WO2021177447A1 - 認知症又は脳機能の判定のためのキット及び方法 - Google Patents

認知症又は脳機能の判定のためのキット及び方法 Download PDF

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WO2021177447A1
WO2021177447A1 PCT/JP2021/008722 JP2021008722W WO2021177447A1 WO 2021177447 A1 WO2021177447 A1 WO 2021177447A1 JP 2021008722 W JP2021008722 W JP 2021008722W WO 2021177447 A1 WO2021177447 A1 WO 2021177447A1
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judgment
base sequence
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absence
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明彦 田口
由紀子 竹内
由佳 沖中
友野　潤
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株式会社カネカ
公益財団法人神戸医療産業都市推進機構
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
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    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
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    • 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
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    • 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
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • 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
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    • 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
    • C12Q1/6813—Hybridisation assays
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor

Definitions

  • the present invention relates to a method for obtaining an index for determining dementia or determining brain function in a subject.
  • the present invention also relates to a kit for determining dementia or determining brain function.
  • the present invention also relates to a method for obtaining an index for determining the degree of aging phenomenon in a subject.
  • the present invention also relates to a kit for determining the degree of aging.
  • Alzheimer's disease has the largest number of patients, and it is reported that it accounts for more than 60% of the total.
  • donepezil hydrochloride (trade name: Aricept, etc.) for Alzheimer-type dementia (improved memory and cognitive impairment and suppressed the progression of the disease)
  • galantamine the same Reminyl
  • rivastigmine the same Ixeron patch, rivastigmine patch.
  • Memantine the same Memary
  • PET positron emission tomography
  • SPECT single photon emission tomography
  • cognitive function tests such as the Hasegawa simple intelligence evaluation scale for discriminating dementia are known, but the cognitive function test is a test used after the patient himself / herself recognizes the progression of symptoms, and is used in the early stage of onset. It cannot be used to judge dementia and brain function. There is also the problem that the results of cognitive function tests are ambiguous.
  • a method for comprehensive analysis of blood microRNAs in subjects has also been developed for predicting the onset of dementia.
  • Patent Documents 1 to 3 can be exemplified as patent documents that disclose a method for determining brain dysfunction based on the gene expression level (biomarker) of a specific protein.
  • Patent Document 1 examines Alzheimer's disease, which comprises detecting in vitro a decrease in the level or function of at least one factor in the insulin / IGF signaling pathway, such as insulin, in a subject-derived central nervous system tissue. The method is described.
  • Patent Document 2 detects phosphorylation of at least one substrate protein such as MARCKS in a subject, and when the degree of phosphorylation is higher than that of a normal sample, the subject suffers from or is at risk of developing Alzheimer's disease. A method for determining that the substance has sex is described.
  • Patent Document 3 describes a biomarker for detecting a cognitive dysfunction disease.
  • the biomarker described in Patent Document 3 is described as a protein that depends on Complement C4, Prothrombin, Complement C3, Gelsolin, and the like.
  • Patent Documents 4 to 6 can be exemplified as patent documents that disclose a method for determining the degree of aging phenomenon based on the gene expression level (biomarker) of a specific protein.
  • Patent Document 4 is a method for determining the degree of aging of the skin, which comprises measuring the expression of secreted proteins and / or intracellular proteins in skin cells and / or skin tissues and / or the gene expression thereof. The above-mentioned method is described in which the expression of a protein and / or an intracellular protein changes depending on the degree of skin aging.
  • the biomarkers described in Patent Document 4 are described as Kallikrein 7, Keratin 7, and the like.
  • Patent Document 5 the degree of aging of cells and / or individuals is evaluated using the expression level of the GREM1 protein and / or the GREM2 protein, or the gene encoding the GREM1 protein and / or the gene encoding the GREM2 protein in a biological sample as an index. How to do it is described.
  • Patent Document 6 describes a method for evaluating the cell senescence state of fibroblasts, which comprises a step of determining the expression level e of the gene product of OLFML2A and / or CRLF1 in a biological sample. ..
  • One or more embodiments of the present invention provide a method and a kit for early determination of dementia by a simple operation, a method and a kit for determining brain function by a simple operation.
  • the purpose is.
  • Another one or more embodiments of the present invention are aimed at providing a method capable of determining the degree of an aging phenomenon at an early stage by a simple operation and a kit for the same.
  • a method for obtaining an index for determining dementia or brain function in a subject comprising measuring the gene expression or enzymatic activity of one or more proteins involved in an energy metabolism reaction in a sample derived from a subject.
  • the method according to (1) wherein the determination of the dementia is a determination of the presence or absence of dementia, a determination of the risk of developing dementia, or a determination of the severity of dementia.
  • the sample is a body fluid sample or a bone marrow sample. It further comprises comparing the measured values of gene expression or enzyme activity of one or more proteins with reference values.
  • a measurement value of gene expression or enzyme activity of one or more proteins, which is higher than the reference value, indicates that the subject suffers from dementia and that the subject has a risk of developing dementia.
  • the method according to (2) which indicates the severity of dementia in the subject.
  • the method according to (3) wherein the ratio of the measured value of gene expression or enzyme activity of one or more proteins to the reference value is 1.1 or more and 50.0 or less.
  • the sample is a central nervous system tissue sample. It further comprises comparing the measured values of gene expression or enzyme activity of one or more proteins with reference values. A measurement of gene expression or enzyme activity of one or more proteins, which is lower than the reference value, indicates that the subject suffers from dementia and that the subject has a risk of developing dementia.
  • the determination of the brain function is as follows: determination of the presence or absence of deterioration of brain function, determination of the risk of deterioration of brain function, determination of the degree of deterioration of brain function, determination of presence or absence of disorder of the autonomic nervous system, autonomic nerve Judgment of risk of system disorder, judgment of degree of disorder of autonomic nervous system, judgment of presence or absence of memory disorder, judgment of risk of memory disorder, judgment of degree of memory disorder, judgment of presence or absence of motor disorder, judgment of movement disorder Judgment of risk of , Judgment of the risk of involuntary movement disorder, judgment of the degree of involuntary movement disorder, judgment of the presence or absence of sensory disorder, judgment of the risk of sensory disorder, judgment of the degree of sensory disorder, presence or absence of visual disorder Judgment, judgment of the risk of visual impairment, judgment of the degree of visual impairment, judgment of the presence or absence of olfactory disorder, judgment of the risk
  • a kit for determining dementia or brain function A kit containing a reagent for measuring gene expression or enzyme activity of one or more proteins involved in an energy metabolism reaction in a sample derived from a subject.
  • the determination of the dementia is a determination of the presence or absence of dementia, a determination of the risk of developing dementia, or a determination of the severity of dementia.
  • the determination of the brain function includes determination of the presence or absence of deterioration of brain function, determination of the risk of deterioration of brain function, determination of the degree of deterioration of brain function, determination of presence or absence of disorder of the autonomic nervous system, and autonomic nerve.
  • Judgment of risk of system disorder judgment of degree of disorder of autonomic nervous system, judgment of presence or absence of memory disorder, judgment of risk of memory disorder, judgment of degree of memory disorder, judgment of presence or absence of motor disorder, judgment of movement disorder Judgment of risk of , Judgment of the risk of involuntary movement disorder, judgment of the degree of involuntary movement disorder, judgment of the presence or absence of sensory disorder, judgment of the risk of sensory disorder, judgment of the degree of sensory disorder, presence or absence of visual disorder Judgment, judgment of the risk of visual impairment, judgment of the degree of visual impairment, judgment of the presence or absence of olfactory disorder, judgment of the risk of olfactory disorder, judgment of the degree of olfactory disorder, presence or absence of hearing disorder Judgment, judgment of the risk of hearing disorder, judgment of the degree of hearing disorder, judgment of the presence or absence of balance disorder, judgment of the risk of balance disorder, judgment of the degree of balance disorder, sleep disorder Judgment of presence / absence, judgment of risk of sleep
  • the reagent is specific to a primer pair for amplifying a nucleic acid containing a gene of one or more proteins, a probe that hybridizes with a nucleic acid containing a gene of one or more proteins, or the protein of one or more.
  • a method comprising measuring the gene expression or enzymatic activity of one or more proteins involved in an energy metabolism reaction in a sample derived from a subject.
  • the determination of the degree of the aging phenomenon is a determination of age, a determination of the degree of aging phenomenon of brain tissue, a determination of the degree of aging phenomenon of peripheral blood, or a determination of the degree of aging phenomenon of bone marrow cells. 13).
  • the method according to (13) or (14), wherein the sample is a body fluid sample, a bone marrow sample, or a central nervous system tissue sample.
  • the one or more proteins are one or more proteins selected from Glut1, Glut3, MCT4, PHD3 and PDK1.
  • a kit for determining the degree of aging in a subject A kit containing a reagent for measuring gene expression or enzyme activity of one or more proteins involved in an energy metabolism reaction in a sample derived from a subject.
  • the determination of the degree of the aging phenomenon is a determination of age, a determination of the degree of aging phenomenon of brain tissue, a determination of the degree of aging phenomenon of peripheral blood, or a determination of the degree of aging phenomenon of bone marrow cells. 17) The kit according to. (19) The kit according to (17) or (18), wherein the sample is a body fluid sample, a bone marrow sample, or a central nervous system tissue sample.
  • the reagent is specific to a primer pair for amplifying a nucleic acid containing a gene of one or more proteins, a probe that hybridizes with a nucleic acid containing a gene of one or more proteins, or the protein of one or more.
  • Measuring the gene expression or enzymatic activity of one or more proteins involved in the energy metabolism reaction in the sample derived from the subject includes (a) and (b) shown below, (1).
  • a method for determining dementia or brain function in a subject To measure the gene expression or enzymatic activity of one or more proteins involved in the energy metabolism reaction in a sample derived from a subject, and A method comprising determining dementia or brain function in a subject based on measurements of gene expression or enzyme activity of one or more proteins.
  • the subject is preferably a human or non-human animal.
  • the sample is preferably a sample isolated from the subject.
  • the method according to (24), wherein the determination of the dementia is a determination of the presence or absence of dementia, a determination of the risk of developing dementia, or a determination of the severity of dementia.
  • the sample is a body fluid sample or a bone marrow sample.
  • the sample is a central nervous system tissue sample. It further comprises comparing the measured values of gene expression or enzyme activity of one or more proteins with reference values.
  • the subject is preferably a human or non-human animal.
  • the sample is preferably a sample isolated from the subject.
  • Measuring the gene expression or enzymatic activity of one or more proteins involved in the energy metabolism reaction in the sample derived from the subject includes (a) and (b) shown below (24).
  • the method according to any one of (33) (A) Obtaining a mononuclear cell-containing sample by separating mononuclear cells from the sample. (B) To measure the gene expression or enzyme activity of one or more proteins in the mononuclear cell-containing sample.
  • a biomarker for determining dementia or brain function which comprises one or more proteins involved in an energy metabolism reaction or a nucleic acid containing a base sequence encoding the amino acid sequence of the one or more proteins.
  • a biomarker for determining the degree of aging phenomenon which comprises one or more proteins involved in an energy metabolism reaction or a nucleic acid containing a base sequence encoding the amino acid sequence of the one or more proteins.
  • the biomarker according to (35) or (36), wherein the one or more proteins are one or more proteins selected from Glut1, Glut3, MCT4, PHD3 and PDK1.
  • the biomarker according to any one of (35) to (37), wherein the nucleic acid is mRNA or cDNA prepared from the mRNA.
  • a kit for determining dementia or determining brain function In the manufacture of a kit for determining dementia or determining brain function. Use of reagents to measure gene expression or enzymatic activity of one or more proteins involved in energy metabolism reactions.
  • the one or more proteins are one or more proteins selected from Glut1, Glut3, MCT4, PHD3 and PDK1.
  • the reagent is specific to a primer pair for amplifying a nucleic acid containing a gene of one or more proteins, a probe that hybridizes with a nucleic acid containing a gene of one or more proteins, or the protein of one or more.
  • the use according to (39) or (40) which is an antibody that binds specifically.
  • reagents to measure gene expression or enzymatic activity of one or more proteins involved in energy metabolism reactions.
  • the reagent is specific to a primer pair for amplifying a nucleic acid containing a gene of one or more proteins, a probe that hybridizes with a nucleic acid containing a gene of one or more proteins, or the protein of one or more.
  • a method for measuring gene expression or enzymatic activity of one or more proteins involved in an energy metabolism reaction in a subject comprising measuring the gene expression or enzymatic activity of one or more proteins involved in an energy metabolism reaction in a sample derived from a subject.
  • the subject is preferably a human or a non-human animal, and more preferably a human.
  • the subject preferably desires or needs to determine a subject suspected of having dementia, a subject suffering from dementia, a subject treated for dementia, and a risk of developing dementia.
