the total RNA sample obtained from the blood sample of (4-1-4-1-1) above as the starting material, cDNA synthesis was performed using Oligo (dT) 24 with T7RNA Polymerase Promoter sequence as a primer. Furthermore, mRNA was amplified with T7 RNA polymerase. By using Cyanine [Cy] 3-CTP (before or before lecture) or Cy5-CTP (after or after lecture) as the substrate for the amplification reaction at this time, fluorescently labeled cRNA was obtained for DNA chip analysis. Provided. DNA chip analysis was performed as follows. Equal amounts of RNA samples fluorescently labeled with each cyanine dye are mixed, and hybridization reaction is performed at 65 ° C for 17 hours on a DNA chip (Agilent (approximately 41,000 oligo DNAs), Whole Human Genome DNA Microarray 4 × 44K). The unreacted molecules were washed and removed after the hybridization reaction, and the fluorescence intensity derived from each dye was measured with a confocal laser scanner (Agilent, G2565). After background correction and global normalization, the Cy5 / Cy3 ratio was determined as a change in gene expression.
4-1-4-1-4．リアルタイム定量PCR解析法
　上記DNAチップ解析結果より、歯周病罹患者群（解析10）で発現変動した遺伝子群で免疫・炎症・生体防御に関連する4つの遺伝子CTSA(cathepsin A)、CTSB(cathepsin B)、IL8RB(interleukin 8 receptor, beta)、TLR7(toll-like receptor 7)について、リアルタイム定量PCR解析を実施した。内部標準はDNAチップ解析で発現変動の少なかったGUSB(glucuronidase, beta)を用いた。使用するリアルタイム定量PCRのプライマー・プローブはDNAチップ解析で使用したAgilentのプローブIDと配列位置が近いものを使用した。 4-1-4-1-4. Real-time quantitative PCR analysis From the above DNA chip analysis results, four genes CTSA (cathepsin A) and CTSB (cathepsin) that are related to immunity, inflammation, and biological defense in the gene group whose expression was changed in the group with periodontal disease (analysis 10) B), IL8RB (interleukin 8 receptor, beta), and TLR7 (toll-like receptor 7) were subjected to real-time quantitative PCR analysis. As the internal standard, GUSB (glucuronidase, beta), whose expression fluctuation was small in DNA chip analysis, was used. The primers and probes used for real-time quantitative PCR were those that were close in sequence to the Agilent probe ID used in DNA chip analysis.
　解析を実施したサンプルは、糖尿病罹患者群で歯周病スコア(CPITN値)が高い被験者と低い被験者(上位3名、下位2名)、糖尿病非罹患者群で歯周病スコア(CPITN値)が高い被験者と低い被験者(上位3名、下位3名)、合計11名の被験者を対象とした。検量線を用いた相対定量を行うために、3種類のcDNA反応溶液（被験者サンプル : 被験者サンプルのtotal RNA から逆転写反応で合成したcDNA サンプル。Standard : 検量線作成用として被験者番号114の笑い後のcDNAサンプル「114 P2-3」の希釈系列。Negative Control : cDNA の代わりに水を加えたサンプル）を使用した。PCR 反応溶液はTaqMan プローブとプライマーがセットになった反応液とcDNA反応溶液をTaqMan Universal PCR Master Mix に加えて調製した。測定には各サンプルにつき3 wells使用し、その内蛍光シグナルを観察できた2 wells以上の平均(Replicate) をとることでデータ解析の信頼性を高めた。PCRの条件は、50°Cで2分間(dUを含むPCR産物からの再PCR増幅を防止する為のUNG[AmpErase uracil-N-glycosylase]の活性化)、95°Cで10分間(AmpliTaq Gold(登録商標) DNA polymerase の活性化)の前反応の後、95°Cで15秒(cDNAの変性)と60°Cで1分間(プライマーとプローブのアニーリング、伸長反応)のサイクルを40回繰り返して行った。上述の検量線作成用のサンプル「114 P2-3」の希釈系列(total RNA 換算；50 ng、10 ng、2 ng、0.4 ng、0.08 ng、0.016 ng、0.0032 ng)を用い、各遺伝子の検量線を以下の方法で作成し、各サンプルの定量値を得た。検量線は、PCR反応のサイクル数に対して、増幅されたPCR産物に相当する蛍光シグナル(ΔRn)をプロットした増幅曲線を作成し、増殖曲線が指数関数的に増幅されている領域にベースラインを引きThresholdとし、各増幅曲線上でこのThresholdの値に相当するサイクル数をThreshold Cycle (CT)値とし、既知の濃度と対応するCT値をプロットして作成した。ま
た、各遺伝子の発現データの補正は、被験者サンプルの内在性コントロール定量値のサンプル「114 P2-3」の定量値に対する比を乗じて行った。リアルタイム定量PCR 反応の操作、および解析は、ABI PRISM(登録商標)7900HT Sequence Detection System と付属のソフトウェアであるSDS 2.2 を用いて行った。 Samples analyzed were those with high and low periodontal disease score (CPITN value) in the diabetes affected group (upper 3 and lower 2), periodontal disease score (CPITN value) in the non-diabetic group The subjects were 11 subjects with a high and low subjects (top 3 and low 3). Three types of cDNA reaction solution (subject sample: cDNA sample synthesized by reverse transcription reaction from total RNA of the subject sample for standard quantification using a standard curve. Standard: After laughter of subject number 114 for creating a standard curve A dilution series of the cDNA sample “114 P2-3” of (Negative Control: a sample to which water was added instead of cDNA). A PCR reaction solution was prepared by adding a TaqMan probe and primer set reaction solution and a cDNA reaction solution to TaqMan Universal PCR Master Mix. Three wells were used for each sample, and the reliability of data analysis was improved by taking an average (replicate) of 2 wells or more in which the fluorescence signal could be observed. PCR conditions are 2 minutes at 50 ° C (activation of UNG [AmpErase uracil-N-glycosylase] to prevent re-PCR amplification from PCR products containing dU), 10 minutes at 95 ° C (AmpliTaq Gold After the pre-reaction of (registered trademark DNA polymerase activation), repeat the cycle of 15 seconds at 95 ° C (denaturation of cDNA) and 1 minute at 60 ° C (primer and probe annealing, extension reaction) 40 times. I went. Use the dilution series (total RNA equivalent: 50 ng, 10 ng, 2 ng, 0.4 ng, 0.08 ng, 0.016 ng, 0.0032 ng) of the sample “114 P2-3” for preparing the above-mentioned calibration curve. A line was created by the following method to obtain a quantitative value for each sample. A calibration curve is created by plotting the fluorescence signal (ΔRn) corresponding to the amplified PCR product against the number of cycles of the PCR reaction, and the baseline is in the region where the growth curve is exponentially amplified. And Threshold, and the number of cycles corresponding to this Threshold value on each amplification curve was taken as the Threshold Cycle (CT) value, and the CT values corresponding to known concentrations were plotted. The expression data of each gene was corrected by multiplying the ratio of the endogenous control quantitative value of the subject sample to the quantitative value of the sample “114 P2-3”. The operation and analysis of the real-time quantitative PCR reaction was performed using ABI PRISM (registered trademark) 7900HT Sequence Detection System and the attached software SDS 2.2.
4-1-4-2．結果
　末梢血白血球細胞を対象に遺伝子発現解析を行った。なお、本結果で用いる「プローブ」と「遺伝子(トランスクリプトを含む)」の用語は同義である。 4-1-4-2. Results Gene expression analysis was performed on peripheral blood white blood cells. The terms “probe” and “gene (including transcript)” used in this result are synonymous.
4-1-4-2-1．発現変化を認めた遺伝子
　解析2、4～10において1.5倍以上あるいは1/1.5倍以下の発現変化を認めた遺伝子の数を示した結果を表105に示した。
Figure JPOXMLDOC01-appb-T000105
 4-1-4-2-1. Table 105 shows the results showing the number of genes whose expression changes were 1.5 times or more or 1 / 1.5 times or less in gene analysis 2, 4 to 10 in which expression changes were observed.
Figure JPOXMLDOC01-appb-T000105
　A＋B群で講義聴講によりUp-regulateされた遺伝子は149、Down-regulateされた遺伝子は369あった。C＋D群では講義聴講によりUp-regulateされた遺伝子は553、Down-regulateされた遺伝子は673あった。A＋B群で笑いにより発現変化した遺伝子は多く、Up-regulateされた遺伝子は969、Down-regulateされた遺伝子は867あった。 In the A + B group, 149 genes were up-regulated by lecture and 369 genes were down-regulated. In the C + D group, there were 553 genes that were up-regulated and 673 genes that were down-regulated. In the A + B group, there were many genes whose expression was changed by laughter, 969 were up-regulated and 867 were down-regulated.
　C＋D群では、A＋B群より発現変化した遺伝子は少なくUp-regulateされた遺伝子は204、Down-regulateされた遺伝子は416であった。歯周病の有無による解析では、B＋D群で発現変化した遺伝子は群間比較中で最も多く、Up-regulateされた遺伝子は1,998、Down-regulateされた遺伝子は2,592であった。これに対しA＋C群では発現変化した遺伝子群は少なくUp-regulateされた遺伝子は157、Down-regulateされた遺伝子は415であった。この傾向は糖尿病罹病者群、糖尿病非罹病者群の中での歯周病の有無においても同様であり、D群で発現変化した遺伝子が1,771、B群で1,949であるのに対し、C群は808、A群では812であった。 In the C + D group, there were few genes whose expression was changed compared to the A + B group, 204 were up-regulated genes and 416 were down-regulated genes. In the analysis based on the presence or absence of periodontal disease, the number of genes whose expression was changed in the B + D group was the largest among the group comparisons, 1,998 were up-regulated and 2,592 were down-regulated. On the other hand, in the A + C group, the number of genes whose expression was changed was small, 157 were up-regulated and 415 were down-regulated. This trend is the same in the presence or absence of periodontal disease in the diabetic group and non-diabetic group. The genes whose expression was changed in group D were 1,771 and 1,949 in group B, whereas group C Was 808 and 812 in group A.
4-1-4-2-2．ヒストグラム、ベン図
　解析2、4～10において、1.5倍以上あるいは1/1.5倍以下の発現変化を認める遺伝子のヒストグラムを図123に示した。縦軸に発現変化倍数のLog2値を表し、横軸にサンプル名を表した。歯周病罹病者群の解析、すなわち解析6(B群の笑い前後)、解析8(D群の笑い前後)、解析10(B＋D群の笑い前後)では、発現変化幅の大きい遺伝子が多い。また、対応する解析ごとの発現変化の相対差を図124に比較して示した。縦軸に発現変化倍数のLog2値を表し、横軸に解析組合せを表した。糖尿病非罹病者群では講義前後と笑い前後では各遺伝子の発現変化の差が大きいが、糖尿病罹病者群では比較的小さい。また、A＋C群とB＋D群間では発現変化の差が大きく、A群とB群でも大きかった。また、C群とD群間の比較では、発現が減少した遺伝子の変化幅が比較的大きかった。 4-1-4-2-2. FIG. 123 shows a histogram of genes in which expression changes of 1.5 times or more or 1 / 1.5 times or less are observed in histograms and Venn diagram analysis 2, 4 to 10. The vertical axis represents the Log2 value of the expression change fold, and the horizontal axis represents the sample name. In the analysis of the group suffering from periodontal disease, that is, analysis 6 (before and after laughing in group B), analysis 8 (before and after laughing in group D), and analysis 10 (before and after laughing in group B + D), there are many genes whose expression change width is large. In addition, the relative difference in expression change for each corresponding analysis is shown in FIG. The vertical axis represents the log2 value of the expression change fold, and the horizontal axis represents the analysis combination. In the non-diabetic group, the difference in the expression change of each gene is large before and after the lecture and before and after laughing, but it is relatively small in the diabetic group. Moreover, the difference in expression change was large between the A + C group and the B + D group, and the A group and the B group were also large. Moreover, in the comparison between the C group and the D group, the change width of the gene whose expression was decreased was relatively large.