  • Subjects to be treated subjects with suspected deterioration of brain function, subjects with decreased brain function, subjects who have been treated for treatment of deterioration of brain function, subjects who desire or need to determine brain function , A subject suspected of having an aging phenomenon, a subject having an aging phenomenon, a subject having been treated to recover from the aging phenomenon, or a subject who desires or needs to determine the degree of the aging phenomenon.
  • the sample is preferably a sample isolated from the subject.
  • the method according to (47), wherein the sample is a body fluid sample, a bone marrow sample, or a central nervous system tissue sample. (49) The method according to (47) or (48), further comprising comparing a measured value of gene expression or enzyme activity of one or more proteins with a reference value.
  • (50) The method according to any one of (47) to (49), wherein the one or more proteins are one or more proteins selected from Glut1, Glut3, MCT4, PHD3 and PDK1.
  • Measuring the gene expression or enzymatic activity of one or more proteins involved in the energy metabolism reaction in the sample derived from the subject includes (a) and (b) shown below, (47).
  • a method for determining dementia or brain function in a subject and improving or treating dementia or brain function in the subject To measure the gene expression or enzymatic activity of one or more proteins involved in the energy metabolism reaction in a sample derived from a subject. Dementia or brain function in the subject is determined based on the measured values of gene expression or enzyme activity of one or more proteins, and A method comprising performing a treatment for improving or treating dementia or brain function with respect to the subject whose result of the determination indicates that it is necessary to improve or treat dementia or brain function.
  • the subject is preferably a human or non-human animal.
  • the sample is preferably a sample isolated from the subject.
  • the method according to (52), wherein the determination of dementia is a determination of the presence or absence of dementia, a determination of the risk of developing dementia, or a determination of the severity of dementia.
  • the sample is a body fluid sample or a bone marrow sample. It further comprises comparing the measured values of gene expression or enzyme activity of one or more proteins with reference values. A measurement value of gene expression or enzyme activity of one or more proteins, which is higher than the reference value, indicates that the subject suffers from dementia and that the subject has a risk of developing dementia. Or, the method according to (53), which indicates the severity of dementia in the subject.
  • the sample is a central nervous system tissue sample. It further comprises comparing the measured values of gene expression or enzyme activity of one or more proteins with reference values. A measurement of gene expression or enzyme activity of one or more proteins, which is lower than the reference value, indicates that the subject suffers from dementia and that the subject has a risk of developing dementia. Or, the method according to (53), which indicates the severity of dementia in the subject. (57) The method according to (52), wherein the determination of the brain function is the determination according to (6).
  • a method for determining the degree of aging phenomenon in a subject and improving or treating the aging phenomenon in the subject To measure the gene expression or enzymatic activity of one or more proteins involved in the energy metabolism reaction in a sample derived from a subject. Based on the measured values of gene expression or enzyme activity of one or more proteins, the degree of aging phenomenon in the subject is determined, and A method comprising performing a treatment for improving or treating the aging phenomenon with respect to the subject whose result of the determination indicates that it is necessary to improve or treat the aging phenomenon.
  • the subject is preferably a human or non-human animal.
  • the sample is preferably a sample isolated from the subject.
  • the method according to (58), wherein the determination of the degree of the aging phenomenon is the determination according to (14).
  • the sample is a body fluid sample, a bone marrow sample, or a central nervous system tissue sample.
  • Measuring the gene expression or enzymatic activity of one or more proteins involved in the energy metabolism reaction in the sample derived from the subject includes (a) and (b) shown below, (52).
  • the method according to any one of (61) (A) Obtaining a mononuclear cell-containing sample by separating mononuclear cells from the sample. (B) To measure the gene expression or enzyme activity of one or more proteins in the mononuclear cell-containing sample.
  • dementia can be determined by a simple operation. Early determination of dementia is possible.
  • brain function can be determined by a simple operation.
  • the degree of the aging phenomenon can be determined by a simple operation.
  • FIG. 1 shows the average relative expression level of each target gene with respect to the expression level of the 18S ribosomal RNA gene in the body fluid samples of the mice of Example 1 and Comparative Example 1.
  • FIG. 2 shows the average relative expression level of each target gene with respect to the expression level of the 18S ribosomal RNA gene in the body fluid sample of each mouse of Example 2.
  • FIG. 3 shows the relationship between the average relative expression level of each target gene in the body fluid sample of the mouse of Example 2 and the age of the mouse.
  • FIG. 3A shows the relationship between the average relative expression level of the Glut1 gene in the body fluid sample of the mouse of Example 2 and the age of the mouse.
  • FIG. 1 shows the average relative expression level of each target gene with respect to the expression level of the 18S ribosomal RNA gene in the body fluid samples of the mice of Example 1 and Comparative Example 1.
  • FIG. 2 shows the average relative expression level of each target gene with respect to the expression level of the 18S ribosomal RNA gene in the body fluid sample
  • FIG. 3B shows the relationship between the average relative expression level of the Glut3 gene in the body fluid sample of the mouse of Example 2 and the age of the mouse.
  • FIG. 3C shows the relationship between the average relative expression level of the MCT4 gene in the body fluid sample of the mouse of Example 2 and the age of the mouse.
  • FIG. 3D shows the relationship between the average relative expression level of the PHD3 gene in the body fluid sample of the mouse of Example 2 and the age of the mouse.
  • FIG. 3E shows the relationship between the average relative expression level of the PDK1 gene in the body fluid sample of the mouse of Example 2 and the age of the mouse.
  • Term> ⁇ 1.1.
  • Sample derived from the subject> refers to a human or non-human animal that is the subject of determination of the degree of dementia, brain function, or aging phenomenon.
  • non-human animals include non-human mammals such as primates, rats, mice, gerbils, guinea pigs, hamsters, ferrets, rabbits, cows, horses, pigs, goats, dogs, and cats.
  • body fluid samples such as peripheral blood, saliva, urine, sputum, sweat, pharyngeal swab, and nasal swab, bone marrow sample, and central nervous tissue sample such as brain tissue are used. be able to.
  • a body fluid sample or a bone marrow sample is preferable, and a body fluid sample, particularly a peripheral blood sample, is most preferable.
  • peripheral blood sample various forms of peripheral blood samples such as serum, plasma, and whole blood can be used.
  • a bone marrow fluid sample can be used as the bone marrow sample.
  • the sample derived from the subject is preferably a sample isolated from the subject.
  • the gene expression level or enzyme activity of the protein described later determines the subject suffering from dementia, the subject who may develop dementia in the future, the subject whose brain function is deteriorated, or the subject. , It has been confirmed that in subjects whose brain function may decline in the future, it is higher in body fluids such as peripheral blood or bone marrow than in normal specimens, and although not shown in Examples, brain tissue It has been confirmed that it is low in central nervous tissue such as. Further, as shown in Examples, it has been confirmed that the gene expression level or enzyme activity of the protein described later is higher in body fluids such as peripheral blood or bone marrow in older subjects than in younger specimens. Although not shown in the examples, it has been confirmed to be low in central nervous system tissues such as brain tissue.
  • the gene expression level or enzyme activity of the protein described later is a subject in which the aging phenomenon of brain tissue is observed, a subject in which the aging phenomenon of brain tissue may progress, and aging of peripheral blood.
  • a subject in which the phenomenon is observed a subject in which the aging phenomenon of peripheral blood may progress, a subject in which the aging phenomenon of bone marrow cells is observed, or a subject in which the aging phenomenon of bone marrow cells may progress.
  • body fluids such as peripheral blood or bone marrow as compared with normal samples
  • central nervous tissue such as brain tissue
  • a mononuclear cell-containing sample containing mononuclear cells separated from the body fluid or bone marrow of the subject can be used.
  • MNC mononuclear Cells
  • lymphocytes and / or monocytes lymphocytes and / or monocytes.
  • white blood cell refers to a general term for neutrophils, basophils, eosinophils, lymphocytes and monocytes.
  • the term “dementia” may be any of Alzheimer's disease, vascular dementia, Lewy body dementia, and frontotemporal dementia, but Alzheimer's disease is particularly preferable. be.
  • To determine dementia determine the presence or absence of dementia in the subject, determine the risk of developing dementia in the subject, and determine the severity of dementia in the subject. Etc. are included.
  • Brain function refers to the function of brain tissue involved in memory, movement, sensation, sleep, language, and the like.
  • To determine brain function determine whether or not there is a decrease in brain function in the subject, determine the risk of decrease in brain function in the subject, and determine the degree of decrease in brain function in the subject.
  • To determine the presence or absence of autonomic dysfunction in the subject to determine the risk of autonomic dysfunction in the subject, to determine the degree of autonomic dysfunction in the subject, to determine the degree of autonomic dysfunction in the subject
  • To determine the presence or absence of memory impairment in the subject to determine the risk of memory impairment in the subject, to determine the degree of memory impairment in the subject, to determine the presence or absence of movement disorder in the subject, in the subject
  • To determine the risk of movement disorder to determine the degree of motor disorder in the subject, to determine the presence or absence of coordinated movement disorder in the subject, to determine the risk of coordinated movement disorder in the subject.
  • Determining the degree of impaired cooperative movement in the subject determining the presence or absence of impaired involuntary movement in the subject, determining the risk of impaired involuntary movement in the subject, non-compliance in the subject Judging the degree of voluntary movement disorder, determining the presence or absence of sensory impairment in the subject, determining the risk of sensory impairment in the subject, determining the degree of sensory impairment in the subject, determining the subject To determine the presence or absence of visual impairment in the subject, to determine the risk of visual impairment in the subject, to determine the degree of visual impairment in the subject, to determine the presence or absence of olfactory impairment in the subject.
  • aging phenomenon refers to physical and / or physiological changes associated with aging. Examples of aging phenomena include skin changes such as spots, wrinkles, sagging, and dullness, hair changes such as white hair, thinning hair, and hair loss, changes in brain morphology, changes in brain tissue such as brain atrophy, and changes in peripheral blood. , Changes in bone marrow cells, changes in bone, changes in muscle, etc. can be exemplified.
  • degree of aging phenomenon refers to the degree of physical and / or physiological changes associated with aging. In the present specification, “degree of aging phenomenon” may be described as “degree of aging”.
  • determination of the degree of aging phenomenon refers to determination of the degree of physical and physiological changes associated with aging.
  • To determine the degree of aging phenomenon for example, to determine the age of the subject, to determine the degree of aging phenomenon of brain tissue, to determine the degree of aging phenomenon of peripheral blood, and to determine the degree of aging phenomenon of bone marrow cells. It includes determining the degree of the phenomenon.
  • one or more proteins involved in energy metabolism reactions include glucose transport, lactic acid transport, energy metabolism regulation, mitochondrial biosynthesis, glycolysis, pentose phosphate pathway, TCA cycle, electron transport chain, fatty acid metabolism, and the like.
  • proteins involved in gap binding or sodium-potassium pumps can be exemplified.
  • proteins involved in glucose transport include proteins that constitute membrane proteins (glucose transporters) involved in glucose transport that transport glucose used for energy metabolism in cells from outside the cell to the inside of the cell.
  • proteins belonging to the Glucose transporter family Glucose 1 to 12.
  • gene expression or enzymatic activity of Glut1, Glut2, Glut3 and Glut4, particularly Glut1 and Glut3 is preferable.
  • the nucleotide sequence of mouse Glut1 is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2.
  • the base sequence of human Glut1 is shown in SEQ ID NO: 3, and the amino acid sequence is shown in SEQ ID NO: 4.
  • the nucleotide sequence of mouse Glut3 is shown in SEQ ID NO: 5, and the amino acid sequence is shown in SEQ ID NO: 6.
  • the base sequence of human Glut3 is shown in SEQ ID NO: 7, and the amino acid sequence is shown in SEQ ID NO: 8.
  • the nucleotide sequences of SEQ ID NOs: 1, 3, 5 and 7 all have a nucleotide sequence encoding the amino acid sequence of a protein (CDS) and a nucleotide sequence of an untranslated region (UTR) located upstream and downstream of the CDS. include.
  • a protein constituting a membrane protein (lactic acid transporter) involved in lactic acid transport that transports lactic acid produced by energy metabolism in the cell from the inside of the cell to the outside of the cell can be exemplified.
  • examples include proteins belonging to the MCT (monocarboxylate transport protein) family.
  • MCT1 monocarboxylate transport protein
  • MCT2 monocarboxylate transport protein
  • MCT4 gene expression or enzymatic activity of MCT1, MCT2, MCT3 and MCT4, particularly MCT4, is preferable.
  • the nucleotide sequence of mouse MCT4 is shown in SEQ ID NO: 9, and the amino acid sequence is shown in SEQ ID NO: 10.