　次に、笑いにより発現変化を認めた各群の遺伝子の数をベン図に表した。A＋B群とC＋D群で1.5倍以上の発現変化を認めた遺伝子のベン図を図125に示す。A＋B群のみでUp-regulateした遺伝子は928、C＋D群のみでは163、両群共通では41あった。1/1.5倍以下の発現変化を認めた遺伝子のベン図を図126に示す。A＋B群のみでDown-regulateした遺伝子は804、C＋D群のみでは353、両群共通では63あった。同様にA群とB群で1.5倍以上の発現変化を認めた遺伝子のベン図を図127に示す。A群のみでUp-regulateした遺伝子は161、B群のみでは886、両群共通では36あった。1/1.5倍以下の発現変化を認めた遺伝子のベン図を図128に示す。A群のみでDown-regulateした遺伝子は577、B群のみでは989、両群共通では38あった。A群とC群で1.5倍以上の発現変化を認めた遺伝子のベン図を図129に示す。A群のみでUp-regulateした遺伝子は176、C群のみでは153、両群共通では21あった。1/1.5倍以下の発現変化を認めた遺伝子のベン図を図130に示す。A群のみでDown-regulateした遺伝子は564、C群のみでは583、両群共通では51あった。B群とD群で1.5倍以上の発現変化を認めた遺伝子のベン図を図131に示す。B群のみでUp-regulateした遺伝子は819、D群のみでは531、両群共通では103あった。1/1.5倍以下の発現変化を認めた遺伝子のベン図を図132に示す。B群のみでDown-regulateした遺伝子は771、D群のみでは881、両群共通では256あった。C群とD群で1.5倍以上の発現変化を認めた遺伝子のベン図を図133に示す。C群のみでUp-regulateした遺伝子は129、D群のみでは589、両群共通では45あった。1/1.5倍以下の発現変化を認めた遺伝子のベン図を図134に示す。C群のみでDown-regulateした遺伝子は481、D群のみでは984、両群共通では153あった。A＋C群とB＋D群で1.5倍以上の発現変化を認めた遺伝子のベン図を図135に示す。A＋C群のみでUp-regulateした遺伝子は361、B＋D群のみでは2,538、両群共通では54あった。1/1.5倍以下の発現変化を認めた遺伝子のベン図を図136に示す。A＋C群のみでDown-regulateした遺伝子は103、B＋D群のみでは1,944、両群共通では54あった。 Next, the Venn diagram shows the number of genes in each group whose expression was changed by laughter. FIG. 125 shows a Venn diagram of genes in which an expression change of 1.5 times or more was observed in the A + B group and the C + D group. There were 928 up-regulated genes in the A + B group alone, 163 in the C + D group alone, and 41 in both groups. FIG. 126 shows a Venn diagram of genes in which expression changes of 1 / 1.5 times or less were observed. There were 804 down-regulated genes in the A + B group alone, 353 in the C + D group alone, and 63 in both groups. Similarly, FIG. 127 shows a Venn diagram of genes in which expression changes of 1.5 times or more were observed in the A group and the B group. There were 161 up-regulated genes in group A alone, 886 in group B alone, and 36 in both groups. FIG. 128 shows a Venn diagram of genes in which an expression change of 1 / 1.5 times or less was observed. There were 577 down-regulated genes in group A alone, 989 in group B alone, and 38 in both groups. FIG. 129 shows a Venn diagram of genes that showed an expression change of 1.5 times or more in the A group and the C group. There were 176 genes up-regulated only in group A, 153 in group C alone, and 21 in both groups. FIG. 130 shows a Venn diagram of genes in which expression changes of 1 / 1.5-fold or less were observed. There were 564 down-regulated genes in group A alone, 583 in group C alone, and 51 in both groups. FIG. 131 shows a Venn diagram of genes in which expression changes of 1.5 times or more were observed in the B group and the D group. There were 819 up-regulated genes in group B alone, 531 in group D alone, and 103 in both groups. FIG. 132 shows a Venn diagram of genes in which an expression change of 1 / 1.5 times or less was observed. There were 771 down-regulated genes in group B alone, 881 in group D alone, and 256 in both groups. FIG. 133 shows a Venn diagram of genes in which expression changes of 1.5 times or more were observed in the C group and the D group. There were 129 genes up-regulated in group C alone, 589 in group D alone, and 45 in both groups. FIG. 134 shows a Venn diagram of genes in which an expression change of 1 / 1.5 times or less was observed. There were 481 down-regulated genes in group C alone, 984 in group D alone, and 153 in both groups. FIG. 135 shows a Venn diagram of genes in which an expression change of 1.5 times or more was observed in the A + C group and the B + D group. There were 361 genes that were up-regulated only in the A + C group, 2,538 in the B + D group alone, and 54 in both groups. FIG. 136 shows a Venn diagram of genes in which an expression change of 1 / 1.5-fold or less was observed. There were 103 down-regulated genes in the A + C group alone, 1,944 in the B + D group alone, and 54 in both groups.
　解析の結果、A＋C群とB＋D群では、1.5倍以上、1.5倍以下発現変動した遺伝子数が多いにも関わらず、共通で発現変動した遺伝子数は少なかった。また、D群／B群間では共通で発現変動した遺伝子数は比較的多かった。 As a result of the analysis, in the A + C group and the B + D group, although the number of genes whose expression was changed 1.5 times or more and 1.5 times or less was large, the number of genes whose expression was commonly changed was small. In addition, the number of genes whose expression was changed in common between Group D and Group B was relatively large.
4-1-4-2-3．オントロジー解析（GO解析）
　当解析は、解析2、4～10において、1.5倍以上あるいは1/1.5倍以下の発現変化を認める遺伝子を対象として行った。抽出された遺伝子数 (プローブ数)とGO term数は表106、107に示した。
Figure JPOXMLDOC01-appb-T000106
Figure JPOXMLDOC01-appb-T000107
 4-1-4-2-3. Ontology analysis (GO analysis)
This analysis was performed on genes that showed an expression change of 1.5-fold or more or 1 / 1.5-fold or less in Analysis 2, 4 to 10. Tables 106 and 107 show the number of extracted genes (number of probes) and the number of GO terms.
Figure JPOXMLDOC01-appb-T000106
Figure JPOXMLDOC01-appb-T000107
　DNAチップ上のプローブとGO termとの対応付けは、Agilent社の提供するプローブ情報とNCBI Entrez(http://www.ncbi.nlm.nih.gov/)のGeneID情報に基づいて行い、Gene IDとGOの対応付けはBiNGO (http://www.psb.ugent.be/cbd/papers/BiNGO/)を用いて行った。各GO termに割り当てられる遺伝子の出現頻度が、抽出遺伝子の方がDNAチップ搭載遺伝子より有意に大きいか、フィッシャーの正確確率検定により求め、有意水準p-Value<0.05で検定した。該検定で有意と判定されたGO termを抽出遺伝子群に特徴的なGOとした。p-Value<0.01で有意と判定されたGO termを表108から表123に示す。
Figure JPOXMLDOC01-appb-T000108
 The probe on the DNA chip is associated with the GO term based on the probe information provided by Agilent and the GeneID information of NCBI Entrez (http://www.ncbi.nlm.nih.gov/). The association between GO and GO was done using BiNGO (http://www.psb.ugent.be/cbd/papers/BiNGO/). The appearance frequency of the gene assigned to each GO term was determined by Fisher's exact test to determine whether the extracted gene was significantly larger than the DNA chip-mounted gene, and the test was performed with a significance level of p-Value <0.05. A GO term determined to be significant by the test was defined as a GO characteristic of the extracted gene group. Tables 108 to 123 show GO terms determined to be significant when p-Value <0.01.
Figure JPOXMLDOC01-appb-T000108
Figure JPOXMLDOC01-appb-I000209
Figure JPOXMLDOC01-appb-T000109
Figure JPOXMLDOC01-appb-T000110
Figure JPOXMLDOC01-appb-T000111
Figure JPOXMLDOC01-appb-T000112
Figure JPOXMLDOC01-appb-T000113
Figure JPOXMLDOC01-appb-T000114
Figure JPOXMLDOC01-appb-I000209
Figure JPOXMLDOC01-appb-T000109
Figure JPOXMLDOC01-appb-T000110
Figure JPOXMLDOC01-appb-T000111
Figure JPOXMLDOC01-appb-T000112
Figure JPOXMLDOC01-appb-T000113
Figure JPOXMLDOC01-appb-T000114
Figure JPOXMLDOC01-appb-I000210
Figure JPOXMLDOC01-appb-I000210
Figure JPOXMLDOC01-appb-I000211
Figure JPOXMLDOC01-appb-I000211
Figure JPOXMLDOC01-appb-I000212
Figure JPOXMLDOC01-appb-T000115
Figure JPOXMLDOC01-appb-I000212
Figure JPOXMLDOC01-appb-T000115
Figure JPOXMLDOC01-appb-I000213
Figure JPOXMLDOC01-appb-T000116
Figure JPOXMLDOC01-appb-I000213
Figure JPOXMLDOC01-appb-T000116
Figure JPOXMLDOC01-appb-I000214
Figure JPOXMLDOC01-appb-T000117
Figure JPOXMLDOC01-appb-T000118
Figure JPOXMLDOC01-appb-I000214
Figure JPOXMLDOC01-appb-T000117
Figure JPOXMLDOC01-appb-T000118
Figure JPOXMLDOC01-appb-I000215
Figure JPOXMLDOC01-appb-T000119
Figure JPOXMLDOC01-appb-I000215
Figure JPOXMLDOC01-appb-T000119
Figure JPOXMLDOC01-appb-I000216
Figure JPOXMLDOC01-appb-T000120
Figure JPOXMLDOC01-appb-T000121
Figure JPOXMLDOC01-appb-T000122
Figure JPOXMLDOC01-appb-I000216
Figure JPOXMLDOC01-appb-T000120
Figure JPOXMLDOC01-appb-T000121
Figure JPOXMLDOC01-appb-T000122
Figure JPOXMLDOC01-appb-I000217
Figure JPOXMLDOC01-appb-I000217
Figure JPOXMLDOC01-appb-I000218
Figure JPOXMLDOC01-appb-I000218
Figure JPOXMLDOC01-appb-I000219
Figure JPOXMLDOC01-appb-T000123
Figure JPOXMLDOC01-appb-I000219
Figure JPOXMLDOC01-appb-T000123
Figure JPOXMLDOC01-appb-I000220
Figure JPOXMLDOC01-appb-I000220
Figure JPOXMLDOC01-appb-I000221
Figure JPOXMLDOC01-appb-I000221
Figure JPOXMLDOC01-appb-I000222
Figure JPOXMLDOC01-appb-I000222
　生物学的経路で注目されるTermとしては、糖尿病非罹病者群(A＋B群)で笑いによりUp-regulateされる遺伝子の解析において、抗原受容体シグナル伝達経路関連(GO ID: 50854、50857)に割り当てられる3遺伝子(表124)、Down-regulateされる遺伝子の解析において、グルコース輸送(GO ID: 15758)に割り当てられる5遺伝子(表125)、リポ蛋白質代謝(GO ID: 42157)に割り当てられる5遺伝子(表126)、pH調整(GO ID: 6885)に割り当てられる4遺伝子(表127)があった。糖尿病罹病者群（C＋D群）では、笑いによりDown-regulateされる遺伝子の解析において、プログラムされた細胞死の制御(GO ID: 12502)に割り当てられる8遺伝子(表128)があった。
Figure JPOXMLDOC01-appb-T000124
Figure JPOXMLDOC01-appb-T000125
Figure JPOXMLDOC01-appb-T000126
Figure JPOXMLDOC01-appb-T000127
Figure JPOXMLDOC01-appb-T000128
 Term that is attracting attention in biological pathways is related to antigen receptor signaling pathway (GO ID: 50854, 50857) in the analysis of genes up-regulated by laughter in non-diabetic group (A + B group) 3 genes assigned (Table 124), 5 genes assigned to glucose transport (GO ID: 15758) (Table 125), 5 assigned to lipoprotein metabolism (GO ID: 42157) in analysis of down-regulated genes There were 4 genes (Table 127) assigned to genes (Table 126), pH adjustment (GO ID: 6885). In the diabetic group (C + D group), in the analysis of genes down-regulated by laughter, there were 8 genes (Table 128) assigned to programmed cell death control (GO ID: 12502).
Figure JPOXMLDOC01-appb-T000124
Figure JPOXMLDOC01-appb-T000125
Figure JPOXMLDOC01-appb-T000126
Figure JPOXMLDOC01-appb-T000127
Figure JPOXMLDOC01-appb-T000128
　A群（糖尿病非罹病かつ歯周病非罹病者群）では、笑いよりUp-regulateされる遺伝子の解析において、抗細胞死（GO ID:0006916）に割り当てられる1遺伝子（遺伝子セット１No.3）、IgE受容体活性（GO ID:0019767）に割り当てられる1遺伝子（遺伝子セット１No.4）、レスポンス防御GO:0006952(defense response)に割り当てられる1遺伝子（遺伝子セット１No.5）、セロトニン輸送関連(GO ID: 6837または15222または5335)に割り当てられる1遺伝子（遺伝子セット１No.7）、γグルタミン酸カルボキシラーゼ活性（GOID:0008488）に割り当てられる1遺伝子（遺伝子セット１No.8）、酸素輸送（GOID:0015671）に割り当てられる1遺伝子（遺伝子セット１No.9）、Down-regulateされる遺伝子の解析において、癌関連の7遺伝子（遺伝子セット１No.11-17）、自然免疫反応（GOID:0045087）に割り当てられる2遺伝子（遺伝子セット１No.18-19）、免疫反応（GOID:0006955）に割り当てられる1遺伝子（遺伝子セット1No.20）、レスポンス防御（GO:0006952）に割り当てられる1遺伝子（遺伝子セット１No.22）、転写因子（GOIID:0003700）に割り当てられる1遺伝子（遺伝子セット１No.25）、細胞死（GOID:0006915）に割り当てられる1遺伝子（遺伝子セット１No.27）があった。なお、遺伝子セット1については下記表164を参照のこと。 In group A (non-diabetic and periodontal disease non-affected group), in the analysis of genes that are up-regulated from laughter, 1 gene assigned to anti-cell death (GO ID: 0006916) (gene set 1 No. 3) 1 gene assigned to IgE receptor activity (GO ID: 0019767) (gene set 1 No. 4), 1 gene assigned to response defense GO: 0006952 (defense response) (gene set 1 No. 5), serotonin transport related ( 1 gene (gene set 1 No. 7) assigned to GO222ID: 6837 or 15222 or 5335), 1 gene (gene set 1 No. 8) assigned to γ-glutamate carboxylase activity (GOID: 0008488), oxygen transport (GOID: 0015671) ) Assigned to 1 gene (Gene set 1 No. 9), down-regulated genes in cancer-related 7 genes (Gene set 1 No. 11-17), innate immune response (GOID: 0045087) 2 genes (Gene set 1 No. 18-19), 1 gene (Gene set 1 No. 20) assigned to immune response (GOID: 0006955), 1 gene (Gene set 1 No. 1) assigned to response defense (GO: 0006952) 22), there was 1 gene (gene set 1 No. 25) assigned to transcription factors (GOIID: 0003700) and 1 gene (gene set 1 No. 27) assigned to cell death (GOID: 0006915). See Table 164 below for gene set 1.