  • the nucleotide sequence of human MCT4 is shown in SEQ ID NO: 11, and the amino acid sequence is shown in SEQ ID NO: 12.
  • the nucleotide sequences of SEQ ID NOs: 9 and 11 both include the nucleotide sequence encoding the amino acid sequence of the protein (CDS) and the nucleotide sequence of the untranslated region (UTR) located upstream and downstream of the CDS.
  • proteins involved in the regulation of energy metabolism include proteins belonging to the PHD (procollagen-contining protein) family.
  • PHD procollagen-contining protein
  • gene expression or enzymatic activity of PHD2 and PHD3, particularly PHD3, is preferable.
  • the nucleotide sequence of mouse PHD3 is shown in SEQ ID NO: 13, and the amino acid sequence is shown in SEQ ID NO: 14.
  • the nucleotide sequence of human PHD3 is shown in SEQ ID NO: 15, and the amino acid sequence is shown in SEQ ID NO: 16.
  • the nucleotide sequences of SEQ ID NOs: 13 and 15 both include the nucleotide sequence encoding the amino acid sequence of the protein (CDS) and the nucleotide sequence of the untranslated region (UTR) located upstream and downstream of the CDS.
  • CDS amino acid sequence of the protein
  • UTR untranslated region
  • HIF1a hyperoxia-inducible factoror 1 alpha
  • Sirtuin 1 sirtuin 1
  • PPARa peroxisome proliferator-activated receptor
  • PGC1a peroxisome proliferators-active receptor-ganma co-activator-1 alpha
  • proteins involved in glycolysis include LDHa (lactate dehydrogenase A), LDHb (lactate dehydrogenase B), HK1 (hexokinase 1), PFK (phosphofructase kinase), and PKR. Can also be exemplified.
  • Proteins involved in the pentose phosphate pathway include G6PD (glucose-6-phosphate dehydrange), RPI (ribose-5-phosphate isomerase), RPE (ribose-5-phosphate epimerase), and 6PGLose (6-PGLose. ) Can be exemplified.
  • proteins involved in the TCA cycle include proteins belonging to the PDK (pyruvate dehydogenesis kinase) family.
  • proteins belonging to the PDK family PDK1, PDK3 and PDK4, particularly PDK1 gene expression or enzymatic activity is preferable.
  • the nucleotide sequence of mouse PDK1 is shown in SEQ ID NO: 17, and the amino acid sequence is shown in SEQ ID NO: 18.
  • the nucleotide sequence of human PDK1 is shown in SEQ ID NO: 19, and the amino acid sequence is shown in SEQ ID NO: 20.
  • the nucleotide sequences of SEQ ID NOs: 17 and 19 both include the nucleotide sequence encoding the amino acid sequence of the protein (CDS) and the nucleotide sequence of the untranslated region (UTR) located upstream and downstream of the CDS.
  • CDS amino acid sequence of the protein
  • UTR untranslated region
  • IDH2 isocitrate dehydrogenase 2
  • OGDH oxoglute dehydrogenase
  • CS citrate synthase
  • AMPK AMP-activated protein kinase
  • proteins involved in fatty acid metabolism include FABP1 (fatty acid binding protein 1), FABP4 (fatty acid binding protein 4), CD36 (fatty acid transit protein: FAT), CPT1 (carnitine palmitoyl), and CPT1 (carnitine palmitoyl).
  • -Transphase 1) and ACC protein-CoA fatty acid
  • proteins involved in gap junctions include Cx37 (connexin 37) and Cx43 (connexin 43).
  • Proteins involved in the sodium-potassium pump include ATP1A1 (sodium / potassium-transporting TAPase subunit alpha-1), ATP1A2 (sodium / potassium-transporting TAPase subunitAtropa 3) can be exemplified.
  • One or more embodiments of the present invention It is a method of acquiring an index for determining dementia or brain function in a subject.
  • the present invention relates to a method comprising measuring the gene expression or enzymatic activity of one or more proteins involved in an energy metabolism reaction in a sample derived from a subject.
  • One or more embodiments of the present invention A method for determining dementia or brain function in a subject. To measure the gene expression or enzymatic activity of one or more proteins involved in the energy metabolism reaction in a sample derived from a subject, and The present invention relates to a method comprising determining dementia or brain function in the subject based on the measured values of gene expression or enzyme activity of one or more proteins.
  • the "reference value" refers to the gene expression or enzyme activity of the one or more proteins in a sample derived from a normal sample, and the gene expression or enzyme activity of the one or more proteins in a sample derived from a subject. It may be a measured value when measured under the same conditions or a reference value set from the measured value, or the above-mentioned one or more proteins in a sample derived from a patient whose presence or absence or severity of dementia is known. Even if the gene expression or enzyme activity is measured under the same conditions as the gene expression or enzyme activity of the one or more proteins in the sample derived from the subject, or even if it is a reference value set from the measured value. good.
  • gene expression or enzymatic activity of one or more proteins involved in an energy metabolism response is a subject suffering from dementia, a subject who may develop dementia in the future.
  • Subjects with reduced brain function, or subjects with a possibility of decreased brain function in the future are higher in body fluids such as peripheral blood, urine, saliva, or bone marrow than normal samples, and brain tissue Based on the unexpected finding that it is low in central nervous tissue such as.
  • the gene expression or enzyme activity of one or more proteins involved in the energy metabolism reaction is higher in body fluids such as peripheral blood, urine, saliva or bone marrow as the severity of dementia in the subject is higher, and in brain tissues and the like. Low in central nervous tissue.
  • the gene expression or enzyme activity of one or more proteins involved in the energy metabolism reaction is higher in body fluids such as peripheral blood, urine, saliva or bone marrow as the brain function in the subject is lower, and the central nervous tissue such as brain tissue. Low in. Therefore, in the embodiment of the present invention, when the sample is a body fluid sample or a bone marrow sample, particularly a mononuclear cell-containing sample, the subject recognizes that the measured value is higher than the reference value.
  • the subject has a disease, the subject has a risk of developing dementia, the subject has a higher degree of dementia, and the subject's brain function is impaired.
  • the sample is a central nervous system sample
  • the measured value lower than the reference value means that the subject suffers from dementia.
  • the sample has a risk of developing dementia, the severity of dementia of the subject is higher, the brain function of the subject is deteriorated, and the brain function of the subject is deteriorated. It is an index showing that the subject has sex or that the degree of deterioration of the brain function of the subject is more remarkable.
  • the lower limit of the ratio of the measured value to the reference value is 1.1 or more and 1. .2 or more, 1.3 or more, 1.4 or more, 1.5 or more, 1.6 or more, 1.7 or more, 1.8 or more, 1.9 or more, 2.0 or more, 2.1 or more, 2 .2 or more, 2.3 or more, 2.4 or more, 2.5 or more, 2.6 or more, 2.7 or more, 2.8 or more, 2.9 or more, 3.0 or more, 3.2 or more, 3 4.4 or higher, 3.6 or higher, 3.8 or higher, 4.0 or higher, 4.2 or higher, 4.4 or higher, 4.6 or higher, 4.8 or higher, 5.0 or higher, 5.2 or higher, 5 It is preferably .4 or more, 5.6 or more, 5.8 or more, or 6.0 or more.
  • the upper limit of the ratio of the measured value to the reference value is not particularly limited, but is, for example, 50.0 or less, 45.0. 40.0 or less, 35.0 or less, 30.0 or less, 25.0 or less, 20.0 or less, 15.0 or less, 14.0 or less, 13.0 or less, 12.0 or less, 11.0 Below, it can be 10.0 or less, 9.8 or less, 9.6 or less, 9.4 or less, 9.2 or less, or 9.0 or less.
  • ratios are the same as the gene expression or enzyme activity of the one or more proteins in the sample derived from the normal sample, and the gene expression or enzyme activity of the one or more proteins in the sample derived from the subject. It is particularly preferable that it is a measured value when measured under the conditions of (1) or a reference value set from the measured value.
  • the sample is a body fluid sample or a bone marrow sample, particularly a mononuclear cell-containing sample, and the gene expression or enzyme activity of 2 or more and 5 or less of the five proteins is measured, half of the measured proteins are used.
  • the measured value of gene expression or enzyme activity of the above for example, 1/2 or more, 2/3 or more, 2/4 or more, 3/5 or more
  • the subject has dementia.
  • the subject has a risk of developing dementia, the subject has a higher severity of dementia, the subject's brain function is impaired, the subject's brain It is preferable to determine that there is a risk of functional deterioration, or that the degree of deterioration of the brain function of the subject is more remarkable.
  • only one of the gene expression and the enzyme activity of the one or more proteins may be measured, or both may be measured. More preferably, the gene expression of one or more proteins is measured.
  • the gene expression of the one or more proteins may be measured by detecting the mRNA (including the coding region and the untranslated region) of the gene of the one or more proteins in the sample, or the gene expression in the sample may be measured. It may be measured by detecting the protein amount of one or more proteins.
  • the measured value of the gene expression of the one or more proteins may be the relative expression level of the gene expression of the one or more proteins with respect to the gene expression level of the one or more endogenous controls in the sample.
  • endogenous controls TBP (TATA-binding protein), GAPDH (glyceraldehyde-3-phosphate dehydogenase), ⁇ -actin, ⁇ 2M ( ⁇ 2 microglobuline), HPRT1 (hypoxanthine), HPRT1 (hypoxanthine), HPRT1 (hypoxanthine), HPRT1 (hypoxanthine)
  • a representative housekeeping gene can be used.
  • the nucleotide sequence of mouse 18S ribosomal RNA is shown in SEQ ID NO: 21.
  • the nucleotide sequence of human 18S ribosomal RNA is shown in SEQ ID NO: 22.
  • Northern blotting Northern blotting, RT-PCR method, real-time RT-PCR method, DNA microarray method (method using DNA chip), dot blotting method, RN for detection of mRNA of the gene encoding the amino acid sequence of one or more proteins.
  • An ase protection assay method or the like can be used. These methods can be performed by known methods.
  • the protein amount of the one or more proteins can be detected by an immunoassay method using an antibody that specifically recognizes and binds to the one or more proteins.
  • the antibody can be prepared by a known method.
  • the immunoassay method include a method using a solid-phase carrier on which an antibody that specifically binds to the one or more proteins to be detected is immobilized, flow cytometry, Western blotting, and the like.
  • the method using a solid phase carrier include, but are not limited to, an enzyme-linked immunosorbent assay (ELISA) using an immobilized microtiter plate and an agglutination method (immunoprecipitation method) using immobilized particles.
  • ELISA enzyme-linked immunosorbent assay
  • a known immunoassay can be used to detect the protein content of one or more of the above proteins in a sample. Further, the detection of the protein amount of the above-mentioned one or more proteins can also be performed by a method using LC-MS / MSMRM or the like, which is a protein mass spectrometry technique that does not use an antibody. These detection methods can also be carried out by a conventional protocol.
  • the enzymatic activity of the one or more proteins in the sample can be measured by a method according to the enzymatic activity of the protein to be measured.
  • the ratio of mononuclear cells in the sample is 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more. , 98% or more, 99% or more, or 100% is preferable.
  • the term "ratio of mononuclear cells in a sample” refers to the ratio of the number of mononuclear cells to the total number of leukocytes contained in the sample.
  • the ratio of mononuclear cells in the sample can be measured by, for example, a blood cell analyzer, a hemocytometer, flow cytometry, or the like, but is not limited thereto.
  • measuring the gene expression or enzyme activity of one or more proteins involved in an energy metabolism reaction in a sample derived from the subject is shown in (a) and (b) below. ) Is preferably included.
  • (A) Obtaining a mononuclear cell-containing sample by separating mononuclear cells from the sample.
  • (B) To measure the gene expression or enzyme activity of one or more proteins in the mononuclear cell-containing sample.
  • the sample derived from the subject may contain granulocytes, and the granulocytes in the sample become a factor that lowers the measurement accuracy of the measured values of gene expression or enzyme activity of one or more proteins. Sometimes. Therefore, a mononuclear cell-containing sample in which the proportion of mononuclear cells is increased by separating mononuclear cells or removing granulocytes from the sample is obtained, and the obtained mononuclear cell-containing sample is used for measurement. Therefore, a highly accurate measured value can be obtained.
  • the ratio of mononuclear cells in the mononuclear cell-containing sample is 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 98% or more, 99% or more or 100%. It is preferable to have. Further, the ratio of granulocytes in the mononuclear cell-containing sample may be less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, less than 2% or 0%. preferable.
  • the ratio of mononuclear cells or granulocytes in the mononuclear cell-containing sample is defined by the number of mononuclear cells or granulocytes with respect to the total number of cells contained in the mononuclear cell-containing sample. Refers to the ratio.