　B群(糖尿病非罹病・歯周病罹病者群)では、笑いによりDown-regulateされる遺伝子の解析において、サイトカイン・ケモカインシグナル伝達(GO ID: 19221)に割り当てられる6遺伝子(表129)、リポ蛋白質異化(GO ID: 42159、45192)に割り当てられる2遺伝子(表130)、脂質輸送(GO ID: 6869)に割り当てられる10遺伝子(表131)、インスリン受容体シグナル伝達関連(GO ID: 8286または46627)に割り当てられる6遺伝子(表132)、インターロイキン6生合成関連(GO ID: 45408、42226)に割り当てられる3遺伝子(表133)があった。C群(糖尿病罹病・歯周病なし群)で笑いによりDown-regulateされる遺伝子の解析において、抗生物質生合成・代謝(GO ID: 17000、16999)に割り当てられる2遺伝子(表134)、アルギニン合成(GO ID: 6526)や尿酸回路・代謝(GO ID: 19627、50)に割り当てられる2遺伝子(表135)、リンパ球分化(GO ID: 30098)に割り当てられる5遺伝子(表136)があった。D群(糖尿病罹病・歯周病罹病者群)で笑いによりUp-regulateされる遺伝子の解析において、ウイルスレスポンス防御(GO ID: 51607)に割り当てられる3遺伝子(表137)、インターフェロン生合成関連(GO ID: 45356、45359、45357、45350、45351)、インターロイキン8生合成(GO ID: 45354、45414、45416)に割り当てられる2遺伝子(表138)、Down-regulateされる遺伝子の解析において、脂質代謝制御(GO ID: 19216)に割り当てられる4遺伝子(表139)、I-kappaBリン酸基転移(GO ID: 7252)に割り当てられる2遺伝子(表140)があった。A＋C群(歯周病非罹病者群)で笑いによりDown-regulateされる遺伝子の解析において、カテコールアミン代謝(GO ID:6584)に割り当てられる3遺伝子(表141)、インスリン制御(GO ID: 46676)として割り当てられる1遺伝子(表142)があった。B＋D群(歯周病罹病者群)で笑いによってUp-regulateされる遺伝子の解析において、抗原授与(GO ID: 19882)に割り当てられる16遺伝子(表143)、NF-KappaB関連(GO ID: 43122、43123、7249)に割り当てられる27遺伝子(表144)、脂質分泌(GO ID: 19915)に割り当てられる6遺伝子(表145)、NK細胞分化(GO ID; 1779)、B細胞分化(GO ID: 45579)、T細胞恒常性(GO ID: 43029)に割り当てられる3遺伝子(表146)、糖脂質代謝(GO ID: 6664)として割り当てられる8遺伝子(表147)があった。
Figure JPOXMLDOC01-appb-T000129
Figure JPOXMLDOC01-appb-T000130
Figure JPOXMLDOC01-appb-T000131
Figure JPOXMLDOC01-appb-T000132
Figure JPOXMLDOC01-appb-T000133
Figure JPOXMLDOC01-appb-T000134
Figure JPOXMLDOC01-appb-T000135
Figure JPOXMLDOC01-appb-T000136
Figure JPOXMLDOC01-appb-T000137
Figure JPOXMLDOC01-appb-T000138
Figure JPOXMLDOC01-appb-T000139
Figure JPOXMLDOC01-appb-T000140
Figure JPOXMLDOC01-appb-T000141
Figure JPOXMLDOC01-appb-T000142
Figure JPOXMLDOC01-appb-T000143
Figure JPOXMLDOC01-appb-T000144
Figure JPOXMLDOC01-appb-T000145
Figure JPOXMLDOC01-appb-T000146
Figure JPOXMLDOC01-appb-T000147
 In group B (non-diabetic and periodontal disease group), in the analysis of genes down-regulated by laughter, 6 genes (Table 129) assigned to cytokine / chemokine signaling (GO ID: 19221), lipo 2 genes assigned to protein catabolism (GO ID: 42159, 45192) (Table 130), 10 genes assigned to lipid transport (GO ID: 6869) (Table 131), insulin receptor signaling related (GO ID: 8286 or There were 6 genes (Table 132) assigned to 46627) and 3 genes (Table 133) assigned to interleukin 6 biosynthesis related (GO ID: 45408, 42226). Two genes (Table 134) assigned to antibiotic biosynthesis and metabolism (GO ID: 17000, 16999) in the analysis of genes that are down-regulated by laughter in group C (group with diabetes and no periodontal disease), arginine There are 2 genes (Table 135) assigned to synthesis (GO ID: 6526) and uric acid cycle / metabolism (GO ID: 19627, 50), and 5 genes (Table 136) assigned to lymphocyte differentiation (GO ID: 30098). It was. In the analysis of genes up-regulated by laughter in group D (groups with diabetes and periodontal disease), 3 genes (Table 137) assigned to virus response protection (GO ID: 51607), related to interferon biosynthesis ( GO ID: 45356, 45359, 45357, 45350, 45351), 2 genes assigned to interleukin 8 biosynthesis (GO ID: 45354, 45414, 45416) (Table 138), analysis of down-regulated genes There were 4 genes (Table 139) assigned to metabolic control (GO ID: 19216) and 2 genes (Table 140) assigned to I-kappaB phosphate transfer (GO ID: 7252). In the analysis of genes down-regulated by laughter in the A + C group (non-periodontal disease group), 3 genes (Table 141) assigned to catecholamine metabolism (GO ID: 6584), insulin regulation (GO ID: 46676) There was one gene assigned as (Table 142). In the analysis of genes up-regulated by laughter in B + D group (periodontal disease affected group), 16 genes (Table 143) assigned to antigen conferring (GO ID: 19882), NF-KappaB related (GO ID: 43122) , 43123, 7249), 27 genes (Table 144), 6 genes assigned to lipid secretion (GO ID: 19915) (Table 145), NK cell differentiation (GO ID; 1779), B cell differentiation (GO ID: 45579), 3 genes assigned to T cell homeostasis (GO ID: 43029) (Table 146), 8 genes assigned to glycolipid metabolism (GO ID: 6664) (Table 147).
Figure JPOXMLDOC01-appb-T000129
Figure JPOXMLDOC01-appb-T000130
Figure JPOXMLDOC01-appb-T000131
Figure JPOXMLDOC01-appb-T000132
Figure JPOXMLDOC01-appb-T000133
Figure JPOXMLDOC01-appb-T000134
Figure JPOXMLDOC01-appb-T000135
Figure JPOXMLDOC01-appb-T000136
Figure JPOXMLDOC01-appb-T000137
Figure JPOXMLDOC01-appb-T000138
Figure JPOXMLDOC01-appb-T000139
Figure JPOXMLDOC01-appb-T000140
Figure JPOXMLDOC01-appb-T000141
Figure JPOXMLDOC01-appb-T000142
Figure JPOXMLDOC01-appb-T000143
Figure JPOXMLDOC01-appb-T000144
Figure JPOXMLDOC01-appb-T000145
Figure JPOXMLDOC01-appb-T000146
Figure JPOXMLDOC01-appb-T000147
4-1-4-2-4．リアルタイム定量PCR解析
　被験者ごとの各遺伝子の笑い体験前のPCR定量値に対する笑い体験後の定量値の比を表148に示す。今回の結果ではDNAチップ解析結果とリアルタイムPCR解析結果では、対象被験者により不一致が見られ、確実な相関は得られなかった。
Figure JPOXMLDOC01-appb-T000148
 4-1-4-2-4. Real-time quantitative PCR analysis Table 148 shows the ratio of the quantitative value after laughter experience to the PCR quantitative value before laughter experience of each gene for each subject. In this result, there was a discrepancy between the DNA chip analysis result and the real-time PCR analysis result depending on the subject, and no reliable correlation was obtained.
Figure JPOXMLDOC01-appb-T000148
4-1-4-3．考察・結論
　今回の実験について、以下の2つの観点から考察した。 4-1-4-3. Discussion / Conclusion We examined this experiment from the following two viewpoints.
(1) 健康な状態の人において、笑い刺激により発現変動する遺伝子群
(2) 歯周病罹病者において、笑い刺激により発現変動する遺伝子群
(1) 健康な状態の人において、笑い刺激により発現変動する遺伝子群
　A群では、免疫系を始め、生体防御、抗細胞死など様々な機能の遺伝子が発現変化し、B群、A+B群においても同様に免疫系を始めとする多くの遺伝子の発現変化がみられた。A群、B群、A+B群においては、笑いによりs-IgA(唾液中の分泌型免疫グロブリンA)の増加が認められた。 (1) Genes whose expression changes due to laughter in healthy people
(2) Genes whose expression changes due to laughter stimulation in patients with periodontal disease
(1) In a healthy group of individuals, the gene group A whose expression changes due to laughter stimulation, the genes of various functions such as immune system, biological defense and anti-cell death change in expression, group B, A + B The group also showed changes in the expression of many genes including the immune system. In groups A, B, and A + B, s-IgA (secretory immunoglobulin A in saliva) was increased by laughter.
　このことは、健康な状態の人において、笑い刺激により、肺への酸素取り込みや、筋肉への酸素運搬量が増加し、様々な生体機能の活性化が起こり、免疫系や生体防御などの健康維持・増進を担う多くの遺伝子の発現変化が起こったのではないかと示唆される。具体的な遺伝子については以下に示す。 This means that in healthy people, laughter stimulation increases oxygen uptake into the lungs and increases oxygen transport to muscles, which activates various biological functions, and promotes health such as the immune system and biological defenses. This suggests that changes in the expression of many genes responsible for maintenance and enhancement have occurred. Specific genes are shown below.
　A群において、Up-regulateされる遺伝子としては、GO解析において抗細胞死（GO ID:0006916）に割り当てられる遺伝子PROK2（遺伝子セット１No.3）があり、IgE受容体活性（GO ID:0019767）に割り当てられる遺伝子FCER1A（遺伝子セット１No.4）、レスポンス防御GO:0006952(defense response)に割り当てられる遺伝子CD69（遺伝子セット１No.5）、セロトニン輸送関連 (GO ID: 6837または15222または5335) に割り当てられる遺伝子SLC6A4（遺伝子セット１No.7）、γグルタミン酸カルボキシラーゼ活性（GOID:0008488）に割り当てられる遺伝子GGCX（遺伝子セット１No.8）、酸素輸送（GOID:0015671）に割り当てられる遺伝子HBA2（遺伝子セット１No.9）、アルコール脱水素酵素活性（GOID :0004022）に割り当てられる遺伝子ADH5があった。逆にDown-regulateされる遺伝子としては、癌関連の遺伝子JUN、TP53、RAB5B、RAB8A、RAB12、RAB20、RAB30（遺伝子セット１No.11-17）、自然免疫反応（GOID:0045087）に割り当てられる遺伝子VISA、CD55（遺伝子セット１No.18-19）、免疫反応（GOID:0006955）に割り当てられる遺伝子IGHG1（遺伝子セット１No.20）、レスポンス防御（GO:0006952）に割り当てられる遺伝子CD84（遺伝子セット１No.22）、転写因子（GOIID:0003700）に割り当てられる遺伝子NFAT5（遺伝子セット１No.25）、細胞死（GOID:0006915）に割り当てられる遺伝子TNFAIP3（遺伝子セット１No.27）があった。 In group A, the up-regulated gene is the gene PROK2 (gene set 1 No. 3) assigned to anti-cell death (GO ID: 0006916) in GO analysis, and IgE receptor activity (GO ID: 0019767) Assigned to the gene FCER1A (Gene set 1 No. 4), the gene CD69 (Gene set 1 No. 5) assigned to the response defense GO: 0006952 (defense response), and the serotonin transport related (GO ID: 6837 or 15222 or 5335) Gene SLC6A4 (gene set 1 No. 7), gene GGCX (gene set 1 No. 8) assigned to γ-glutamate carboxylase activity (GOID: 0008488), gene HBA2 (gene set 1 No. 1) assigned to oxygen transport (GOID: 0015671) 9) There was a gene ADH5 assigned to alcohol dehydrogenase activity (GOID 0004022). Conversely, genes that are down-regulated include cancer-related genes JUN, TP53, RAB5B, RAB8A, RAB12, RAB20, RAB30 (gene set 1 No. 11-17), genes assigned to innate immune responses (GOID: 0045087) VISA, CD55 (gene set 1 No. 18-19), gene IGHG1 (gene set 1 No. 20) assigned to immune response (GOID: 0006955), gene CD84 (gene set 1 No. 1) assigned to response defense (GO: 0006952) 22), gene NFAT5 (gene set 1 No. 25) assigned to transcription factors (GOIID: 0003700), and gene TNFAIP3 (gene set 1 No. 27) assigned to cell death (GOID: 0006915).
　B群では、笑いによりDown-regulateされる遺伝子の解析において、サイトカイン・ケモカインシグナル伝達(GO ID: 19221)に割り当てられる遺伝子SOCS1、CCR1、CSF2RB、STAT5A、
STAT3、TNFRSF1B (表129)、リポ蛋白質異化(GO ID: 42159、45192)に割り当てられる遺伝子SNX17、SCARF1 (表130)、脂質輸送(GO ID: 6869)に割り当てられる脂質輸送(GO ID: 6869)に割り当てられるLDLR、STARD3、CLN8、APOL1、ATP10A、PSAP、OSBP2、SLC27A1、APOL6(表131)、インスリン受容体シグナル伝達関連(GO ID: 8286または46627)に割り当てられるSOCS1、STXBP4、SOCS3、AKT1、MLLT7、INSIGF(表132)、インターロイキン6生合成関連(GO ID: 45408、42226)に割り当てられる遺伝子CEBPB、NLRP12 (表133)があった。 In group B, in the analysis of genes that are down-regulated by laughter, the genes SOCS1, CCR1, CSF2RB, STAT5A, which are assigned to cytokine chemokine signaling (GO ID: 19221),
STAT3, TNFRSF1B (Table 129), genes assigned to lipoprotein catabolism (GO ID: 42159, 45192), SNX17, SCARF1 (Table 130), lipid transport assigned to lipid transport (GO ID: 6869) (GO ID: 6869) LDLR, STARD3, CLN8, APOL1, ATP10A, PSAP, OSBP2, SLC27A1, APOL6 (Table 131), SOCS1, STXBP4, SOCS3, AKT1, assigned to insulin receptor signaling related (GO ID: 8286 or 46627) There were genes CEBPB and NLRP12 (Table 133) assigned to MLLT7, INSIGF (Table 132), and interleukin 6 biosynthesis related (GO ID: 45408, 42226).
　A+B群においては、Up-regulateされる遺伝子の解析において、抗原受容体シグナル伝達経路関連(GO ID: 50854、50857)に割り当てられる3遺伝子PTPRC、PAWR、TRAT1 (表124)、Down-regulateされる遺伝子の解析において、グルコース輸送(GO ID: 15758)に割り当てられる5遺伝子STXBP4、SLC2A5、SLC2A3、SORBS1、AKT1 (表126)、リポ蛋白質代謝(GO ID: 42157)に割り当てられる遺伝子APOL6、ATG10、SNX17、PIGS、FNTB (表126)、pH調整(GO ID: 6885)に割り当てられる遺伝子SLC9A5、LOC133308、CLN5、CLN3 (表127)があった。 In the A + B group, in the analysis of up-regulated genes, the three genes PTPRC, PAWR, TRAT1 (Table 124), Down-regulate assigned to antigen receptor signaling pathway-related (GO ID: 50854, 50857) 5 genes STXBP4, SLC2A5, SLC2A3, SORBS1, AKT1 (Table 126), genes APOL6, ATG10 assigned to lipoprotein metabolism (GO ID: 42157) assigned to glucose transport (GO ID: 15758) , SNX17, PIGS, FNTB (Table 126), and genes SLC9A5, LOC133308, CLN5, and CLN3 (Table 127) assigned to pH adjustment (GO ID: 6885).
(2) 歯周病罹病者において、笑い刺激により発現変動する遺伝子群
　B＋D群(歯周病罹病者群)において、笑いにより多くの遺伝子発現変化が認められた。この結果は、C群とD群との比較でも、A群のB群の比較でも同様であった。このことは、歯周病が慢性炎症疾患であり、常に細菌などの刺激にさらされ、免疫系を始めとする様々な機能が活性された状態であるため、笑いという刺激が多くの遺伝子の発現変化を引き起こす要因となったのではないかと示唆される。 (2) Among the patients with periodontal disease, many gene expression changes were observed due to laughter in the gene group B + D group (periodontal disease affected group) whose expression was changed by laughter stimulation . This result was the same in the comparison between group C and group D and in the comparison between group A and group B. This is because periodontal disease is a chronic inflammatory disease, and is constantly exposed to stimuli such as bacteria, and various functions including the immune system are activated. It is suggested that it may have caused change.
　歯周病は糖尿病の第6の合併症といわれ、糖尿病による末梢血管の脆弱性の亢進は歯周病発症や増悪の原因となり、また、慢性炎症疾患である歯周病では腫瘍壊死因子(TNF-α)などのサイトカインが産生されることによりインスリン抵抗性が増し、糖尿病の増悪因子となることが報告されている。更に、糖尿病と歯周病の相関は、近年多くのことがわかってきており、糖尿病患者の歯周炎の組織では、多形核白血球機能（走化能、貪食能、殺菌能）の低下、歯周結合組織のコラーゲン代謝異常、終末糖化産物(AGE;advanced endproduct)の歯肉組織への蓄積による炎症、創傷治癒の遅延などが認められる。 Periodontal disease is said to be the sixth complication of diabetes, and the increased vulnerability of peripheral blood vessels due to diabetes causes the onset and exacerbation of periodontal disease, and tumor necrosis factor (TNF) in periodontal disease, which is a chronic inflammatory disease. It has been reported that production of cytokines such as -α) increases insulin resistance and becomes an exacerbation factor of diabetes. Furthermore, the correlation between diabetes and periodontal disease has been known a lot in recent years. In the periodontitis tissue of diabetic patients, polymorphonuclear leukocyte function (chemotactic, phagocytic, bactericidal) decreases, Abnormal collagen metabolism in periodontal connective tissue, inflammation due to accumulation of advanced glycation end products (AGE) in gingival tissues, delayed wound healing, etc. are observed.
　今回の研究では、歯周病罹病者群(B＋D群)が歯周病非罹病者群(A＋C群)に比較して多くの遺伝子の発現変化がみられ、これらの遺伝子が多くのGO termに割り当てられた。Up-regulateされる遺伝子としてはGOカテゴリー上比較的上位である代謝や生合成に関するGO termに割り当てられ、特異的な下位のGO termには割り当てられなかった。逆にDown-regulateされる遺伝子としてはGOカテゴリー上、特異的な下位のGO termにも割り与えられた。これらのGO termに含まれる遺伝子としてはHLA-A 、HLA-B、HLA-C、HLA-E、HLA-H、CD1D、CD74、VISA、CASP8、CFLAR、APOL、LTBR、CTSA、CTSB、LOC391803、KRTAP3-3、SESN2など免疫・炎症・自己防衛などに関わる遺伝子やSTAT5A、STAT5Bなど脂質分泌に関わる遺伝子、FFAR2など脂質代謝に関わる遺伝子があった。 In this study, the number of genes in the periodontal disease affected group (B + D group) was changed compared to the periodontal disease non-affected group (A + C group). Assigned. Up-regulated genes were assigned to GO-terms related to metabolism and biosynthesis, which are relatively higher in the GO category, and were not assigned to specific lower-level GO-terms. Conversely, down-regulated genes were also assigned to specific GO-terms in the GO category. The genes contained in these GO terms include HLA-A, HLA-B, HLA-C, HLA-E, HLA-H, CD1D, CD74, VISA, CASP8, CFLAR, APOL, LTBR, CTSA, CTSB, LOC391803, There were genes related to immunity / inflammation / self-protection such as KRTAP3-3 and SESN2, genes related to lipid secretion such as STAT5A and STAT5B, and genes related to lipid metabolism such as FFAR2.
　また、D群(糖尿病罹病・歯周病罹病者群)では、笑いによりUp-regulateされた遺伝子としては、GO解析においてウイルスレスポンス防御(GO ID: 51607)、インターフェロン生合成関連(GO ID: 45356、45359、45357、45350、45351)、インターロイキン8生合成(GO ID: 45354、45414、45416)に割り当てられる遺伝子TLR7、TLR8、BCL2があり、Down-regulateされた遺伝子としては、GO解析において脂質代謝制御(GO ID: 19216)に割り当てられるPPARA、NR5A1、C1QTNF、I-kappaB、リン酸基転移(GO ID:7252)割り当てられるERC1があった。 In addition, in group D (diabetes and periodontal disease group), as a gene up-regulated by laughter, virus response protection in GO analysis (GO ID: 51607), interferon biosynthesis related (GO ID: 45356) 45359, 45357, 45350, 45351), genes TLR7, TLR8 and BCL2 assigned to interleukin 8 biosynthesis (GO ID: 45354, 45414, 45416). Down-regulated genes are lipids in GO analysis. There were PPARA, NR5A1, C1QTNF, I-kappaB assigned to metabolic control (GO ID: 19216), and ERC1 assigned to phosphate group transfer (GO ID: 7252).
　同様に、B群(糖尿病非罹病・歯周病罹病者群)では、Up-regulate された遺伝子としては、特異的な機能を有するGOtermは少なく、Down-regulateされた遺伝子としては、GO解析においてサイトカイン・ケモカインシグナル伝達 (GO ID: 19221)に割り当てられるSOCS1、CCR1、CFS2RB、STAT5A、STAT3、TINFRSF1B、リポ蛋白質異化(GO ID: 42159)に割り当てられるSNX17、SCARF1、脂質輸送(GO ID: 6869)に割り当てられるLDLR、STARD3、CLN8、APOL1、ATP10A、PSAP、SLC27A1、APOL6、インスリン受容体シグナル伝達関連(GO ID: 8286または46627)に割り当てられるSOCS1、STXBP4、SOCS3、AKT1、MLLT7、INSIGFがあった。 Similarly, in Group B (non-diabetic / periodontal diseased group), there are few up-regulated genes in GOterm with specific functions, and down-regulated genes in GO analysis. SOCS1, CCR1, CFS2RB, STAT5A, STAT3A, STAT3A, TINFRSF1B, SNX17, SCARF1, lipid transport (GO ID: サ イ ト カ イ ン 6869) assigned to the cytokine-chemokine signaling (GO ID: 19221) There were LDCS, STARD3, CLN8, APOL1, ATP10A, PSAP, SLC27A1, APOL6, SOCS1, STXBP4, SOCS3, AKT1, MLLT7, INSIGF assigned to insulin receptor signaling related (GO ID: 8286 or 46627) .
　B＋D群(歯周病罹病者群)と、D群(糖尿病患者・歯周病罹病者群)で発現変化した遺伝子のオントロジー解析では、主に免疫・炎症・生体防御に関するGOtermに割り当てられた。B群(糖尿病非罹病・歯周病罹病者群)ではこれらTermに割り当てられる遺伝子は少なかった。糖尿病と歯周病との相関には免疫・炎症・生体防御が深く関与しているとされているが、これらに関連する遺伝子の発現が笑いにより変化することが、末梢血白血球の遺伝子発現の網羅的解析で検証される結果となった。 In the ontology analysis of genes whose expression was changed in B + D group (periodontal disease affected group) and D group (diabetic patient / periodontal disease affected group), it was assigned to GOterm mainly related to immunity, inflammation and biological defense. In group B (non-diabetic and periodontal disease group), there were few genes assigned to these terms. The correlation between diabetes and periodontal disease is thought to be closely related to immunity, inflammation, and biological defense, but the expression of genes related to these changes due to laughter is related to the expression of peripheral blood leukocytes. The results were verified by exhaustive analysis.
4-1-5．特定された遺伝子
　上記解析2で笑いにより遺伝子発現の変動が認められた遺伝子のうち、機能面で注目すべき遺伝子、及び特に発現変動の大きかった上位15遺伝子を表149～154に示し、これらを要約した表155を遺伝子セット5の表として示す。
Figure JPOXMLDOC01-appb-T000149
Figure JPOXMLDOC01-appb-T000150
Figure JPOXMLDOC01-appb-T000151
Figure JPOXMLDOC01-appb-T000152
Figure JPOXMLDOC01-appb-T000153
 4-1-5. Specified genes Among the genes whose variation in gene expression was recognized by laughter in analysis 2 above, the genes that should be noted in terms of function, and the top 15 genes with particularly large variation in expression are shown in Tables 149 to 154. A summary table 155 is shown as a table for gene set 5.
Figure JPOXMLDOC01-appb-T000149
Figure JPOXMLDOC01-appb-T000150
Figure JPOXMLDOC01-appb-T000151
Figure JPOXMLDOC01-appb-T000152
Figure JPOXMLDOC01-appb-T000153
Figure JPOXMLDOC01-appb-I000223
Figure JPOXMLDOC01-appb-T000154
Figure JPOXMLDOC01-appb-I000223
Figure JPOXMLDOC01-appb-T000154
Figure JPOXMLDOC01-appb-I000224
Figure JPOXMLDOC01-appb-I000224
Figure JPOXMLDOC01-appb-I000225
Figure JPOXMLDOC01-appb-T000155
Figure JPOXMLDOC01-appb-I000225
Figure JPOXMLDOC01-appb-T000155
　上記解析4で笑いにより遺伝子発現の変動が認められた遺伝子のうち、機能面で注目すべき遺伝子、及び特に発現変動の大きかった上位15遺伝子を表156～158に示し、これらを要約した表159を遺伝子セット6の表として示す。
Figure JPOXMLDOC01-appb-T000156
Figure JPOXMLDOC01-appb-T000157
 Table 156 to 158 show genes that are notable in terms of function among the genes whose variation in gene expression was recognized by laughter in analysis 4 above, and the top 15 genes that have particularly large variation in expression. Is shown as a table for gene set 6.
Figure JPOXMLDOC01-appb-T000156
Figure JPOXMLDOC01-appb-T000157
Figure JPOXMLDOC01-appb-I000226
Figure JPOXMLDOC01-appb-I000226
Figure JPOXMLDOC01-appb-I000227
Figure JPOXMLDOC01-appb-I000227
Figure JPOXMLDOC01-appb-I000228
Figure JPOXMLDOC01-appb-T000158
Figure JPOXMLDOC01-appb-I000228
Figure JPOXMLDOC01-appb-T000158
Figure JPOXMLDOC01-appb-I000229
Figure JPOXMLDOC01-appb-I000229
Figure JPOXMLDOC01-appb-I000230
Figure JPOXMLDOC01-appb-T000159
Figure JPOXMLDOC01-appb-I000230
Figure JPOXMLDOC01-appb-T000159
　上記解析5で笑いにより遺伝子発現の変動が認められた遺伝子のうち、機能面で注目すべき遺伝子、及び特に発現変動の大きかった上位15遺伝子を表160～163に示し、これらを要約した表164を遺伝子セット1の表として示す。
Figure JPOXMLDOC01-appb-T000160
 Of the genes whose variation in gene expression was recognized by laughter in Analysis 5 above, genes notable in terms of function and the top 15 genes with particularly large variation in expression are shown in Tables 160 to 163, which are summarized in Table 164 Is shown as a table of gene set 1.