  • the ratio of mononuclear cells or granulocytes in a mononuclear cell-containing sample can be measured by, for example, a blood cell analyzer, a hemocytometer, flow cytometry, or the like, but is not limited thereto.
  • a method for obtaining a mononuclear cell-containing sample from a sample derived from a subject a method using a reagent and / or a column for separating mononuclear cells is preferable.
  • the reagent and / or column include Ficoll-Paque PLUS (manufactured by GE Healthcare), Lymphoprep (manufactured by Abbott Laboratories Technologies), and Human Peripheral Blood Monologic Laboratory (manufactured by Human Biological Blood Mononulus Technology).
  • CPT mononuclear cell separation blood collection tube manufactured by Becton Dickinson
  • purriMate manufactured by purriSelect
  • SepMate manufactured by STEMCELL Technologies
  • One or more embodiments of the present invention It is a method of obtaining an index for determining the degree of aging phenomenon in a subject.
  • the present invention relates to a method comprising measuring the gene expression or enzymatic activity of one or more proteins involved in an energy metabolism reaction in a sample derived from a subject.
  • One or more embodiments of the present invention It is a method of determining the degree of aging phenomenon in a subject.
  • the gene expression or enzymatic activity of one or more proteins involved in the energy metabolism response is such that in older subjects, in peripheral blood, urine, saliva, etc., as compared to younger specimens. It is based on the unexpected finding that it is high in body fluids or bone marrow and low in central nervous tissue such as brain tissue. Further, in one or more embodiments of the present invention, the gene expression or enzyme activity of one or more proteins involved in the energy metabolism reaction may cause the aging phenomenon of the brain tissue to progress in the subject in which the aging phenomenon of the brain tissue is observed.
  • the subject in which the disease may progress it is higher in body fluids such as peripheral blood, urine, saliva or bone marrow, and lower in central nervous tissue such as brain tissue, as compared with normal specimens.
  • the gene expression or enzyme activity of one or more proteins involved in the energy metabolism reaction is such that the degree of aging phenomenon of brain tissue is large and the degree of aging phenomenon of peripheral blood is high.
  • a large subject or a subject having a large degree of aging phenomenon of bone marrow cells it is higher in body fluids such as peripheral blood, urine, saliva or bone marrow, and lower in central nervous tissue such as brain tissue, as compared with normal samples.
  • the gene expression or enzyme activity of one or more proteins involved in the energy metabolism reaction is higher in body fluids such as peripheral blood, urine, saliva, or bone marrow as the degree of aging phenomenon of the brain tissue of the subject increases.
  • Low in central nervous tissue such as.
  • the gene expression or enzyme activity of one or more proteins involved in the energy metabolism reaction increases in body fluids such as peripheral blood, urine, saliva, or bone marrow as the degree of aging phenomenon of peripheral blood of the subject increases, and brain tissue.
  • the gene expression or enzyme activity of one or more proteins involved in the energy metabolism reaction is higher in body fluids such as peripheral blood, urine, saliva, or bone marrow as the degree of aging phenomenon of the bone marrow cells of the subject is greater, and the brain tissue.
  • body fluids such as peripheral blood, urine, saliva, or bone marrow
  • the measured value is used to determine the age of the subject and the aging of the brain tissue of the subject.
  • the possibility of the phenomenon progressing, the degree of the aging phenomenon of the brain tissue of the subject, the possibility of the aging phenomenon of the peripheral blood of the subject progressing, the degree of the aging phenomenon of the peripheral blood of the subject, or the subject It is an index showing the degree of aging phenomenon of the bone marrow cells of the sample.
  • the sample is a body fluid sample or a bone marrow sample, particularly a mononuclear cell-containing sample, and the gene expression or enzyme activity of 2 or more and 5 or less of the five proteins is measured, half of the measured proteins.
  • the measured value of gene expression or enzyme activity of the above for example, 1/2 or more, 2/3 or more, 2/4 or more, 3/5 or more
  • the aging phenomenon of the subject is preferable to determine that is more advanced.
  • only one of the gene expression and the enzyme activity of the one or more proteins may be measured, or both may be measured. More preferably, the gene expression of one or more proteins is measured.
  • the gene expression of the one or more proteins may be measured by detecting the mRNA (including the coding region and the untranslated region) of the gene of the one or more proteins in the sample, or the gene expression in the sample may be measured. It may be measured by detecting the protein amount of one or more proteins.
  • Northern blotting RT-PCR method, real-time RT-PCR method, DNA microarray method (method using DNA chip), dot blotting method, RNase protection assay method, etc. Can be used. These methods can be performed by known methods.
  • the protein amount of the one or more proteins can be detected by an immunoassay method using an antibody that specifically recognizes and binds to the one or more proteins.
  • the antibody can be prepared by a known method.
  • the immunoassay method include a method using a solid-phase carrier on which an antibody that specifically binds to the one or more proteins to be detected is immobilized, flow cytometry, Western blotting, and the like.
  • the method using a solid phase carrier include, but are not limited to, an enzyme-linked immunosorbent assay (ELISA) using an immobilized microtiter plate and an agglutination method (immunoprecipitation method) using immobilized particles.
  • ELISA enzyme-linked immunosorbent assay
  • a known immunoassay can be used to detect the protein content of one or more of the above proteins in a sample. Further, the detection of the protein amount of the above-mentioned one or more proteins can also be performed by a method using LC-MS / MS MRM or the like, which is a protein mass spectrometry technique that does not use an antibody. These detection methods can also be carried out by a conventional protocol.
  • the enzymatic activity of the one or more proteins in the sample can be measured by a method according to the enzymatic activity of the protein to be measured.
  • Kit for determining dementia or brain function Another embodiment of the present invention A kit for determining dementia or brain function.
  • the present invention relates to a kit containing a reagent for measuring gene expression or enzymatic activity of one or more proteins involved in an energy metabolism reaction in a sample derived from a subject.
  • a reagent that can be used in the above-mentioned method for measuring gene expression or enzyme activity of one or more proteins is preferable.
  • a primer pair for amplifying a nucleic acid (genome DNA, mRNA or cDNA prepared based on mRNA) containing a gene of the one or more proteins to be measured, and the one or more proteins to be measured.
  • a probe that hybridizes with a nucleic acid containing the gene (genomic DNA, mRNA or cDNA prepared based on the mRNA)
  • an antibody that specifically binds to the one or more proteins and the enzyme activity of the one or more proteins.
  • the reaction substrate of the above can be exemplified.
  • the nucleic acid may also include an untranslated region, an intron region, a signal sequence region, and the like.
  • the nucleic acid is mRNA or cDNA.
  • the mRNA or cDNA may contain at least a part of the base sequence (CDS) encoding the amino acid sequence of the one or more proteins and the base sequence of the untranslated region (UTR) located upstream and downstream thereof.
  • CDS base sequence
  • UTR untranslated region
  • An example of the primer pair is a primer pair capable of amplifying at least a part of the base sequences of CDS and UTR.
  • An example of the probe is a probe that can hybridize to at least a part of the base sequences of CDS and UTR.
  • 3 is a primer pair for amplifying a nucleic acid containing the Glut1 gene, which has the same or homologous base sequence Af as any of the base sequences shown in (A1) to (A4) below.
  • An example includes a Glut1 primer pair containing.
  • (A1) Consecutive 10 or more base sequences contained in the base sequences of positions 101 to 180 among the base sequences of SEQ ID NO: 1
  • (A2) Bases of SEQ ID NO: 3 among the base sequences of SEQ ID NO: 3.
  • the base sequence corresponding to the base sequence of (A1) is changed to the base sequence of positions 256 to 336 among the base sequences of (A3) SEQ ID NO: 3.
  • Consecutive base sequence of 10 or more bases (A4) Of the base sequences of SEQ ID NO: 1, when the base sequence of SEQ ID NO: 1 and the base sequence of SEQ ID NO: 3 are aligned, the base sequence of (A3) is obtained.
  • the "base sequence at positions 101 to 180” is preferably the “base sequence at positions 111 to 170", and more preferably “base sequence at positions 121 to 160". It is a “base sequence”, more preferably “base sequence at positions 121 to 155”, and more preferably “base sequence at positions 126 to 150”.
  • the "base sequence at positions 256 to 336” is preferably the “base sequence at positions 266 to 326", and more preferably “base sequence at positions 276 to 316". It is a “base sequence”, more preferably “base sequence at positions 276 to 311”, and more preferably “base sequence at positions 281 to 306”.
  • the base sequence Af may be the same as or homologous to the base sequence of (A1), the base sequence of (A2), the base sequence of (A3), or the base sequence of (A4). From the 3'end, preferably 2 bases or more, more preferably 3 bases or more, more preferably 5 bases or more, more preferably 10 bases or more, more preferably 15 bases or more, more preferably 17 bases or more, more preferably
  • the continuous base sequence of 19 bases or more is the same as the base sequence of (A1), (A2), (A3) or (A4), and the rest of the base sequence Af is It is the same as or homologous to the base sequence of (A1), the base sequence of (A2), the base sequence of (A3), or the base sequence of (A4).
  • the range of "homology" is as described later.
  • the base sequence Af is the same as the base sequence of (A1), the base sequence of (A2), the base sequence of (A3), or the base sequence of (A4).
  • the Glut1 forward primer may be one containing a polynucleotide containing the base sequence Af at the 3'end, and may further contain another base sequence on the 5'end side of the base sequence Af.
  • the number of bases of the polynucleotide contained in the Glut1 forward primer is not particularly limited, but is preferably 50 bases or less, more preferably 40 bases or less, and more preferably 30 bases or less.
  • the base sequence of (A1) the base sequence of SEQ ID NO: 25 can be exemplified.
  • the base sequence of SEQ ID NO: 25 can be exemplified.
  • the base sequence of SEQ ID NO: 37 can be exemplified.
  • the base sequence of SEQ ID NO: 37 can be exemplified.
  • the "base sequence at positions 213 to 292” is preferably the “base sequence at positions 223 to 282", and more preferably “base sequence at positions 233 to 272". It is a “base sequence”, more preferably “base sequence at positions 238 to 272”, and more preferably “base sequence at positions 243 to 267”.
  • the "base sequence at positions 339 to 418” is preferably the “base sequence at positions 349 to 408", and more preferably “base sequence at positions 359 to 398". It is a “base sequence”, more preferably “base sequence at positions 364 to 398”, and more preferably “base sequence at positions 369 to 393”.
  • the base sequence Ar may be the same as or homologous to the base sequence of (A5), the base sequence of (A6), the base sequence of (A7), or the complementary base sequence of the base sequence of (A8), but the base From the 3'end of the sequence Ar, preferably 2 bases or more, more preferably 3 bases or more, more preferably 5 bases or more, more preferably 10 bases or more, more preferably 15 bases or more, more preferably 17 bases or more. , More preferably, the continuous base sequence of 19 bases or more is the same as the base sequence of (A5), the base sequence of (A6), the base sequence of (A7), or the complementary base sequence of the base sequence of (A8).
  • the rest of the base sequence Ar is the same as or homologous to the base sequence of (A5), the base sequence of (A6), the base sequence of (A7), or the complementary base sequence of the base sequence of (A8).
  • the range of "homology” is as described later.
  • the base sequence Ar is the same as the base sequence of (A5), the base sequence of (A6), the base sequence of (A7), or the complementary base sequence of the base sequence of (A8).
  • the Glut1 reverse primer may contain a polynucleotide containing the base sequence Ar at the 3'end, and may further contain another base sequence on the 5'end side of the base sequence Ar.
  • the number of bases of the polynucleotide contained in the Glut1 reverse primer is not particularly limited, but is preferably 50 bases or less, more preferably 40 bases or less, and more preferably 30 bases or less.
  • a complementary base sequence of the base sequence of SEQ ID NO: 26 can be exemplified.
  • the base sequence of SEQ ID NO: 26 can be exemplified.
  • the complementary base sequence of the base sequence of SEQ ID NO: 38 can be exemplified.
  • the base sequence of SEQ ID NO: 38 can be exemplified.
  • 3 is a primer pair for amplifying a nucleic acid containing the Glut3 gene, which has the same or homologous base sequence Bf as any of the base sequences shown in (B1) to (B4) below.
  • An example includes a Glut3 primer pair containing.
  • (B1) Consecutive 10 or more base sequences contained in the base sequences of positions 250 to 329 of the base sequence of SEQ ID NO: 5 (B2) Base of SEQ ID NO: 7 among the base sequences of SEQ ID NO: 7.
  • the base sequence corresponding to the base sequence of (B1) (B3) is the base sequence of positions 224 to 303 among the base sequences of SEQ ID NO: 7.
  • Consecutive base sequence of 10 or more bases (B4) Among the base sequences of SEQ ID NO: 5, when the base sequence of SEQ ID NO: 5 and the base sequence of SEQ ID NO: 7 are aligned, the base sequence of (B3) is obtained.