Figure JPOXMLDOC01-appb-T000160
Figure JPOXMLDOC01-appb-I000231
Figure JPOXMLDOC01-appb-T000161
Figure JPOXMLDOC01-appb-I000231
Figure JPOXMLDOC01-appb-T000161
Figure JPOXMLDOC01-appb-I000232
Figure JPOXMLDOC01-appb-I000232
Figure JPOXMLDOC01-appb-I000233
Figure JPOXMLDOC01-appb-I000233
Figure JPOXMLDOC01-appb-I000234
Figure JPOXMLDOC01-appb-T000162
Figure JPOXMLDOC01-appb-I000234
Figure JPOXMLDOC01-appb-T000162
Figure JPOXMLDOC01-appb-I000235
Figure JPOXMLDOC01-appb-I000235
Figure JPOXMLDOC01-appb-I000236
Figure JPOXMLDOC01-appb-I000236
Figure JPOXMLDOC01-appb-I000237
Figure JPOXMLDOC01-appb-I000237
Figure JPOXMLDOC01-appb-I000238
Figure JPOXMLDOC01-appb-T000164
Figure JPOXMLDOC01-appb-I000238
Figure JPOXMLDOC01-appb-T000164
　上記解析6で笑いにより遺伝子発現の変動が認められた遺伝子のうち、機能面で注目すべき遺伝子、及び特に発現変動の大きかった上位15遺伝子を表165～171に示し、これらを要約した表172を遺伝子セット2の表として示す。
Figure JPOXMLDOC01-appb-T000165
 Table 165 to 171 show the genes that should be noted in terms of function and the top 15 genes that have the most significant fluctuations in expression among the genes whose fluctuations in gene expression were recognized by laughter in Analysis 6 above. Is shown as a table of gene set 2.
Figure JPOXMLDOC01-appb-T000165
Figure JPOXMLDOC01-appb-I000239
Figure JPOXMLDOC01-appb-T000166
Figure JPOXMLDOC01-appb-T000167
Figure JPOXMLDOC01-appb-I000239
Figure JPOXMLDOC01-appb-T000166
Figure JPOXMLDOC01-appb-T000167
Figure JPOXMLDOC01-appb-I000240
Figure JPOXMLDOC01-appb-T000168
Figure JPOXMLDOC01-appb-T000169
Figure JPOXMLDOC01-appb-T000170
Figure JPOXMLDOC01-appb-I000240
Figure JPOXMLDOC01-appb-T000168
Figure JPOXMLDOC01-appb-T000169
Figure JPOXMLDOC01-appb-T000170
Figure JPOXMLDOC01-appb-I000241
Figure JPOXMLDOC01-appb-I000241
Figure JPOXMLDOC01-appb-I000242
Figure JPOXMLDOC01-appb-T000171
Figure JPOXMLDOC01-appb-I000242
Figure JPOXMLDOC01-appb-T000171
Figure JPOXMLDOC01-appb-I000243
Figure JPOXMLDOC01-appb-I000243
Figure JPOXMLDOC01-appb-I000244
Figure JPOXMLDOC01-appb-T000172
Figure JPOXMLDOC01-appb-I000244
Figure JPOXMLDOC01-appb-T000172
　上記解析7で笑いにより遺伝子発現の変動が認められた遺伝子のうち、機能面で注目すべき遺伝子、及び特に発現変動の大きかった上位15遺伝子を表173～177に示し、これらを要約した表178を遺伝子セット3の表として示す。
Figure JPOXMLDOC01-appb-T000173
Figure JPOXMLDOC01-appb-T000174
Figure JPOXMLDOC01-appb-T000175
Figure JPOXMLDOC01-appb-T000176
 Table 173 to 177 show the genes that should be noted in terms of function and the top 15 genes with the most significant fluctuation in expression among the genes whose fluctuations in gene expression were recognized by laughter in Analysis 7 above. Is shown as a table of gene set 3.
Figure JPOXMLDOC01-appb-T000173
Figure JPOXMLDOC01-appb-T000174
Figure JPOXMLDOC01-appb-T000175
Figure JPOXMLDOC01-appb-T000176
Figure JPOXMLDOC01-appb-I000245
Figure JPOXMLDOC01-appb-I000245
Figure JPOXMLDOC01-appb-I000246
Figure JPOXMLDOC01-appb-T000177
Figure JPOXMLDOC01-appb-I000246
Figure JPOXMLDOC01-appb-T000177
Figure JPOXMLDOC01-appb-I000247
Figure JPOXMLDOC01-appb-I000247
Figure JPOXMLDOC01-appb-I000248
Figure JPOXMLDOC01-appb-T000178
Figure JPOXMLDOC01-appb-I000248
Figure JPOXMLDOC01-appb-T000178
　上記解析8で笑いにより遺伝子発現の変動が認められた遺伝子のうち、機能面で注目すべき遺伝子、及び特に発現変動の大きかった上位15遺伝子を表179～184に示し、これらを要約した表185を遺伝子セット4の表として示す。
Figure JPOXMLDOC01-appb-T000179
Figure JPOXMLDOC01-appb-T000180
Figure JPOXMLDOC01-appb-T000181
Figure JPOXMLDOC01-appb-T000182
Figure JPOXMLDOC01-appb-T000183
 Table 179 to 184 show the genes that should be noted in terms of function, and the top 15 genes that have the most significant fluctuations in expression among the genes whose fluctuations in gene expression were recognized by laughter in Analysis 8 above. Is shown as a table of gene set 4.
Figure JPOXMLDOC01-appb-T000179
Figure JPOXMLDOC01-appb-T000180
Figure JPOXMLDOC01-appb-T000181
Figure JPOXMLDOC01-appb-T000182
Figure JPOXMLDOC01-appb-T000183
Figure JPOXMLDOC01-appb-I000249
Figure JPOXMLDOC01-appb-I000249
Figure JPOXMLDOC01-appb-I000250
Figure JPOXMLDOC01-appb-I000250
Figure JPOXMLDOC01-appb-I000251
Figure JPOXMLDOC01-appb-T000184
Figure JPOXMLDOC01-appb-I000251
Figure JPOXMLDOC01-appb-T000184
Figure JPOXMLDOC01-appb-I000252
Figure JPOXMLDOC01-appb-I000252
Figure JPOXMLDOC01-appb-I000253
Figure JPOXMLDOC01-appb-T000185
Figure JPOXMLDOC01-appb-I000253
Figure JPOXMLDOC01-appb-T000185
　上記解析9で笑いにより遺伝子発現の変動が認められた遺伝子のうち、機能面で注目すべき遺伝子、及び特に発現変動の大きかった上位15遺伝子を表186～189に示し、これらを要約した表190を遺伝子セット7の表として示す。
Figure JPOXMLDOC01-appb-T000186
Figure JPOXMLDOC01-appb-T000187
Figure JPOXMLDOC01-appb-T000188
 Of the genes whose variation in gene expression was recognized by laughter in Analysis 9 above, the genes that should be noted in terms of function and the top 15 genes that were particularly large in variation in expression are shown in Tables 186 to 189, and these are summarized in Table 190. Is shown as a table in gene set 7.
Figure JPOXMLDOC01-appb-T000186
Figure JPOXMLDOC01-appb-T000187
Figure JPOXMLDOC01-appb-T000188
Figure JPOXMLDOC01-appb-I000254
Figure JPOXMLDOC01-appb-I000254
Figure JPOXMLDOC01-appb-I000255
Figure JPOXMLDOC01-appb-T000189
Figure JPOXMLDOC01-appb-I000255
Figure JPOXMLDOC01-appb-T000189
Figure JPOXMLDOC01-appb-I000256
Figure JPOXMLDOC01-appb-I000256
Figure JPOXMLDOC01-appb-I000257
Figure JPOXMLDOC01-appb-T000190
Figure JPOXMLDOC01-appb-I000257
Figure JPOXMLDOC01-appb-T000190
　上記解析10で笑いにより遺伝子発現の変動が認められた遺伝子のうち、機能面で注目すべき遺伝子、及び特に発現変動の大きかった上位15遺伝子を表191～197に示し、これらを要約した表198を遺伝子セット8の表として示す。
Figure JPOXMLDOC01-appb-T000191
 Of the genes whose variation in gene expression was recognized by laughter in Analysis 10 above, the genes that should be noted in terms of function and the top 15 genes with the greatest variation in expression are shown in Tables 191 to 197, and these are summarized in Table 198. Is shown as a table for gene set 8.
Figure JPOXMLDOC01-appb-T000191
Figure JPOXMLDOC01-appb-I000258
Figure JPOXMLDOC01-appb-I000258
Figure JPOXMLDOC01-appb-I000259
Figure JPOXMLDOC01-appb-T000192
Figure JPOXMLDOC01-appb-I000259
Figure JPOXMLDOC01-appb-T000192
Figure JPOXMLDOC01-appb-I000260
Figure JPOXMLDOC01-appb-I000260
Figure JPOXMLDOC01-appb-I000261
Figure JPOXMLDOC01-appb-I000261
Figure JPOXMLDOC01-appb-I000262
Figure JPOXMLDOC01-appb-T000193
Figure JPOXMLDOC01-appb-T000194
Figure JPOXMLDOC01-appb-T000195
Figure JPOXMLDOC01-appb-T000196
Figure JPOXMLDOC01-appb-I000262
Figure JPOXMLDOC01-appb-T000193
Figure JPOXMLDOC01-appb-T000194
Figure JPOXMLDOC01-appb-T000195
Figure JPOXMLDOC01-appb-T000196
Figure JPOXMLDOC01-appb-I000263
Figure JPOXMLDOC01-appb-I000263
Figure JPOXMLDOC01-appb-I000264
Figure JPOXMLDOC01-appb-T000197
Figure JPOXMLDOC01-appb-I000264
Figure JPOXMLDOC01-appb-T000197
Figure JPOXMLDOC01-appb-I000265
Figure JPOXMLDOC01-appb-I000265
Figure JPOXMLDOC01-appb-I000266
Figure JPOXMLDOC01-appb-T000198
Figure JPOXMLDOC01-appb-I000266
Figure JPOXMLDOC01-appb-T000198
Figure JPOXMLDOC01-appb-I000267
Figure JPOXMLDOC01-appb-I000267
4-2．陽性刺激で発現変化するラット／ヒト間で共通する遺伝子（群）の特定
4-2-1．解析法
　ラットを対象とした陽性刺激モデル実験・短期刺激(上記2-2)時と、ヒトを対象とした「笑いと健康Part4」の末梢血液における遺伝子発現解析で得られたデータの比較解析を行った。 4-2. Identification of common gene (s) between rats / humans whose expression changes with positive stimulation
4-2-1. Analysis method Comparison analysis of data obtained by gene expression analysis in the peripheral blood of positive stimulation model experiment / short-term stimulation (2-2 above) for rats and “Laughter and Health Part 4” for humans went.
4-2-2．結果・考察
　解析2、4～10において、ラット短期陽性刺激(Tickling刺激)負荷時と同方向に発現変化した遺伝子（1.5倍以上、1/1.5倍以下）、逆方向に発現変化した遺伝子（1.5倍以上、1/1.5倍以下）を表199に示す。
Figure JPOXMLDOC01-appb-T000199
 4-2-2. Results / Discussion In Analysis 2 and 4-10, genes that changed in the same direction (1.5 times or more, 1 / 1.5 times or less) in the same direction as when rats were loaded with short-term positive stimulation (Tickling stimulation), genes that changed expression in the opposite direction (1.5 199 times or more and 1 / 1.5 times or less).
Figure JPOXMLDOC01-appb-T000199
Figure JPOXMLDOC01-appb-I000268
Figure JPOXMLDOC01-appb-I000268
　また表200及び201には、ラット短期陽性刺激(Tickling刺激)負荷時と同方向に発現変化したヒト遺伝子(ヒト共通遺伝子)40種のアノテーションを示し、表202にはその要約を示す。
Figure JPOXMLDOC01-appb-T000200
 Tables 200 and 201 show 40 types of annotations of human genes (human common genes) whose expression changes in the same direction as when rats were loaded with a short-term positive stimulus (Tickling stimulus), and Table 202 shows a summary thereof.
Figure JPOXMLDOC01-appb-T000200
Figure JPOXMLDOC01-appb-I000269
Figure JPOXMLDOC01-appb-I000269
Figure JPOXMLDOC01-appb-I000270
Figure JPOXMLDOC01-appb-T000201
Figure JPOXMLDOC01-appb-I000270
Figure JPOXMLDOC01-appb-T000201
Figure JPOXMLDOC01-appb-I000271
Figure JPOXMLDOC01-appb-I000271
Figure JPOXMLDOC01-appb-I000272
Figure JPOXMLDOC01-appb-I000272
Figure JPOXMLDOC01-appb-I000273
Figure JPOXMLDOC01-appb-T000202
Figure JPOXMLDOC01-appb-I000273
Figure JPOXMLDOC01-appb-T000202
　また表203及び204には、ヒトの笑いにより発現変動した遺伝子と同方向に発現変化した短期陽性刺激(Tickling刺激)負荷ラットの遺伝子(ラット共通遺伝子)40種のアノテーションを示し、表205にはその要約を示す。
Figure JPOXMLDOC01-appb-T000203
 Tables 203 and 204 show annotations of 40 types of short-term positive stimulation (tickling stimulation) loaded rat genes (rat common genes) whose expression was changed in the same direction as the genes whose expression was changed by human laughter, and Table 205 Here is a summary.