  • the "base sequence at positions 250 to 329” is preferably the “base sequence at positions 260 to 319", and more preferably “base sequence at positions 270 to 309". It is a “base sequence”, more preferably “base sequence at positions 270 to 304", and more preferably “base sequence at positions 275 to 299”.
  • the "base sequence at positions 224 to 303” is preferably the “base sequence at positions 234 to 293", and more preferably “base sequence at positions 244 to 283". It is a “base sequence”, more preferably “base sequence at positions 244 to 278”, and more preferably “base sequence at positions 249 to 273”.
  • the base sequence Bf may be the same as or homologous to the base sequence of (B1), the base sequence of (B2), the base sequence of (B3), or the base sequence of (B4). From the 3'end, preferably 2 bases or more, more preferably 3 bases or more, more preferably 5 bases or more, more preferably 10 bases or more, more preferably 15 bases or more, more preferably 17 bases or more, more preferably
  • the continuous base sequence of 19 bases or more is the same as the base sequence of (B1), (B2), (B3) or (B4), and the rest of the base sequence Bf is It is the same as or homologous to the base sequence of (B1), the base sequence of (B2), the base sequence of (B3), or the base sequence of (B4).
  • the range of "homology" is as described later.
  • the base sequence Bf is the same as the base sequence of (B1), the base sequence of (B2), the base sequence of (B3), or the base sequence of (B4).
  • the Glut3 forward primer may be one containing a polynucleotide containing the base sequence Bf at the 3'end, and may further contain another base sequence on the 5'end side of the base sequence Bf.
  • the number of bases of the polynucleotide contained in the Glut3 forward primer is not particularly limited, but is preferably 50 bases or less, more preferably 40 bases or less, and more preferably 30 bases or less.
  • the base sequence of (B1) the base sequence of SEQ ID NO: 27 can be exemplified.
  • the base sequence of SEQ ID NO: 27 can be exemplified.
  • the base sequence of (B3) the base sequence of SEQ ID NO: 39 can be exemplified.
  • the base sequence of SEQ ID NO: 39 can be exemplified.
  • the "base sequence at positions 375 to 454" is preferably the “base sequence at positions 385 to 444", and more preferably “base sequence at positions 395 to 434". It is a “base sequence”, more preferably “base sequence at positions 400 to 434", and more preferably “base sequence at positions 405 to 429”.
  • the "base sequence at positions 288 to 367” is preferably the “base sequence at positions 298 to 357”, and more preferably “base sequence at positions 308 to 347". It is a “base sequence”, more preferably “base sequence at positions 313 to 347”, and more preferably “base sequence at positions 318 to 342”.
  • the base sequence Br may be the same as or homologous to the base sequence of (B5), the base sequence of (B6), the base sequence of (B7), or the complementary base sequence of the base sequence of (B8), but the base From the 3'end of the sequence Br, preferably 2 bases or more, more preferably 3 bases or more, more preferably 5 bases or more, more preferably 10 bases or more, more preferably 15 bases or more, more preferably 17 bases or more. , More preferably, the continuous base sequence of 19 bases or more is the same as the base sequence of (B5), the base sequence of (B6), the base sequence of (B7), or the complementary base sequence of the base sequence of (B8).
  • the rest of the base sequence Br is the same as or homologous to the base sequence of (B5), the base sequence of (B6), the base sequence of (B7), or the complementary base sequence of the base sequence of (B8).
  • the range of "homology" is as described later.
  • the base sequence Br is the same as the base sequence of (B5), the base sequence of (B6), the base sequence of (B7), or the complementary base sequence of the base sequence of (B8).
  • the Glut3 reverse primer may be one containing a polynucleotide containing the base sequence Br at the 3'end, and may further contain another base sequence on the 5'end side of the base sequence Br.
  • the number of bases of the polynucleotide contained in the Glut3 reverse primer is not particularly limited, but is preferably 50 bases or less, more preferably 40 bases or less, and more preferably 30 bases or less.
  • the complementary base sequence of the base sequence of SEQ ID NO: 28 can be exemplified.
  • the base sequence Br the base sequence of SEQ ID NO: 28 can be exemplified.
  • the complementary base sequence of the base sequence of SEQ ID NO: 40 can be exemplified.
  • the base sequence Br the base sequence of SEQ ID NO: 40 can be exemplified.
  • 3 is a primer pair for amplifying a nucleic acid containing the MCT4 gene, which has the same or homologous base sequence Cf as any of the base sequences shown in (C1) to (C4) below.
  • An example includes an MCT4 primer pair containing.
  • (C1) Consecutive base sequence of 10 or more bases contained in the base sequence of positions 40 to 118 of the base sequence of SEQ ID NO: 9
  • the "base sequence at positions 40 to 118" is preferably the “base sequence at positions 50 to 108", and more preferably “base sequence at positions 60 to 98". It is a “base sequence”, more preferably a “base sequence at positions 60 to 93”, and more preferably a "base sequence at positions 65 to 88".
  • the "base sequence at positions 146 to 225” is preferably the “base sequence at positions 156 to 215", and more preferably “base sequence at positions 166 to 205". It is a “base sequence”, more preferably “base sequence at positions 166 to 200”, and more preferably “base sequence at positions 171 to 195”.
  • the base sequence Cf may be the same as or homologous to the base sequence of (C1), the base sequence of (C2), the base sequence of (C3), or the base sequence of (C4). From the 3'end, preferably 2 bases or more, more preferably 3 bases or more, more preferably 5 bases or more, more preferably 10 bases or more, more preferably 15 bases or more, more preferably 17 bases or more, more preferably
  • the continuous base sequence of 19 bases or more is the same as the base sequence of (C1), (C2), (C3) or (C4), and the rest of the base sequence Cf is It is the same as or homologous to the base sequence of (C1), the base sequence of (C2), the base sequence of (C3), or the base sequence of (C4).
  • the range of "homology" is as described later.
  • the base sequence Cf is the same as the base sequence of (C1), the base sequence of (C2), the base sequence of (C3), or the base sequence of (C4).
  • the MCT4 forward primer may be one containing a polynucleotide containing the base sequence Cf at the 3'end, and may further contain another base sequence on the 5'end side of the base sequence Cf.
  • the number of bases of the polynucleotide contained in the MCT4 forward primer is not particularly limited, but is preferably 50 bases or less, more preferably 40 bases or less, and more preferably 30 bases or less.
  • the base sequence of (C1) the base sequence of SEQ ID NO: 29 can be exemplified.
  • the base sequence of SEQ ID NO: 29 can be exemplified.
  • the base sequence of SEQ ID NO: 41 can be exemplified.
  • the base sequence of SEQ ID NO: 41 can be exemplified.
  • the "base sequence at positions 107 to 186” is preferably the “base sequence at positions 117 to 176", and more preferably “base sequence at positions 127 to 166". It is a “base sequence”, more preferably “base sequence at positions 132 to 166”, and more preferably “base sequence at positions 137 to 161".
  • the "base sequence at positions 225 to 304" is preferably the “base sequence at positions 235 to 294", and more preferably “base sequence at positions 245 to 284". It is a “base sequence”, more preferably “a base sequence at positions 250 to 284", and more preferably “a base sequence at positions 255 to 279”.
  • the base sequence Cr may be the same as or homologous to the base sequence of (C5), the base sequence of (C6), the base sequence of (C7), or the complementary base sequence of the base sequence of (C8). From the 3'end of the sequence Cr, preferably 2 bases or more, more preferably 3 bases or more, more preferably 5 bases or more, more preferably 10 bases or more, more preferably 15 bases or more, more preferably 17 bases or more. , More preferably, the continuous base sequence of 19 bases or more is the same as the base sequence of (C5), the base sequence of (C6), the base sequence of (C7), or the complementary base sequence of the base sequence of (C8).
  • the rest of the base sequence Cr is the same as or homologous to the base sequence of (C5), the base sequence of (C6), the base sequence of (C7), or the complementary base sequence of the base sequence of (C8).
  • the range of "homology” is as described later.
  • the base sequence Cr is the same as the base sequence of (C5), the base sequence of (C6), the base sequence of (C7), or the complementary base sequence of the base sequence of (C8).
  • the MCT4 reverse primer may contain a polynucleotide containing the base sequence Cr at the 3'end, and may further contain another base sequence on the 5'end side of the base sequence Cr.
  • the number of bases of the polynucleotide contained in the MCT4 reverse primer is not particularly limited, but is preferably 50 bases or less, more preferably 40 bases or less, and more preferably 30 bases or less.
  • the complementary base sequence of the base sequence of SEQ ID NO: 30 can be exemplified.
  • the base sequence of SEQ ID NO: 30 can be exemplified.
  • the complementary base sequence of the base sequence of SEQ ID NO: 42 can be exemplified.
  • the base sequence of SEQ ID NO: 42 can be exemplified.
  • 3 is a primer pair for amplifying a nucleic acid containing a PHD3 gene, which has the same or homologous base sequence Df as any of the base sequences shown in (D1) to (D4) below.
  • An example includes a pair of PHD3 primers.
  • (D1) Consecutive 10 or more base sequences contained in the base sequences of positions 864 to 943 among the base sequences of SEQ ID NO: 13
  • (D2) Bases of SEQ ID NO: 15 among the base sequences of SEQ ID NO: 15.
  • the base sequence corresponding to the base sequence of (D1) is changed to the base sequence of positions 863 to 942 among the base sequences of (D3) SEQ ID NO: 15.
  • Consecutive base sequence of 10 or more bases (D4) Among the base sequences of SEQ ID NO: 13, when the base sequence of SEQ ID NO: 13 and the base sequence of SEQ ID NO: 15 are aligned, the base sequence of (D3) is obtained.
  • the "base sequence at positions 864 to 943” is preferably the “base sequence at positions 874 to 933", and more preferably “base sequence at positions 884 to 923". It is a “base sequence”, more preferably “base sequence at positions 884 to 918”, and more preferably “base sequence at positions 889 to 913".
  • the "base sequence at positions 863 to 942” is preferably the “base sequence at positions 873 to 932", and more preferably “base sequence at positions 883 to 922". It is a “base sequence”, more preferably “base sequence at positions 883 to 917”, and more preferably “base sequence at positions 888 to 912".
  • the base sequence Df may be the same as or homologous to the base sequence of (D1), the base sequence of (D2), the base sequence of (D3), or the base sequence of (D4). From the 3'end, preferably 2 bases or more, more preferably 3 bases or more, more preferably 5 bases or more, more preferably 10 bases or more, more preferably 15 bases or more, more preferably 17 bases or more, more preferably
  • the continuous base sequence of 19 bases or more is the same as the base sequence of (D1), (D2), (D3) or (D4), and the rest of the base sequence Df is It is the same as or homologous to the base sequence of (D1), the base sequence of (D2), the base sequence of (D3), or the base sequence of (D4).
  • the range of "homology" is as described later.
  • the base sequence Df is the same as the base sequence of (D1), the base sequence of (D2), the base sequence of (D3), or the base sequence of (D4).
  • the PHD3 forward primer may be one containing a polynucleotide containing the base sequence Df at the 3'end, and may further contain another base sequence on the 5'end side of the base sequence Df.
  • the number of bases of the polynucleotide contained in the PHD3 forward primer is not particularly limited, but is preferably 50 bases or less, more preferably 40 bases or less, and more preferably 30 bases or less.
  • the base sequence of (D1) the base sequence of SEQ ID NO: 31 can be exemplified.
  • the base sequence Df the base sequence of SEQ ID NO: 31 can be exemplified.
  • the base sequence of (D3) the base sequence of SEQ ID NO: 43 can be exemplified.
  • the base sequence of SEQ ID NO: 43 can be exemplified.
  • the "base sequence at positions 991 to 1072” is preferably the “base sequence at positions 1001 to 1062", and more preferably “base sequence at positions 1011 to 1052". It is a “base sequence”, more preferably “base sequence at positions 1016 to 1047”, and more preferably “base sequence at positions 1021 to 1042”.
  • the "base sequence at positions 1004 to 1083” is preferably the “base sequence at positions 1014 to 1073”, and more preferably “base sequence at positions 1024 to 1063". It is a “base sequence”, more preferably “base sequence at positions 1029 to 1063”, and more preferably “base sequence at positions 1034 to 1058”.
  • the base sequence Dr may be the same as or homologous to the base sequence of (D5), the base sequence of (D6), the base sequence of (D7), or the complementary base sequence of the base sequence of (D8). From the 3'end of the sequence Dr, preferably 2 bases or more, more preferably 3 bases or more, more preferably 5 bases or more, more preferably 10 bases or more, more preferably 15 bases or more, more preferably 17 bases or more. , More preferably, the continuous base sequence of 19 bases or more is the same as the base sequence of (D5), the base sequence of (D6), the base sequence of (D7), or the complementary base sequence of the base sequence of (D8).
  • the rest of the base sequence Dr is the same as or homologous to the base sequence of (D5), the base sequence of (D6), the base sequence of (D7), or the complementary base sequence of the base sequence of (D8).
  • the range of "homology” is as described later.
  • the base sequence Dr is the same as the base sequence of (D5), the base sequence of (D6), the base sequence of (D7), or the complementary base sequence of the base sequence of (D8).
  • the PHD3 reverse primer may be one containing a polynucleotide containing the base sequence Dr at the 3'end, and may further contain another base sequence on the 5'end side of the base sequence Dr.
  • the number of bases of the polynucleotide contained in the PHD3 reverse primer is not particularly limited, but is preferably 50 bases or less, more preferably 40 bases or less, and more preferably 30 bases or less.
  • the complementary base sequence of the base sequence of SEQ ID NO: 32 can be exemplified.
  • the base sequence of SEQ ID NO: 32 can be exemplified.
  • the complementary base sequence of the base sequence of SEQ ID NO: 44 can be exemplified.
  • the base sequence of SEQ ID NO: 44 can be exemplified.
  • 3 is a primer pair for amplifying a nucleic acid containing a PDK1 gene, which has the same or homologous base sequence Ef as any of the base sequences shown in (E1) to (E4) below.
  • Ef the base sequences shown in (E1) to (E4) below.
  • An example includes a PDK1 primer pair containing.
  • (E1) Consecutive 10 or more base sequences contained in the base sequences of positions 445 to 528 of the base sequence of SEQ ID NO: 17 (E2) Base of SEQ ID NO: 19 among the base sequences of SEQ ID NO: 19.
  • the base sequence corresponding to the base sequence of (E1) (E3) is the base sequence of positions 981 to 1060 among the base sequences of SEQ ID NO: 19.
  • Consecutive base sequence of 10 or more bases (E4) Among the base sequences of SEQ ID NO: 17, when the base sequence of SEQ ID NO: 17 and the base sequence of SEQ ID NO: 19 are aligned, the base sequence of (E3) is obtained.
  • the "base sequence at positions 445 to 528” is preferably the “base sequence at positions 455 to 518", and more preferably “base sequence at positions 465 to 508". It is a “base sequence”, more preferably “base sequence at positions 465 to 503”, and more preferably “base sequence at positions 470 to 498".
  • the "base sequence at positions 981 to 1060” is preferably the “base sequence at positions 991 to 1050", and more preferably “base sequence at positions 1001 to 1040". It is a “base sequence”, more preferably “base sequence at positions 1001 to 1035”, and more preferably “base sequence at positions 1006 to 1030”.
  • the base sequence Ef may be the same as or homologous to the base sequence of (E1), the base sequence of (E2), the base sequence of (E3), or the base sequence of (E4). From the 3'end, preferably 2 bases or more, more preferably 3 bases or more, more preferably 5 bases or more, more preferably 10 bases or more, more preferably 15 bases or more, more preferably 17 bases or more, more preferably
  • the continuous base sequence of 19 bases or more is the same as the base sequence of (E1), (E2), (E3) or (E4), and the rest of the base sequence Ef is It is the same as or homologous to the base sequence of (E1), the base sequence of (E2), the base sequence of (E3), or the base sequence of (E4).
  • the range of "homology" is as described later.
  • the base sequence Ef is the same as the base sequence of (E1), the base sequence of (E2), the base sequence of (E3), or the base sequence of (E4).
  • the PDK1 forward primer may be one containing a polynucleotide containing the base sequence Ef at the 3'end, and may further contain another base sequence on the 5'end side of the base sequence Ef.
  • the number of bases of the polynucleotide contained in the PDK1 forward primer is not particularly limited, but is preferably 50 bases or less, more preferably 40 bases or less, and more preferably 30 bases or less.
  • the base sequence of (E1) the base sequence of SEQ ID NO: 33 can be exemplified.
  • the base sequence of SEQ ID NO: 33 can be exemplified.
  • the base sequence of (E3) the base sequence of SEQ ID NO: 45 can be exemplified.
  • the base sequence of SEQ ID NO: 45 can be exemplified.
  • the "base sequence at positions 545 to 624" is preferably the “base sequence at positions 555 to 614", and more preferably “base sequence at positions 565 to 604". It is a “base sequence”, more preferably “base sequence at positions 570 to 604", and more preferably “base sequence at positions 575 to 599”.
  • the "base sequence at positions 1078 to 1157” is preferably the “base sequence at positions 1088 to 1147”, and more preferably “base sequence at positions 1098 to 1137”. It is a “base sequence”, more preferably “base sequence at positions 1103 to 1137”, and more preferably “base sequence at positions 1108 to 1132”.
  • the base sequence Er may be the same as or homologous to the base sequence of (E5), the base sequence of (E6), the base sequence of (E7), or the complementary base sequence of the base sequence of (E8), but the base From the 3'end of the sequence Er, preferably 2 bases or more, more preferably 3 bases or more, more preferably 5 bases or more, more preferably 10 bases or more, more preferably 15 bases or more, more preferably 17 bases or more. , More preferably, the continuous base sequence of 19 bases or more is the same as the base sequence of (E5), the base sequence of (E6), the base sequence of (E7), or the complementary base sequence of the base sequence of (E8).
  • the rest of the base sequence Er is the same as or homologous to the base sequence of (E5), the base sequence of (E6), the base sequence of (E7), or the complementary base sequence of the base sequence of (E8).
  • the range of "homology” is as described later.
  • the base sequence Er is the same as the base sequence of (E5), the base sequence of (E6), the base sequence of (E7), or the complementary base sequence of the base sequence of (E8).
  • the PDK1 reverse primer may be one containing a polynucleotide containing the base sequence Er at the 3'end, and may further contain another base sequence on the 5'end side of the base sequence Er.
  • the number of bases of the polynucleotide contained in the PDK1 reverse primer is not particularly limited, but is preferably 50 bases or less, more preferably 40 bases or less, and more preferably 30 bases or less.
  • the complementary base sequence of the base sequence of SEQ ID NO: 34 can be exemplified.
  • the base sequence of SEQ ID NO: 34 can be exemplified.
  • the complementary base sequence of the base sequence of SEQ ID NO: 46 can be exemplified.
  • the base sequence of SEQ ID NO: 46 can be exemplified.
  • the kit can include, as a primer pair, a primer pair for amplifying the nucleic acid of the endogenous control gene.
  • a primer pair for amplifying the nucleic acid of the endogenous control gene.
  • it is a primer pair for amplifying a nucleic acid encoding 18S ribosome RNA, and has the same or homologous base as any of the base sequences shown in (F1) to (F4) below.
  • the 18S ribosome RNA forward primer containing the sequence Ff at the 3'end and the base sequence Fr which is the same as or homologous to the complementary base sequence of any of the base sequences shown in (F5) to (F8) below are contained at the 3'end.
  • Consecutive base sequence of 10 or more bases (F4) Of the base sequences of SEQ ID NO: 21, when the base sequence of SEQ ID NO: 21 and the base sequence of SEQ ID NO: 22 are aligned, the base sequence of (F3) is obtained.
  • the "base sequence at positions 1216 to 1297” is preferably the “base sequence at positions 1226 to 1287”, and more preferably “base sequence at positions 1236 to 1277”. It is a “base sequence”, more preferably “base sequence at positions 1241 to 1272”, and more preferably “base sequence at positions 1241 to 1267”.
  • the "base sequence at positions 1216 to 1297” is preferably the “base sequence at positions 1226 to 1287”, and more preferably “base sequence at positions 1236 to 1277”. It is a “base sequence”, more preferably “base sequence at positions 1236 to 1272”, and more preferably “base sequence at positions 1241 to 1267”.
  • the base sequence Ff may be the same as or homologous to the base sequence of (F1), the base sequence of (F2), the base sequence of (F3), or the base sequence of (F4). From the 3'end, preferably 2 bases or more, more preferably 3 bases or more, more preferably 5 bases or more, more preferably 10 bases or more, more preferably 15 bases or more, more preferably 17 bases or more, more preferably
  • the continuous base sequence of 19 bases or more is the same as the base sequence of (F1), (F2), (F3) or (F4), and the rest of the base sequence Ff is It is the same as or homologous to the base sequence of (F1), the base sequence of (F2), the base sequence of (F3), or the base sequence of (F4).
  • the range of "homology" is as described later.
  • the base sequence Ff is the same as the base sequence of (F1), the base sequence of (F2), the base sequence of (F3), or the base sequence of (F4).
  • the 18S ribosomal RNA forward primer may contain a polynucleotide containing the base sequence Ff at the 3'end, and may further contain another base sequence on the 5'end side of the base sequence Ff. ..
  • the number of bases of the polynucleotide contained in the 18S ribosomal RNA forward primer is not particularly limited, but is preferably 50 bases or less, more preferably 40 bases or less, and more preferably 30 bases or less.
  • the base sequence of (F1) the base sequence of SEQ ID NO: 23 can be exemplified.
  • the base sequence of SEQ ID NO: 23 can be exemplified.
  • the base sequence of (F3) the base sequence of SEQ ID NO: 35 can be exemplified.
  • the base sequence Ff the base sequence of SEQ ID NO: 35 can be exemplified.
  • the "base sequence at positions 1317 to 1396” is preferably the “base sequence at positions 1327 to 1386", and more preferably “base sequence at positions 1337 to 1376". It is a “base sequence”, more preferably “base sequence at positions 1342 to 1376”, and more preferably “base sequence at positions 1347 to 1371”.
  • the "base sequence at positions 1317 to 1396” is preferably the “base sequence at positions 1327 to 1386", and more preferably “base sequence at positions 1337 to 1376". It is a “base sequence”, more preferably “base sequence at positions 1342 to 1376”, and more preferably “base sequence at positions 1347 to 1371”.
  • the base sequence Fr may be the same as or homologous to the base sequence of (F5), the base sequence of (F6), the base sequence of (F7), or the complementary base sequence of the base sequence of (F8), but the base From the 3'end of the sequence Fr, preferably 2 bases or more, more preferably 3 bases or more, more preferably 5 bases or more, more preferably 10 bases or more, more preferably 15 bases or more, more preferably 17 bases or more. , More preferably, the continuous base sequence of 19 bases or more is the same as the base sequence of (F5), the base sequence of (F6), the base sequence of (F7), or the complementary base sequence of the base sequence of (F8).
  • the rest of the base sequence Fr is the same as or homologous to the base sequence of (F5), the base sequence of (F6), the base sequence of (F7), or the complementary base sequence of the base sequence of (F8).
  • the range of "homology" is as described later.
  • the base sequence Fr is the same as the base sequence of (F5), the base sequence of (F6), the base sequence of (F7), or the complementary base sequence of the base sequence of (F8).
  • the 18S ribosomal RNA reverse primer may contain a polynucleotide containing the base sequence Fr at the 3'end, and may further contain another base sequence on the 5'end side of the base sequence Fr. ..
  • the number of bases of the polynucleotide contained in the 18S ribosomal RNA reverse primer is not particularly limited, but is preferably 50 bases or less, more preferably 40 bases or less, and more preferably 30 bases or less.
  • the complementary base sequence of the base sequence of SEQ ID NO: 24 can be exemplified.
  • the base sequence Fr the base sequence of SEQ ID NO: 24 can be exemplified.
  • the complementary base sequence of the base sequence of SEQ ID NO: 36 can be exemplified.
  • the base sequence of SEQ ID NO: 36 can be exemplified.
  • base sequence Y homologous to base sequence X or "base sequence X and base sequence Y are homologous"
  • a polynucleotide consisting of a complementary sequence of base sequence X and a base sequence As long as the polynucleotide consisting of Y is a combination capable of hybridizing under the annealing conditions of the nucleic acid amplification reaction and forming a hydrogen bond sufficient to form a stable double strand, the nucleotide sequences X and Y May be partially different.
  • a polynucleotide consisting of a complementary sequence of the base sequence X and a polynucleotide consisting of the base sequence Y have several mismatches such as 1 mismatch in 10 nucleotides, 1 mismatch in 20 nucleotides, or 1 mismatch in 30 nucleotides. There may be a mismatch.
  • the base sequence Y is "homologous" to the base sequence X, it means that the base sequences X and Y satisfy any of the following relationships.
  • the base sequence Y is a base sequence in which one or several bases are deleted, substituted, added and / or inserted in the base sequence X.
  • the base sequence Y is a base sequence having 70% or more identity with the base sequence X.
  • a polynucleotide having a base sequence Y can hybridize with a polynucleotide having a base sequence complementary to SEQ ID NO: X under stringent conditions.