Figure JPOXMLDOC01-appb-T000203
Figure JPOXMLDOC01-appb-I000274
Figure JPOXMLDOC01-appb-I000274
Figure JPOXMLDOC01-appb-I000275
Figure JPOXMLDOC01-appb-I000275
Figure JPOXMLDOC01-appb-I000276
Figure JPOXMLDOC01-appb-T000204
Figure JPOXMLDOC01-appb-I000276
Figure JPOXMLDOC01-appb-T000204
Figure JPOXMLDOC01-appb-I000277
Figure JPOXMLDOC01-appb-I000277
Figure JPOXMLDOC01-appb-I000278
Figure JPOXMLDOC01-appb-I000278
Figure JPOXMLDOC01-appb-I000279
Figure JPOXMLDOC01-appb-I000279
Figure JPOXMLDOC01-appb-I000280
Figure JPOXMLDOC01-appb-I000280
Figure JPOXMLDOC01-appb-I000281
Figure JPOXMLDOC01-appb-I000281
Figure JPOXMLDOC01-appb-I000282
Figure JPOXMLDOC01-appb-T000205
Figure JPOXMLDOC01-appb-I000282
Figure JPOXMLDOC01-appb-T000205
4-3．総括
　陽性感情で活性化される健康増進遺伝子の特定のために、糖尿病の第6の合併症である歯周病に注目して、笑いによる遺伝子の発現変化を解析した。末梢血白血球による網羅的な遺伝子発現解析を実施し、笑いにより特異的に発現変化する遺伝子群を抽出することができた。また、ラットの短期陽性刺激（Tickling刺激）と、ヒトの「笑い」体験により、共通に発現変化した遺伝子を特定できた。 4-3. In order to identify health-promoting genes that are activated by overall positive emotions, we focused on periodontal disease, the sixth complication of diabetes, and analyzed gene expression changes due to laughter. We conducted a comprehensive gene expression analysis using peripheral blood leukocytes, and extracted genes that specifically change expression due to laughter. In addition, we were able to identify genes whose expression was changed in common by short-term positive stimulation (tickling stimulation) in rats and human “laughing” experience.
　本明細書で引用した全ての刊行物、特許および特許出願をそのまま参考として本明細書にとり入れるものとする。 All publications, patents and patent applications cited in this specification shall be incorporated into the present specification as they are.

Claims (25)

  1. 　ラットに評価対象である刺激を負荷する第1のステップと、
    　下記の表1に示す遺伝子群から選択される少なくとも1種の遺伝子の発現量を指定の生体由来試料において測定する第2のステップと、
    　測定した前記遺伝子の発現量に基づいて、対象刺激が陽性刺激であるか否かを評価する第3のステップと
    を含む、所与の刺激がラットに対する陽性刺激であるか否かを評価する方法。
    Figure JPOXMLDOC01-appb-T000206
    Figure JPOXMLDOC01-appb-I000283
    [表中、
    Bcは口腔内細胞であり；Blは血液であり；Hyは視床下部であり；Stは線条体である。]     A first step of loading a rat with a stimulus to be evaluated;
    A second step of measuring the expression level of at least one gene selected from the gene group shown in Table 1 below in a specified biological sample;
    A third step of evaluating whether the target stimulus is a positive stimulus based on the measured expression level of the gene, and a method for evaluating whether the given stimulus is a positive stimulus for the rat .
    Figure JPOXMLDOC01-appb-T000206
    Figure JPOXMLDOC01-appb-I000283
    [In the table,
    Bc is an oral cell; Bl is blood; Hy is the hypothalamus; St is the striatum. ]
  2. 　ラットに評価対象である刺激を負荷する第1のステップと、
    　神経ペプチドホルモン活性を有する表81に示す遺伝子群、血圧調節に関連する表82に示す遺伝子群、食行動に関連する表83に示す遺伝子群、生体リズムに関連する表84に示す遺伝子群、からなる群から選択される少なくとも1種の遺伝子の発現量を指定の生体由来試料において測定する第2のステップと、
    　測定した前記遺伝子の発現量に基づいて、対象刺激が陽性刺激であるか否かを評価する第3のステップと
    を含む、所与の刺激がラットに対する陽性刺激であるか否かを評価する方法。
    Figure JPOXMLDOC01-appb-T000207
    Figure JPOXMLDOC01-appb-I000284
    Figure JPOXMLDOC01-appb-T000208
    Figure JPOXMLDOC01-appb-I000285
    Figure JPOXMLDOC01-appb-I000286
    Figure JPOXMLDOC01-appb-T000209
    Figure JPOXMLDOC01-appb-T000210
         A first step of loading a rat with a stimulus to be evaluated;
    From the gene group shown in Table 81 having neuropeptide hormone activity, the gene group shown in Table 82 related to blood pressure regulation, the gene group shown in Table 83 related to eating behavior, the gene group shown in Table 84 related to biological rhythm, from A second step of measuring the expression level of at least one gene selected from the group consisting of a specified biological sample;
    A third step of evaluating whether the target stimulus is a positive stimulus based on the measured expression level of the gene, and a method for evaluating whether the given stimulus is a positive stimulus for the rat .
    Figure JPOXMLDOC01-appb-T000207
    Figure JPOXMLDOC01-appb-I000284
    Figure JPOXMLDOC01-appb-T000208
    Figure JPOXMLDOC01-appb-I000285
    Figure JPOXMLDOC01-appb-I000286
    Figure JPOXMLDOC01-appb-T000209
    Figure JPOXMLDOC01-appb-T000210
  3. 　ラットに評価対象である刺激を負荷する第1のステップと、
    　下記の表2に示す遺伝子群から選択される少なくとも1種の遺伝子の発現量を指定の生体由来試料において測定する第2のステップと、
    　測定した前記遺伝子の発現量に基づいて、対象刺激が陽性刺激であるか否かを評価する第3のステップと
    を含む、所与の刺激がラットに対する陽性刺激であるか否かを評価する方法。
    Figure JPOXMLDOC01-appb-T000211
    Figure JPOXMLDOC01-appb-I000287
    Figure JPOXMLDOC01-appb-I000288
    Figure JPOXMLDOC01-appb-I000289
    Figure JPOXMLDOC01-appb-I000290
    Figure JPOXMLDOC01-appb-I000291
    Figure JPOXMLDOC01-appb-I000292
    Figure JPOXMLDOC01-appb-I000293
    Figure JPOXMLDOC01-appb-I000294
    [表中、
    Sgは唾液腺であり；Blは血液であり；Hyは視床下部であり；Stは線条体である。]     A first step of loading a rat with a stimulus to be evaluated;
    A second step of measuring the expression level of at least one gene selected from the gene group shown in Table 2 below in a specified biological sample;
    A third step of evaluating whether the target stimulus is a positive stimulus based on the measured expression level of the gene, and a method for evaluating whether the given stimulus is a positive stimulus for the rat .
    Figure JPOXMLDOC01-appb-T000211
    Figure JPOXMLDOC01-appb-I000287
    Figure JPOXMLDOC01-appb-I000288
    Figure JPOXMLDOC01-appb-I000289
    Figure JPOXMLDOC01-appb-I000290
    Figure JPOXMLDOC01-appb-I000291
    Figure JPOXMLDOC01-appb-I000292
    Figure JPOXMLDOC01-appb-I000293
    Figure JPOXMLDOC01-appb-I000294
    [In the table,
    Sg is the salivary gland; Bl is blood; Hy is the hypothalamus; St is the striatum. ]
  4. 　ラットに評価対象である刺激を負荷する第1のステップと、
    　MAPキナーゼ脱リン酸化酵素活性を有する表45に示す遺伝子群、組織カリクレイン活性を有する表46に示す遺伝子群、細胞外成分である表47および表51に示す遺伝子群、脂質代謝に関連する表48に示す遺伝子群、ドーパミン合成に関連する表49に示す遺伝子群、アクチン結合に関連する表50に示す遺伝子群、核酸構成糖の代謝に関連する表52に示す遺伝子群、ホルモン活性を有する表53に示す遺伝子群、モノアミントランスポーターに関連する表54に示す遺伝子群、受容体結合に関連する表55に示す遺伝子群、食行動に関連する表56に示す遺伝子群、生体アミン合成に関連する表57に示す遺伝子群、生体アミン代謝に関連する表58に示す遺伝子群、陽イオン結合に関連する表59に示す遺伝子群からなる群から選択される少なくとも1種の遺伝子の発現量を指定の生体由来試料において測定する第2のステップと、
    　測定した前記遺伝子の発現量に基づいて、対象刺激が陽性刺激であるか否かを評価する第3のステップと
    を含む、所与の刺激がラットに対する陽性刺激であるか否かを評価する方法。
    Figure JPOXMLDOC01-appb-T000212
    Figure JPOXMLDOC01-appb-T000213
    Figure JPOXMLDOC01-appb-I000295
    Figure JPOXMLDOC01-appb-T000214
    Figure JPOXMLDOC01-appb-I000296
    Figure JPOXMLDOC01-appb-I000297
    Figure JPOXMLDOC01-appb-I000298
    Figure JPOXMLDOC01-appb-I000299
    Figure JPOXMLDOC01-appb-I000300
    Figure JPOXMLDOC01-appb-I000301
    Figure JPOXMLDOC01-appb-I000302
    Figure JPOXMLDOC01-appb-I000303
    Figure JPOXMLDOC01-appb-I000304
    Figure JPOXMLDOC01-appb-I000305
    Figure JPOXMLDOC01-appb-I000306
    Figure JPOXMLDOC01-appb-I000307
    Figure JPOXMLDOC01-appb-I000308
    Figure JPOXMLDOC01-appb-I000309
    Figure JPOXMLDOC01-appb-I000310
    Figure JPOXMLDOC01-appb-I000311
    Figure JPOXMLDOC01-appb-I000312
    Figure JPOXMLDOC01-appb-T000215
    Figure JPOXMLDOC01-appb-I000313
    Figure JPOXMLDOC01-appb-I000314
    Figure JPOXMLDOC01-appb-T000216
    Figure JPOXMLDOC01-appb-T000217
    Figure JPOXMLDOC01-appb-I000315
    Figure JPOXMLDOC01-appb-I000316
    Figure JPOXMLDOC01-appb-I000317
    Figure JPOXMLDOC01-appb-T000218
    Figure JPOXMLDOC01-appb-I000318
    Figure JPOXMLDOC01-appb-I000319
    Figure JPOXMLDOC01-appb-I000320
    Figure JPOXMLDOC01-appb-I000321
    Figure JPOXMLDOC01-appb-I000322
    Figure JPOXMLDOC01-appb-I000323
    Figure JPOXMLDOC01-appb-I000324
    Figure JPOXMLDOC01-appb-I000325
    Figure JPOXMLDOC01-appb-I000326
    Figure JPOXMLDOC01-appb-I000327
    Figure JPOXMLDOC01-appb-I000328
    Figure JPOXMLDOC01-appb-I000329
    Figure JPOXMLDOC01-appb-I000330
    Figure JPOXMLDOC01-appb-I000331
    Figure JPOXMLDOC01-appb-T000219
    Figure JPOXMLDOC01-appb-T000220
    Figure JPOXMLDOC01-appb-T000221
    Figure JPOXMLDOC01-appb-T000222
    Figure JPOXMLDOC01-appb-T000223
    Figure JPOXMLDOC01-appb-I000332
    Figure JPOXMLDOC01-appb-T000224
    Figure JPOXMLDOC01-appb-T000225
    Figure JPOXMLDOC01-appb-T000226
    Figure JPOXMLDOC01-appb-I000333
    Figure JPOXMLDOC01-appb-I000334
    Figure JPOXMLDOC01-appb-I000335
    Figure JPOXMLDOC01-appb-I000336
    Figure JPOXMLDOC01-appb-I000337
    Figure JPOXMLDOC01-appb-I000338
    Figure JPOXMLDOC01-appb-I000339
    Figure JPOXMLDOC01-appb-I000340
    Figure JPOXMLDOC01-appb-I000341
    A first step of loading a rat with a stimulus to be evaluated;
    Gene group shown in Table 45 having MAP kinase phosphatase activity, Gene group shown in Table 46 having tissue kallikrein activity, Gene group shown in Table 47 and Table 51 which are extracellular components, Table 48 related to lipid metabolism Gene group shown in Table 49 related to dopamine synthesis, gene group shown in Table 50 related to actin binding, gene group shown in Table 52 related to metabolism of nucleic acid constituent sugar, table 53 having hormone activity The gene group shown in Table 54, the gene group shown in Table 54 related to monoamine transporter, the gene group shown in Table 55 related to receptor binding, the gene group shown in Table 56 related to eating behavior, the table related to biogenic amine synthesis Specifies the expression level of at least one gene selected from the group consisting of the gene group shown in 57, the gene group shown in Table 58 related to biogenic amine metabolism, and the gene group shown in Table 59 related to cation binding. A second step of measuring in a constant biological sample;
    A third step of evaluating whether the target stimulus is a positive stimulus based on the measured expression level of the gene, and a method for evaluating whether the given stimulus is a positive stimulus for the rat .
    Figure JPOXMLDOC01-appb-T000212
    Figure JPOXMLDOC01-appb-T000213
    Figure JPOXMLDOC01-appb-I000295
    Figure JPOXMLDOC01-appb-T000214
    Figure JPOXMLDOC01-appb-I000296
    Figure JPOXMLDOC01-appb-I000297
    Figure JPOXMLDOC01-appb-I000298
    Figure JPOXMLDOC01-appb-I000299
    Figure JPOXMLDOC01-appb-I000300
    Figure JPOXMLDOC01-appb-I000301
    Figure JPOXMLDOC01-appb-I000302
    Figure JPOXMLDOC01-appb-I000303
    Figure JPOXMLDOC01-appb-I000304
    Figure JPOXMLDOC01-appb-I000305
    Figure JPOXMLDOC01-appb-I000306
    Figure JPOXMLDOC01-appb-I000307
    Figure JPOXMLDOC01-appb-I000308
    Figure JPOXMLDOC01-appb-I000309
    Figure JPOXMLDOC01-appb-I000310
    Figure JPOXMLDOC01-appb-I000311
    Figure JPOXMLDOC01-appb-I000312
    Figure JPOXMLDOC01-appb-T000215
    Figure JPOXMLDOC01-appb-I000313
    Figure JPOXMLDOC01-appb-I000314
    Figure JPOXMLDOC01-appb-T000216
    Figure JPOXMLDOC01-appb-T000217
    Figure JPOXMLDOC01-appb-I000315
    Figure JPOXMLDOC01-appb-I000316
    Figure JPOXMLDOC01-appb-I000317
    Figure JPOXMLDOC01-appb-T000218
    Figure JPOXMLDOC01-appb-I000318
    Figure JPOXMLDOC01-appb-I000319
    Figure JPOXMLDOC01-appb-I000320
    Figure JPOXMLDOC01-appb-I000321
    Figure JPOXMLDOC01-appb-I000322
    Figure JPOXMLDOC01-appb-I000323
    Figure JPOXMLDOC01-appb-I000324
    Figure JPOXMLDOC01-appb-I000325
    Figure JPOXMLDOC01-appb-I000326
    Figure JPOXMLDOC01-appb-I000327
    Figure JPOXMLDOC01-appb-I000328
    Figure JPOXMLDOC01-appb-I000329
    Figure JPOXMLDOC01-appb-I000330
    Figure JPOXMLDOC01-appb-I000331
    Figure JPOXMLDOC01-appb-T000219
    Figure JPOXMLDOC01-appb-T000220
    Figure JPOXMLDOC01-appb-T000221
    Figure JPOXMLDOC01-appb-T000222
    Figure JPOXMLDOC01-appb-T000223
    Figure JPOXMLDOC01-appb-I000332
    Figure JPOXMLDOC01-appb-T000224
    Figure JPOXMLDOC01-appb-T000225
    Figure JPOXMLDOC01-appb-T000226
    Figure JPOXMLDOC01-appb-I000333
    Figure JPOXMLDOC01-appb-I000334
    Figure JPOXMLDOC01-appb-I000335
    Figure JPOXMLDOC01-appb-I000336
    Figure JPOXMLDOC01-appb-I000337
    Figure JPOXMLDOC01-appb-I000338
    Figure JPOXMLDOC01-appb-I000339
    Figure JPOXMLDOC01-appb-I000340
    Figure JPOXMLDOC01-appb-I000341
  5. 　第1のステップにおいて、評価対象である刺激を断続的に2分以上、又は一日当たり断続的に5分以上の刺激を1日以上で負荷することを特徴とする請求項1～4のいずれか1項記載の方法。 5. The method according to any one of claims 1 to 4, wherein in the first step, the stimulus to be evaluated is intermittently loaded for 2 minutes or longer, or intermittently for 5 minutes or longer per day for 1 day or longer. The method according to 1.
  6. 　前記第2のステップでは、測定対象の遺伝子の塩基配列の少なくとも一部を含む核酸を固定化した支持体を用いて、当該遺伝子の発現量を測定することを特徴とする請求項1～5のいずれか1項記載の方法。 6. In the second step, the expression level of the gene is measured using a support on which a nucleic acid containing at least a part of the base sequence of the gene to be measured is immobilized. The method according to any one of the above.
  7. 　下記の表1に示す遺伝子の塩基配列の少なくとも一部を含む核酸からなる群から選ばれる少なくとも1種の核酸、又は下記の表1に示す遺伝子によってコードされるタンパク質に対して特異的に結合する分子からなる群から選択される少なくとも1種の分子を含む、ラットの陽性感情を評価するための検査キット。
    Figure JPOXMLDOC01-appb-T000227
    Figure JPOXMLDOC01-appb-I000342
    [表中、
    Bcは口腔内細胞であり；Blは血液であり；Hyは視床下部であり；Stは線条体である。]     It specifically binds to at least one nucleic acid selected from the group consisting of nucleic acids containing at least part of the base sequence of the gene shown in Table 1 below, or a protein encoded by the gene shown in Table 1 below. A test kit for evaluating a rat's positive emotion, comprising at least one molecule selected from the group consisting of molecules.
    Figure JPOXMLDOC01-appb-T000227
    Figure JPOXMLDOC01-appb-I000342
    [In the table,
    Bc is an oral cell; Bl is blood; Hy is the hypothalamus; St is the striatum. ]
  8. 　下記の表2に示す遺伝子の塩基配列の少なくとも一部を含む核酸からなる群から選ばれる少なくとも1種の核酸、又は下記の表2に示す遺伝子によってコードされるタンパク質に対して特異的に結合する分子からなる群から選択される少なくとも1種の分子を含む、ラットの陽性感情を評価するための検査キット。
    Figure JPOXMLDOC01-appb-T000228
    Figure JPOXMLDOC01-appb-I000343
    Figure JPOXMLDOC01-appb-I000344
    Figure JPOXMLDOC01-appb-I000345
    Figure JPOXMLDOC01-appb-I000346
    Figure JPOXMLDOC01-appb-I000347
    Figure JPOXMLDOC01-appb-I000348
    Figure JPOXMLDOC01-appb-I000350
    [表中、
    Sgは唾液腺であり；Blは血液であり；Hyは視床下部であり；Stは線条体である。]     Specifically binds to at least one nucleic acid selected from the group consisting of nucleic acids containing at least part of the base sequence of the gene shown in Table 2 below, or to a protein encoded by the gene shown in Table 2 below. A test kit for evaluating a rat's positive emotion, comprising at least one molecule selected from the group consisting of molecules.
    Figure JPOXMLDOC01-appb-T000228
    Figure JPOXMLDOC01-appb-I000343
    Figure JPOXMLDOC01-appb-I000344
    Figure JPOXMLDOC01-appb-I000345
    Figure JPOXMLDOC01-appb-I000346
    Figure JPOXMLDOC01-appb-I000347
    Figure JPOXMLDOC01-appb-I000348
    Figure JPOXMLDOC01-appb-I000350
    [In the table,
    Sg is the salivary gland; Bl is blood; Hy is the hypothalamus; St is the striatum. ]
  9. 　下記の表81～84に示す遺伝子の塩基配列の少なくとも一部を含む核酸からなる群から選ばれる少なくとも1種の核酸、又は下記の表1に示す遺伝子によってコードされるタンパク質に対して特異的に結合する分子からなる群から選択される少なくとも1種の分子を含む、ラットの陽性感情を評価するための検査キット。
    Figure JPOXMLDOC01-appb-T000229
    Figure JPOXMLDOC01-appb-I000351
    Figure JPOXMLDOC01-appb-T000230
    Figure JPOXMLDOC01-appb-I000352
    Figure JPOXMLDOC01-appb-I000353
    Figure JPOXMLDOC01-appb-T000231
    Figure JPOXMLDOC01-appb-T000232
         Specific to at least one nucleic acid selected from the group consisting of nucleic acids containing at least part of the base sequences of the genes shown in Tables 81 to 84 below, or to proteins encoded by the genes shown in Table 1 below A test kit for evaluating the positive emotion of a rat, comprising at least one molecule selected from the group consisting of molecules that bind.
    Figure JPOXMLDOC01-appb-T000229
    Figure JPOXMLDOC01-appb-I000351
    Figure JPOXMLDOC01-appb-T000230
    Figure JPOXMLDOC01-appb-I000352
    Figure JPOXMLDOC01-appb-I000353
    Figure JPOXMLDOC01-appb-T000231
    Figure JPOXMLDOC01-appb-T000232
  10. 　下記の表45～59に示す遺伝子の塩基配列の少なくとも一部を含む核酸からなる群から選ばれる少なくとも1種の核酸、又は下記の表1に示す遺伝子によってコードされるタンパク質に対して特異的に結合する分子からなる群から選択される少なくとも1種の分子を含む、ラットの陽性感情を評価するための検査キット。
    Figure JPOXMLDOC01-appb-T000233
    Figure JPOXMLDOC01-appb-T000234
    Figure JPOXMLDOC01-appb-I000354
    Figure JPOXMLDOC01-appb-T000235
    Figure JPOXMLDOC01-appb-I000355
    Figure JPOXMLDOC01-appb-I000356
    Figure JPOXMLDOC01-appb-I000357
    Figure JPOXMLDOC01-appb-I000358
    Figure JPOXMLDOC01-appb-I000359
    Figure JPOXMLDOC01-appb-I000360
    Figure JPOXMLDOC01-appb-I000361
    Figure JPOXMLDOC01-appb-I000362
    Figure JPOXMLDOC01-appb-I000363
    Figure JPOXMLDOC01-appb-I000364
    Figure JPOXMLDOC01-appb-I000365
    Figure JPOXMLDOC01-appb-I000366
    Figure JPOXMLDOC01-appb-I000367
    Figure JPOXMLDOC01-appb-I000368
    Figure JPOXMLDOC01-appb-I000369
    Figure JPOXMLDOC01-appb-I000370
    Figure JPOXMLDOC01-appb-I000371
    Figure JPOXMLDOC01-appb-T000236
    Figure JPOXMLDOC01-appb-I000372
    Figure JPOXMLDOC01-appb-I000373
    Figure JPOXMLDOC01-appb-T000237
    Figure JPOXMLDOC01-appb-T000238
    Figure JPOXMLDOC01-appb-I000374
    Figure JPOXMLDOC01-appb-I000375
    Figure JPOXMLDOC01-appb-I000376
    Figure JPOXMLDOC01-appb-T000239
    Figure JPOXMLDOC01-appb-I000377
    Figure JPOXMLDOC01-appb-I000378
    Figure JPOXMLDOC01-appb-I000379
    Figure JPOXMLDOC01-appb-I000380
    Figure JPOXMLDOC01-appb-I000381
    Figure JPOXMLDOC01-appb-I000382
    Figure JPOXMLDOC01-appb-I000383
    Figure JPOXMLDOC01-appb-I000384
    Figure JPOXMLDOC01-appb-I000385
    Figure JPOXMLDOC01-appb-I000386
    Figure JPOXMLDOC01-appb-I000387
    Figure JPOXMLDOC01-appb-I000388
    Figure JPOXMLDOC01-appb-I000389
    Figure JPOXMLDOC01-appb-I000390
    Figure JPOXMLDOC01-appb-T000240
    Figure JPOXMLDOC01-appb-T000241
    Figure JPOXMLDOC01-appb-T000242
    Figure JPOXMLDOC01-appb-T000243
    Figure JPOXMLDOC01-appb-T000244
    Figure JPOXMLDOC01-appb-I000391
    Figure JPOXMLDOC01-appb-T000245
    Figure JPOXMLDOC01-appb-T000246
    Figure JPOXMLDOC01-appb-T000247
    Figure JPOXMLDOC01-appb-I000392
    Figure JPOXMLDOC01-appb-I000393
    Figure JPOXMLDOC01-appb-I000394
    Figure JPOXMLDOC01-appb-I000395
    Figure JPOXMLDOC01-appb-I000396
    Figure JPOXMLDOC01-appb-I000397
    Figure JPOXMLDOC01-appb-I000398
    Figure JPOXMLDOC01-appb-I000399
    Figure JPOXMLDOC01-appb-I000400
    Specific to at least one nucleic acid selected from the group consisting of nucleic acids containing at least part of the base sequences of the genes shown in Tables 45 to 59 below, or proteins encoded by the genes shown in Table 1 below A test kit for evaluating the positive emotion of a rat, comprising at least one molecule selected from the group consisting of molecules that bind.
    Figure JPOXMLDOC01-appb-T000233
    Figure JPOXMLDOC01-appb-T000234
    Figure JPOXMLDOC01-appb-I000354
    Figure JPOXMLDOC01-appb-T000235
    Figure JPOXMLDOC01-appb-I000355
    Figure JPOXMLDOC01-appb-I000356
    Figure JPOXMLDOC01-appb-I000357
    Figure JPOXMLDOC01-appb-I000358
    Figure JPOXMLDOC01-appb-I000359
    Figure JPOXMLDOC01-appb-I000360
    Figure JPOXMLDOC01-appb-I000361
    Figure JPOXMLDOC01-appb-I000362
    Figure JPOXMLDOC01-appb-I000363
    Figure JPOXMLDOC01-appb-I000364
    Figure JPOXMLDOC01-appb-I000365
    Figure JPOXMLDOC01-appb-I000366
    Figure JPOXMLDOC01-appb-I000367
    Figure JPOXMLDOC01-appb-I000368
    Figure JPOXMLDOC01-appb-I000369
    Figure JPOXMLDOC01-appb-I000370
    Figure JPOXMLDOC01-appb-I000371
    Figure JPOXMLDOC01-appb-T000236
    Figure JPOXMLDOC01-appb-I000372
    Figure JPOXMLDOC01-appb-I000373
    Figure JPOXMLDOC01-appb-T000237
    Figure JPOXMLDOC01-appb-T000238
    Figure JPOXMLDOC01-appb-I000374
    Figure JPOXMLDOC01-appb-I000375
    Figure JPOXMLDOC01-appb-I000376
    Figure JPOXMLDOC01-appb-T000239
    Figure JPOXMLDOC01-appb-I000377
    Figure JPOXMLDOC01-appb-I000378
    Figure JPOXMLDOC01-appb-I000379
    Figure JPOXMLDOC01-appb-I000380
    Figure JPOXMLDOC01-appb-I000381
    Figure JPOXMLDOC01-appb-I000382
    Figure JPOXMLDOC01-appb-I000383
    Figure JPOXMLDOC01-appb-I000384
    Figure JPOXMLDOC01-appb-I000385
    Figure JPOXMLDOC01-appb-I000386
    Figure JPOXMLDOC01-appb-I000387
    Figure JPOXMLDOC01-appb-I000388
    Figure JPOXMLDOC01-appb-I000389
    Figure JPOXMLDOC01-appb-I000390
    Figure JPOXMLDOC01-appb-T000240
    Figure JPOXMLDOC01-appb-T000241
    Figure JPOXMLDOC01-appb-T000242
    Figure JPOXMLDOC01-appb-T000243
    Figure JPOXMLDOC01-appb-T000244
    Figure JPOXMLDOC01-appb-I000391
    Figure JPOXMLDOC01-appb-T000245
    Figure JPOXMLDOC01-appb-T000246
    Figure JPOXMLDOC01-appb-T000247
    Figure JPOXMLDOC01-appb-I000392
    Figure JPOXMLDOC01-appb-I000393
    Figure JPOXMLDOC01-appb-I000394
    Figure JPOXMLDOC01-appb-I000395
    Figure JPOXMLDOC01-appb-I000396
    Figure JPOXMLDOC01-appb-I000397
    Figure JPOXMLDOC01-appb-I000398
    Figure JPOXMLDOC01-appb-I000399
    Figure JPOXMLDOC01-appb-I000400
  11. 　前記分子は抗体、リガンドタンパク質又は受容体タンパク質であることを特徴とする請求項7～10のいずれか1項記載の検査キット。 The test kit according to any one of claims 7 to 10, wherein the molecule is an antibody, a ligand protein, or a receptor protein.