  • D Thymine (T) at an arbitrary position in one of the base sequence X and the base sequence Y is replaced with uracil (U) in the other.
  • “1 or several” preferably refers to 1 to 5, more preferably 1 to 4, more preferably 1 to 3, particularly preferably 1 or 2, and most preferably. It is one.
  • “1 or several” refers to the total number of deleted, substituted, added and / or inserted bases.
  • the identity value indicates a value calculated with default settings using software (for example, FASTA, DNASIS, and BLAST) that calculates the identity between a plurality of base sequences.
  • software for example, FASTA, DNASIS, and BLAST
  • the base sequence identity value the number of matching bases when the pair of base sequences are aligned so as to maximize the degree of matching is calculated, and the total number of bases in the compared base sequence of the number of matching bases is calculated. Calculated as a percentage of the number.
  • the total number of bases described above is the number of bases counted with one gap as one base.
  • the identity is more preferably 80% or more, more preferably 90% or more, more preferably 95% or more, more preferably 96% or more, more preferably 97% or more, more preferably 98% or more. , More preferably 99% or more identity.
  • the “stringent condition” means a condition in which a so-called specific hybrid is formed and a non-specific hybrid is not formed.
  • stringent conditions can be set by the temperature at the time of Southern hybridization and the salt concentration contained in the solution, and the temperature at the time of the washing step of Southern hybridization and the salt concentration contained in the solution. More specifically, as stringent conditions, for example, in the hybridization step, the sodium concentration is 25 to 500 mM, preferably 25 to 300 mM, and the temperature is 40 to 68 ° C, preferably 40 to 65 ° C.
  • hybridization can be performed at 1-7 ⁇ SSC, 0.02-3% SDS, and a temperature of 40 ° C-60 ° C.
  • a washing step may be performed after hybridization, and the washing step can be performed, for example, at 0.1 to 2 ⁇ SSC, 0.1 to 0.3% SDS, and a temperature of 50 to 65 ° C.
  • One or both of the above primer pairs may be modified with another substance such as a labeling substance.
  • the probe is a nucleotide sequence of a nucleic acid containing the gene of one or more proteins, for example, the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3 of the Glut1 gene (particularly, positions 131 to 262 of the nucleotide sequence of SEQ ID NO: 1). Nucleotide sequence at position; Of the nucleotide sequence of SEQ ID NO: 3, when the nucleotide sequence of SEQ ID NO: 3 and the nucleotide sequence of SEQ ID NO: 1 are aligned, the nucleotide sequence of SEQ ID NO: 1 is located at positions 131 to 262.
  • the base sequence of SEQ ID NO: 5 or SEQ ID NO: 7 of the Glut3 gene particularly, the base sequence
  • the base sequence of SEQ ID NO: 15 base sequence corresponding to the base sequence of positions 893 to 1053 or the base sequence of SEQ ID NO: 17 or SEQ ID NO: 19 of the PDK1 gene (particularly, SEQ ID NO: Of the 17 base sequences, the base sequences of positions 475 to 594; among the base sequences of SEQ ID NO: 19, when the base sequence of SEQ ID NO: 19 and the base sequence of SEQ ID NO: 17 are aligned, the base sequence of SEQ ID NO: 17 The base sequence corresponding to the base sequence of positions 475 to 594 of the base sequence; the base sequence of positions 1011 to 1127 of the base sequence of SEQ ID NO: 19; or the base sequence of the base sequence of SEQ ID NO: 17 When the base sequence of SEQ ID NO: 17 and the base sequence of SEQ ID NO: 19 are aligned, 10 or more bases contained in the base sequence of positions 1011 to 1127 of the base sequence of SEQ ID NO: 19) It can contain a nucleic acid
  • the upper limit of the length of the partial base sequence is not particularly limited, but may be, for example, 50 bases or less, 40 bases or less, or 30 bases or less.
  • the probe may be one in which the nucleic acid fragment is modified with another substance such as a labeling substance.
  • the antibody that specifically binds to the one or more proteins may be a polyclonal antibody or a monoclonal antibody.
  • the antibody can also be used as a fragment as long as it can specifically bind to the protein to be measured.
  • Examples of the antibody fragment include a Fab fragment, an F (ab') 2 fragment, a single chain antibody (scFv), and the like.
  • the antibody may be immobilized on a solid-phase carrier such as a microtiter plate or particles.
  • the reaction substrate for measuring the enzyme activity of the one or more proteins can be appropriately selected according to the activity of the protein to be measured.
  • the kit according to the above embodiment may further include a buffer solution for dilution or reaction containing components necessary for measurement, a washing solution, a coloring reagent, a reaction vessel, and the like.
  • the kit according to the embodiment preferably further contains a reagent and / or a column for separating mononuclear cells from the sample.
  • a reagent and / or a column for separating mononuclear cells from the sample.
  • the reagent and / or column include Ficoll-Paque PLUS (manufactured by GE Healthcare), Lymphoprep (manufactured by Abbott Laboratories Technologies), and Human Peripheral Blood Monologic Laboratory (manufactured by Human Biological Blood Mononulus Technology). Registered trademark) CPT mononuclear cell separation blood collection tube (manufactured by Becton Dickinson), purriMate (manufactured by purriSelect), SepMate (manufactured by STEMCELL Technologies) and the like can be mentioned.
  • the mononuclear cells and granulocytes in the sample can be separated, and a mononuclear cell-containing sample for measurement can be efficiently obtained.
  • Kit for determining the degree of aging in a subject Another embodiment of the present invention A kit for determining the degree of aging in a subject.
  • the present invention relates to a kit containing a reagent for measuring gene expression or enzymatic activity of one or more proteins involved in an energy metabolism reaction in a sample derived from a subject.
  • the determination of the degree of the aging phenomenon is preferably a determination of age, a determination of the degree of aging phenomenon of brain tissue, a degree of aging phenomenon of peripheral blood, or a determination of the degree of aging phenomenon of bone marrow cells.
  • the bone marrow cells are preferably hematopoietic stem cells, mesenchymal stem cells, and immature cells.
  • a reagent that can be used in the above-mentioned method for measuring gene expression or enzyme activity of one or more proteins is preferable.
  • a primer pair for amplifying a nucleic acid (genomic DNA, mRNA or a cDNA prepared based on mRNA) containing a gene of the one or more proteins to be measured, and the one or more proteins to be measured.
  • a probe that hybridizes with a nucleic acid containing the gene (mRNA or a cDNA prepared based on mRNA), an antibody that specifically binds to the one or more proteins, and a reaction substrate for measuring the enzymatic activity of the one or more proteins.
  • Etc. can be exemplified.
  • the genomic DNA may also include an untranslated region, an intron region, a signal sequence region, and the like.
  • the nucleic acid is mRNA or cDNA.
  • the mRNA or cDNA may contain at least a part of the base sequence (CDS) encoding the amino acid sequence of the one or more proteins and the base sequence of the untranslated region (UTR) located upstream and downstream thereof.
  • CDS base sequence
  • UTR untranslated region
  • An example of the primer pair is a primer pair capable of amplifying at least a part of the base sequences of CDS and UTR.
  • An example of the probe is a probe that can hybridize to at least a part of the base sequences of CDS and UTR.
  • the primer pair can be selected from the same range as the preferred example of the primer pair described in the above "4. Kit for determination of dementia or determination of brain function".
  • the probe can be selected from the same range as the preferred example of the blow part described in "4. Kit for determining dementia or determining brain function" above.
  • the antibody that specifically binds to the one or more proteins may be a polyclonal antibody or a monoclonal antibody.
  • the antibody can also be used as a fragment as long as it can specifically bind to the protein to be measured.
  • Examples of the antibody fragment include a Fab fragment, an F (ab') 2 fragment, a single chain antibody (scFv), and the like.
  • the antibody may be immobilized on a solid-phase carrier such as a microtiter plate or particles.
  • the reaction substrate for measuring the enzyme activity of the one or more proteins can be appropriately selected according to the activity of the protein to be measured.
  • the kit according to the above embodiment may further include a buffer solution for dilution or reaction containing components necessary for measurement, a washing solution, a coloring reagent, a reaction vessel, and the like.
  • Comparative Example 1 ⁇ Method> In Comparative Example 1, an index for determining dementia and brain function in a subject was examined using a body fluid sample derived from a healthy mouse.
  • Behavioral Tests Five behavioral tests (open field test, wire hang test, passive avoidance test, open space swimming test and rotarod test) were performed to evaluate dementia and / or brain function in mice.
  • Open field test uses the average number of mouse movements (the average value of the total number of horizontal movements and vertical movements) in the light and dark states as an index, and the responsiveness of the mouse to changes in light and dark. It is a behavioral test to evaluate. The open field test was carried out by the following method.
  • the mouse was placed in a transparent acrylic box (bottom surface: length 30 cm x width 30 cm).
  • the mouse in the acrylic box was freely searched for 30 minutes with the white light turned on (bright state). The lights were then turned off and kept dark for 30 minutes.
  • the number of horizontal movements of the mouse was evaluated by measuring the number of times the mouse blocked infrared beams provided at intervals of 10 cm along the vertical and horizontal axes at a height of 2 cm from the floor.
  • the number of vertical movements (number of rises) of the mouse was evaluated by measuring the number of times the mouse blocked infrared beams provided at intervals of 3 cm along the vertical axis at a height of 5 cm from the floor. The test was carried out 9 times or more, and the average number of movements was calculated.
  • Wire hang test is a behavioral test that evaluates the muscle strength of a mouse by using the average value (average fall time) of the time until the mouse suspended from the wire net falls as an index.
  • the wire hang test was carried out by the following method.
  • the mouse was placed on a wire mesh with an interval of 12 mm. Next, the mouse was turned over together with the wire mesh and hung at a height of 20 cm. The time until the mouse fell from the wire mesh (falling time) was recorded. The test was carried out 8 times or more, and the average value (average fall time) of those fall times was calculated.
  • Passive avoidance test In the passive avoidance test, learning and memory of mice that have been subjected to aversive stimuli in advance using the average value of the response latency (mean response latency) at the time of this test as an index. It is a behavioral test that evaluates the function of the mouse. The passive avoidance test was conducted by the following method.
  • the passive avoidance test consisted of a light room and a dark room separated by a plate with a hole through which the mouse could pass, and was conducted using a box with an electrical stimulator installed on the floor of the dark room.
  • a mouse was placed in a bright room with white lighting, and the inside of the box was freely explored. Mice gradually entered the darkroom because they preferred darkrooms, but when they entered the darkroom, they were given a light electrical stimulus. The time the mouse stayed in the bright room (ie, the time it took to enter the dark room) was measured and recorded.
  • the reaction latency was calculated as the absolute value of the difference between the time spent in the bright room in this test and the time spent in the bright room in the preliminary stage. The test was carried out 5 times or more, and the average value of their reaction latency was calculated.
  • Open space swimming test uses the average swimming distance (average swimming distance) of mice placed in a large pool of water as an index to measure the coping behavior and depressive tendency of mice. It is a behavioral test to evaluate.
  • the open space swimming test was conducted by the following method.
  • a mouse was placed in a pool with a diameter of 120 cm and allowed to swim freely.
  • the state of swimming was photographed with a video camera, and the swimming distance for 60 seconds was continuously analyzed for 10 minutes using image analysis software.
  • Rotarod test is a behavioral test that evaluates the motor function and sense of balance of a mouse using the average value (average fall time) of the time it takes for the mouse to fall from the rotating rotor rod as an index. ..
  • the rotor rod test was carried out by the following method.
  • the mouse was placed in the lane of the rotor rod that rotates 20 times per minute, and the time (fall time) until the mouse fell from the rotating rotor rod was measured. The test was carried out three times, and the average value (average fall time) of those fall times was calculated.
  • RNA extraction kit Manton (registered trademark) II 1st reverse cDNA Synthesis Kit (Takara Bio Inc./model number: 6210B (A ⁇ 4)) according to the manufacturer's protocol.
  • CDNA was synthesized (reverse transcription) from the total RNA of.
  • a real-time PCR reaction was carried out using a nucleic acid amplification reagent (PowerUp SYBR (registered trademark) Green Master Mix (Thermo Fisher Scientific / model number: A25777)).
  • a real-time PCR system Agilent AliaMx Real-Time PCR System (Agilent) was used.
  • the real-time PCR reaction conditions are 50 ° C. 3 minutes for 1 cycle, 95 ° C. for 3 minutes for 1 cycle, 95 ° C. for 5 seconds and 60 ° C. for 30 seconds for 40 cycles, 95 ° C. for 30 seconds for 1 cycle, and 65 ° C.
  • the 18S ribosomal RNA gene which is a housekeeping gene, was used as the endogenous control, and the relative expression level of each target gene with respect to the endogenous control was analyzed by the Pfaffl method.