  12. 　前記核酸又は分子は支持体に固定されていることを特徴とする請求項7～10のいずれか1項記載の検査キット。 The test kit according to any one of claims 7 to 10, wherein the nucleic acid or molecule is fixed to a support.
  13. 　下記の表155、159、164、172、178、185、190、198又は202に示す遺伝子群中の各遺伝子の発現量をヒト生体由来試料において測定する第1のステップと、
    　測定した各遺伝子の発現量に基づいて、ヒトの陽性感情又は健康度を評価する第2のステップと
    を含む、ヒトの陽性感情又は健康度の評価方法。
    Figure JPOXMLDOC01-appb-T000248
    Figure JPOXMLDOC01-appb-T000249
    Figure JPOXMLDOC01-appb-T000250
    Figure JPOXMLDOC01-appb-T000251
    Figure JPOXMLDOC01-appb-T000252
    Figure JPOXMLDOC01-appb-T000253
    Figure JPOXMLDOC01-appb-T000254
    Figure JPOXMLDOC01-appb-T000255
    Figure JPOXMLDOC01-appb-I000401
    Figure JPOXMLDOC01-appb-T000256
         A first step of measuring the expression level of each gene in the gene group shown in Table 155, 159, 164, 172, 178, 185, 190, 198 or 202 in a human biological sample;
    A method for evaluating human positive emotion or health, comprising: a second step of evaluating human positive emotion or health based on the measured expression level of each gene.
    Figure JPOXMLDOC01-appb-T000248
    Figure JPOXMLDOC01-appb-T000249
    Figure JPOXMLDOC01-appb-T000250
    Figure JPOXMLDOC01-appb-T000251
    Figure JPOXMLDOC01-appb-T000252
    Figure JPOXMLDOC01-appb-T000253
    Figure JPOXMLDOC01-appb-T000254
    Figure JPOXMLDOC01-appb-T000255
    Figure JPOXMLDOC01-appb-I000401
    Figure JPOXMLDOC01-appb-T000256
  14. 　ヒト生体由来試料は血液である、請求項13記載の方法。 14. The method according to claim 13, wherein the human biological sample is blood.
  15. 　前記第1のステップでは、測定対象の遺伝子群中の各遺伝子の塩基配列の少なくとも一部を含む核酸を固定化した支持体を用いて、遺伝子の発現量を測定することを特徴とする請求項13記載の方法。 The gene expression level is measured in the first step using a support on which a nucleic acid containing at least a part of the base sequence of each gene in the gene group to be measured is immobilized. 13. The method according to 13.
  16. 　ヒトに評価対象である刺激を負荷する第1のステップと、
    　下記の表155、159、164、172、178、185、190、198又は202に示す遺伝子群中の各遺伝子の発現量をヒト生体由来試料において測定する第2のステップと、
    　測定した各遺伝子の発現量に基づいて、対象刺激が陽性刺激であるか否かを評価する第3のステップと
    を含む、所与の刺激がヒトに対する陽性刺激であるか否かを評価する方法。
    Figure JPOXMLDOC01-appb-T000257
    Figure JPOXMLDOC01-appb-T000258
    Figure JPOXMLDOC01-appb-T000259
    Figure JPOXMLDOC01-appb-T000260
    Figure JPOXMLDOC01-appb-T000261
    Figure JPOXMLDOC01-appb-T000262
    Figure JPOXMLDOC01-appb-T000263
    Figure JPOXMLDOC01-appb-T000264
    Figure JPOXMLDOC01-appb-I000402
    Figure JPOXMLDOC01-appb-T000265
         A first step of loading a human being with a stimulus to be evaluated;
    A second step of measuring the expression level of each gene in the gene group shown in Table 155, 159, 164, 172, 178, 185, 190, 198 or 202 in a human biological sample;
    A third step of evaluating whether the target stimulus is a positive stimulus based on the measured expression level of each gene, and a method for evaluating whether the given stimulus is a positive stimulus for a human .
    Figure JPOXMLDOC01-appb-T000257
    Figure JPOXMLDOC01-appb-T000258
    Figure JPOXMLDOC01-appb-T000259
    Figure JPOXMLDOC01-appb-T000260
    Figure JPOXMLDOC01-appb-T000261
    Figure JPOXMLDOC01-appb-T000262
    Figure JPOXMLDOC01-appb-T000263
    Figure JPOXMLDOC01-appb-T000264
    Figure JPOXMLDOC01-appb-I000402
    Figure JPOXMLDOC01-appb-T000265
  17. 　ヒト生体由来試料は血液である、請求項16記載の方法。 17. The method according to claim 16, wherein the human biological sample is blood.
  18. 　ラットに評価対象である刺激を負荷する第1のステップと、
    　下記の表205に示す遺伝子群中の各遺伝子の発現量を指定の生体由来試料において測定する第2のステップと、
    　測定した前記遺伝子の発現量に基づいて、対象刺激が陽性刺激であるか否かを評価する第3のステップと、
    　対象刺激がラットに対する陽性刺激であると評価された場合に、該対象刺激をヒト陽性刺激であると認定する第4のステップと、
    を含む、所与の刺激がヒトに対する陽性刺激であるか否かを評価する方法。
    Figure JPOXMLDOC01-appb-T000266
         A first step of loading a rat with a stimulus to be evaluated;
    A second step of measuring the expression level of each gene in the gene group shown in Table 205 below in a specified biological sample;
    A third step of evaluating whether the target stimulus is a positive stimulus based on the measured expression level of the gene;
    A fourth step of qualifying the target stimulus as a human positive stimulus if the target stimulus is evaluated as a positive stimulus for the rat;
    A method for assessing whether a given stimulus is a positive stimulus for a human.
    Figure JPOXMLDOC01-appb-T000266
  19. 　ラットに評価対象である刺激を負荷する第1のステップと、
    　下記の表205に示す遺伝子群中の各遺伝子の発現量を指定の生体由来試料において測定する第2のステップと、
    　測定した前記遺伝子の発現量に基づいて、対象刺激が陽性刺激であるか否かを評価する第3のステップと
    を含む、所与の刺激がラットに対する陽性刺激であるか否かを評価する方法。
    Figure JPOXMLDOC01-appb-T000267
         A first step of loading a rat with a stimulus to be evaluated;
    A second step of measuring the expression level of each gene in the gene group shown in Table 205 below in a specified biological sample;
    A third step of evaluating whether the target stimulus is a positive stimulus based on the measured expression level of the gene, and a method for evaluating whether the given stimulus is a positive stimulus for the rat .
    Figure JPOXMLDOC01-appb-T000267
  20. 　前記第2のステップでは、測定対象の遺伝子群中の各遺伝子の塩基配列の少なくとも一部を含む核酸を固定化した支持体を用いて、遺伝子の発現量を測定することを特徴とする請求項16、18又は19記載の方法。 The gene expression level is measured in the second step using a support on which a nucleic acid containing at least a part of the base sequence of each gene in the gene group to be measured is immobilized. The method according to 16, 18 or 19.
  21. 　下記の表155、159、164、172、178、185、190、198又は202に示す遺伝子群中の各遺伝子の塩基配列の少なくとも一部を含む核酸、又は下記の表155、159、164、172、178、185、190、198又は202に示す遺伝子群中の各遺伝子によってコードされるタンパク質に対して特異的に結合する分子を含む、ヒトの陽性感情又は健康度を評価するための検査キット。
    Figure JPOXMLDOC01-appb-T000268
    Figure JPOXMLDOC01-appb-T000269
    Figure JPOXMLDOC01-appb-T000270
    Figure JPOXMLDOC01-appb-T000271
    Figure JPOXMLDOC01-appb-T000272
    Figure JPOXMLDOC01-appb-T000273
    Figure JPOXMLDOC01-appb-T000274
    Figure JPOXMLDOC01-appb-T000275
    Figure JPOXMLDOC01-appb-I000403
    Figure JPOXMLDOC01-appb-T000276
         Nucleic acids comprising at least a part of the base sequence of each gene in the gene group shown in Table 155, 159, 164, 172, 178, 185, 190, 198 or 202 below, or Tables 155, 159, 164, 172 below 178, 185, 190, 198 or 202, a test kit for assessing human positive emotion or health, comprising a molecule that specifically binds to a protein encoded by each gene in the gene group shown in FIG.
    Figure JPOXMLDOC01-appb-T000268
    Figure JPOXMLDOC01-appb-T000269
    Figure JPOXMLDOC01-appb-T000270
    Figure JPOXMLDOC01-appb-T000271
    Figure JPOXMLDOC01-appb-T000272
    Figure JPOXMLDOC01-appb-T000273
    Figure JPOXMLDOC01-appb-T000274
    Figure JPOXMLDOC01-appb-T000275
    Figure JPOXMLDOC01-appb-I000403
    Figure JPOXMLDOC01-appb-T000276
  22. 　下記の表205に示す遺伝子群中の各遺伝子の塩基配列の少なくとも一部を含む核酸、又は下記の表205に示す遺伝子群中の各遺伝子によってコードされるタンパク質に対して特異的に結合する分子を含む、ラットの陽性感情を評価するための検査キット。
    Figure JPOXMLDOC01-appb-T000277
         A molecule that specifically binds to a nucleic acid containing at least a part of the base sequence of each gene in the gene group shown in Table 205 below or a protein encoded by each gene in the gene group shown in Table 205 below A test kit for evaluating positive emotions in rats.
    Figure JPOXMLDOC01-appb-T000277
  23. 　前記分子は抗体、リガンドタンパク質又は受容体タンパク質であることを特徴とする請求項21又は22記載の検査キット。 23. The test kit according to claim 21 or 22, wherein the molecule is an antibody, a ligand protein, or a receptor protein.
  24. 　前記核酸又は分子は支持体に固定されていることを特徴とする請求項21又は22記載の検査キット。 The test kit according to claim 21 or 22, wherein the nucleic acid or molecule is fixed to a support.
  25. 　ラットの生体由来試料中の遺伝子発現量を網羅的に測定する第1のステップと、
    　ラットに陽性刺激を負荷した後の生体由来試料中の遺伝子発現量を網羅的に測定する第2のステップと、
    　第1のステップで測定した結果と第2のステップで測定した結果とを比較し、発現変動が認められた遺伝子を選択する第3のステップと、
    　第3のステップで選択された遺伝子のヒトホモログをヒト陽性感情マーカー遺伝子であると特定する第4のステップと
    を含む、ヒト陽性感情マーカー遺伝子     A first step of comprehensively measuring the gene expression level in a rat biological sample;
    A second step of comprehensively measuring the gene expression level in a biological sample after loading a positive stimulus to a rat;
    Comparing the result measured in the first step with the result measured in the second step, and selecting a gene in which expression variation was observed, and a third step;
    A human positive emotion marker gene comprising: a human homolog of the gene selected in the third step is identified as a human positive emotion marker gene;
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Non-Patent Citations (11)

* Cited by examiner, † Cited by third party
Title
BURGDORF JEFFREY ET AL.: "Tickling induces reward in adolescent rats", PHYSIOLOGY & BEHAVIOR, vol. 72, 2001, pages 167 - 173 *
FRIDLUND J. ALAN ET AL.: "Relations between tickling and humourous laughter preliminary support for the Darwin-Hecker hypothesis", BIOLOGICAL PSYCHOLOGY, vol. 30, 1990, pages 141 - 150 *
HAYASHI KEIKO ET AL.: "Laughter lowered the increase in postprandial blood glucose", DIABETES CARE, vol. 26, no. 5, 2003, pages 1651 - 1652 *
HAYASHI TAKASHI ET AL.: "Laughter modulates prorenin receptor gene expression in patients with type 2 diabetes", JOURNAL OF PSYCHOSOMATIC RESEARCH, vol. 62, 2007, pages 703 - 706 *
HAYASHI TAKASHI ET AL.: "Laughter up-regulates the genes related to NK cell activity in diabetes", BIOMEDICAL RESEARCH, vol. 28, no. 6, 2007, pages 281 - 285 *
HAYASHI TAKASHI ET AL.: "The effects of laughter on post-prandial glucose levels and gene expression in type 2 diabetic patients", LIFE SCIENCES, vol. 85, 2009, pages 185 - 187 *
KAZUO MURAKAMI ET AL.: "Kokoro to Karada no Kankei o Idenshi de Toku", TENRI MEDICAL BULLETIN, vol. 7, no. 1, 2004, pages 1 - 22 *
KAZUO MURAKAMI: "Kagaku Gijutsu no Saizensen o Tenbo suru Idenshi Kogaku no Saisentan wa Ima -Anata no Nemureru Idenshi o Okosu", GIJUTSU TO KEIZAI, no. 450, 2004, pages 12 - 21 *
LU Z. NICK ET AL.: "Selective Regulation of Bone Cell Apoptosis by Translational Isoforms of the Glucocorticoid Receptor", MOL. CEL. BIOL., vol. 27, no. 20, 2007, pages 7143 - 7160 *
PANKSEPP JAAK ET AL.: "Neuroevolutionary sources of laughter and social joy: Modeling primal human laughter in laboratory rats", BEHAVIOURAL BRAIN RESEARCH, vol. 182, 2007, pages 231 - 244 *
WOLBER K. PAUL ET AL.: "The Agilent In Situ-Synthesized Microarray Platform", METHODS IN ENZYMOLOGY, vol. 410, 2006, pages 28 - 57 *

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