  • the Pfaffl method is a kind of relative quantification method of the comparative Ct method in which the PCR amplification efficiency of the housekeeping gene and the target gene is taken into consideration.
  • Nucleic Acids Res. 2001; 29 (9): e45 The method described in can be used. The test was carried out 5 times or more, and the average value (average relative expression level) of their relative expression levels was calculated. Table 1 shows the base sequences of primers for each gene.
  • Example 1 ⁇ Method> In Example 1, an index for determining dementia and brain function in a subject was examined using a body fluid sample derived from a mouse that developed dementia.
  • Quantitative RT-PCR The relative expression level of each target gene in the body fluid sample of the mouse was analyzed in the same manner as in "1-3. Quantitative RT-PCR" of Comparative Example 1.
  • the average value of the time spent in the bright room in the pre-stage of the mouse of Comparative Example 1 was 31.5 ⁇ 22.9 seconds, whereas the average value of the time spent in the pre-stage of the mouse of Example 1 was 31.5 ⁇ 22.9 seconds.
  • the average time spent in the room was 20.0 ⁇ 14.1 seconds.
  • the average value of the time spent in the bright room of the mouse of Comparative Example 1 in this test was 180.0 ⁇ 0.0 seconds, whereas the mouse of Example 1 stayed in the bright room in the preliminary stage.
  • the average time was 47.8 ⁇ 67.4 seconds.
  • the average response latency of the mouse of Comparative Example 1 was calculated to be 148.5 ⁇ 22.9 seconds, and the average response latency of the mouse of Example 1 was calculated to be 32.8 ⁇ 63.5 seconds. From the above, the mouse of Comparative Example 1 was able to learn and memorize the invasion into the dark room and the fear of aversive stimulus in association with each other, whereas the mouse of Example 1 had a deteriorated learning and memory function. It has been suggested.
  • the average swimming distance of the mouse of Comparative Example 1 was 794 ⁇ 192 cm, whereas the average swimming distance of the mouse of Example 1 was 1193 ⁇ 154 cm.
  • the mouse of Example 1 floats on the water in an immobile posture (that is, without struggling, making only the minimum movement necessary to get the head out of the water surface. There was a tendency to shorten the time to indicate the condition), and there was a tendency to swim around more than necessary. From the above, it was suggested that the coping behavior of the mouse of Example 1 was attenuated as compared with the mouse of Comparative Example 1 and showed a depressive tendency.
  • the average relative expression of the Glut1 gene was 0.56, the average relative expression of the Glut3 gene was 0.67, and the average relative expression of the MCT4 gene.
  • the amount was 0.45, the average relative expression level of the PHD3 gene was 0.58, and the average relative expression level of the PDK1 gene was 0.43.
  • the average relative expression level of the Glut1 gene was 3.39, the average relative expression level of the Glut3 gene was 1.70, the average relative expression level of the MCT4 gene was 3.17, and the PHD3 gene.
  • the average relative expression level of PDK1 gene was 2.33, and the average relative expression level of PDK1 gene was 3.03.
  • the gene expression of the Glut1 gene, the Glut3 gene, the MCT4 gene, the PHD3 gene and the PDK1 gene in the body fluid sample of the mouse of Example 1 was significantly increased. Therefore, as genes whose expression levels change remarkably with the onset of dementia and / or deterioration of brain function, genes of proteins involved in glucose transport (Glut1 gene and Glut3 gene) and genes of proteins involved in lactic acid transport. (MCT4 gene), a gene for a protein involved in the glycolytic system (PHD3 gene), and a gene for a protein involved in the TCA circuit (PDK1 gene) were identified.
  • the possibility of using the above gene and / or the protein encoded by the above gene as an index in determining the onset of dementia and / or the decrease in brain function of the subject was shown.
  • the possibility of using a gene for a protein involved in energy metabolism and / or a protein involved in energy metabolism as an index for determining the onset of dementia and / or a decrease in brain function of a subject was shown. ..
  • the ratio of the average relative expression level of the Glut1 gene to the above reference value was 2.6.
  • the ratio of the average relative expression level of the Glut3 gene to the above reference value was 1.3.
  • the ratio of the average relative expression level of the MCT4 gene to the above reference value was 2.3.
  • the ratio of the average relative expression level of the PHD3 gene to the above reference value was 1.9.
  • the ratio of the average relative expression level of the PDK1 gene to the above reference value was 2.3.
  • the relative expression level of one or more of the above genes as an index higher than the above reference value, it is possible to obtain an index for determining dementia or brain function in a subject. That is, by measuring the relative expression level of one or more of the above genes in the body fluid sample derived from the subject and comparing the relative expression level with the reference value, the determination of dementia in the subject or the brain function can be determined. An index for judgment can be obtained.
  • the gene expression or enzyme activity of one or more proteins involved in the energy metabolism reaction in the sample derived from the subject is measured, and the measured values of the gene expression or enzyme activity of the one or more proteins are used as reference values.
  • an index for determining dementia or determining brain function in a subject can be obtained.
  • Example 2 ⁇ Method> In Example 2, an index for determining the degree of the aging phenomenon in the subject was examined using a body fluid sample derived from a plurality of mice having a known degree of the aging phenomenon.
  • RNA extraction kit Manton (registered trademark) II 1st reverse cDNA Synthesis Kit (Takara Bio Inc./model number: 6210B (A ⁇ 4)) according to the manufacturer's protocol.
  • CDNA was synthesized (reverse transcription) from the total RNA of.
  • a real-time PCR reaction was carried out using a nucleic acid amplification reagent (PowerUp SYBR (registered trademark) Green Master Mix (Thermo Fisher Scientific / model number: A25777)).
  • a real-time PCR system Agilent AliaMx Real-Time PCR System (Agilent) was used.
  • the real-time PCR reaction conditions are 50 ° C. 3 minutes for 1 cycle, 95 ° C. for 3 minutes for 1 cycle, 95 ° C. for 5 seconds and 60 ° C. for 30 seconds for 40 cycles, 95 ° C. for 30 seconds for 1 cycle, and 65 ° C.
  • the 18S ribosomal RNA gene which is a housekeeping gene, was used as the endogenous control, and the relative expression level of each target gene with respect to the endogenous control was analyzed by the Pfaffl method.
  • the Pfaffl method is a kind of relative quantification method of the comparative Ct method in which the PCR amplification efficiency of the housekeeping gene and the target gene is taken into consideration.
  • Nucleic Acids Res. 2001; 29 (9): e45 The method described in can be used. The test was carried out 5 times or more, and the average value (average relative expression level) of their relative expression levels was calculated. Table 3 shows the base sequences of primers for each gene.
  • the average relative expression of the Glut1 gene was 0.42, the average relative expression of the Glut3 gene was 0.27, and the average relative expression of the MCT4 gene.
  • the amount was 0.20, the average relative expression level of the PHD3 gene was 0.35, and the average relative expression level of the PDK1 gene was 0.28.
  • the average relative expression of the Glut1 gene was 0.68, the average relative expression of the Glut3 gene was 0.81, the average relative expression of the MCT4 gene was 0.54, and the average of the PHD3 gene.
  • the relative expression level was 0.62, and the average relative expression level of the PDK1 gene was 0.83.
  • the average relative expression of the Glut1 gene was 1.16, the average relative expression of the Glut3 gene was 1.18, the average relative expression of the MCT4 gene was 1.22, and the average of the PHD3 gene.
  • the relative expression level was 1.12, and the average relative expression level of the PDK1 gene was 1.17.
  • the average relative expression of the Glut1 gene was 1.47, the average relative expression of the Glut3 gene was 1.83, the average relative expression of the MCT4 gene was 2.47, and the average of the PHD3 gene.
  • the relative expression level was 1.74, and the average relative expression level of the PDK1 gene was 1.71.
  • the average relative expression of the Glut1 gene was 1.99
  • the average relative expression of the Glut3 gene was 1.96
  • the average relative expression of the MCT4 gene was 2.82
  • the average of the PHD3 gene was 2.35
  • the average relative expression level of the PDK1 gene was 1.96.
  • Glut1 gene and Glut3 gene the gene of the protein involved in glucose transport
  • MCT4 gene the gene of the gene involved in lactic acid transport
  • a gene for a protein involved in the sugar system (PHD3 gene) and a gene for a protein involved in the TCA circuit (PDK1 gene) were identified.
  • the possibility of using the above gene and / or the protein encoded by the above gene as an index for determining the degree of aging phenomenon in the subject was shown.
  • the possibility of using the gene of the protein involved in energy metabolism and / or the protein involved in energy metabolism as an index for determining the degree of the aging phenomenon in the subject was shown.
  • FIG. 3 shows the relationship between the average relative expression level of each target gene in the body fluid sample of the mouse of Example 2 and the age of the mouse.
  • the relational expression between the average relative expression level of each target gene in the body fluid sample of the mouse of Example 2 and the age of the mouse is represented by the following relational expressions (A) to (E). Was done.
  • the age of the subject can be determined based on the above relational expression using the relative expression level of one or more of the above genes as an index. That is, the age in the subject can be determined by measuring the relative expression level of one or more of the genes in the body fluid sample derived from the subject and using the relative expression level and the relational expression.
  • Example 3 Manufacturing of dementia / brain function judgment kit> A kit for determining dementia or determining brain function (dementia / brain function determination kit) is manufactured by the following procedure.
  • the primer pair for amplifying the Glut1 gene shown in Table 1 was dissolved in TE buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA) to prepare a primer pair solution for Glut1 gene amplification having a final concentration of 10 ⁇ M. do.
  • the primer pair for amplifying the 18S ribosomal RNA gene shown in Table 1 is dissolved in TE buffer to prepare a primer pair solution for amplifying the 18S ribosomal RNA gene at a final concentration of 10 ⁇ M.
  • the primer pair solution for Glut1 gene amplification is used as a reagent for measuring gene expression of Glut1, which is a protein involved in glucose transport.
  • the Glut1 gene amplification primer pair solution the 18S ribosomal RNA gene amplification primer pair solution, an RNA extraction reagent (Mouse RiboPure Blood RNA Isolation Kit (Thermo Fisher Scientific / model number: AM1951)), and a cDNA synthesis reagent.
  • RNA extraction reagent Manton RiboPure Blood RNA Isolation Kit (Thermo Fisher Scientific / model number: AM1951)
  • a cDNA synthesis reagent Combined to form a dementia / brain function determination kit.
  • the dementia / brain function determination kit can be used by the following procedure.
  • RNA is extracted from the body fluid sample using the RNA extraction reagent constituting the dementia / brain function determination kit.
  • cDNA is synthesized using the cDNA synthesis reagent constituting the dementia / brain function determination kit.
  • a real-time PCR reaction is performed using a primer pair solution for Glut1 gene amplification, a primer pair solution for 18S ribosomal RNA gene amplification, and a nucleic acid amplification reagent that constitute the dementia / brain function determination kit.
  • the real-time PCR system uses Agilent AliaMx Real-Time PCR System (Agilent).
  • the real-time PCR reaction conditions are 50 ° C. 3 minutes for 1 cycle, 95 ° C. for 3 minutes for 1 cycle, 95 ° C. for 5 seconds and 60 ° C. for 30 seconds for 40 cycles, 95 ° C. for 30 seconds for 1 cycle, and 65 ° C. for 30 seconds for 1 cycle. , And 95 ° C. for 30 seconds as one cycle.
  • the relative expression level of the Glut1 gene with respect to the 18S ribosomal RNA gene is analyzed by the Pfaffl method. By comparing the relative expression level of the Glut1 gene with respect to the 18S ribosomal RNA gene with 1.30, which is the reference value of the gene expression of the Glut1 gene, the presence or absence of dementia in mice can be determined.
  • Example 4 Manufacturing of aging degree judgment kit> A kit for determining the degree of the aging phenomenon (aging degree determination kit) is manufactured by the following procedure.
  • the primer pair for amplifying the Glut3 gene shown in Table 3 was dissolved in TE buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA) to prepare a primer pair solution for Glut3 gene amplification at a final concentration of 10 ⁇ M. do.
  • the primer pair for amplifying the 18S ribosomal RNA gene shown in Table 3 is dissolved in TE buffer to prepare a primer pair solution for amplifying the 18S ribosomal RNA gene at a final concentration of 10 ⁇ M.
  • the primer pair solution for Glut3 gene amplification is used as a reagent for measuring gene expression of Glut3, which is a protein involved in glucose transport.
  • the Glut3 gene amplification primer pair solution the 18S ribosome RNA gene amplification primer pair solution, an RNA extraction reagent (Mouse RivoPure Blood RNA Isolation Kit (Thermo Fisher Scientific / model number: AM1951)), and a cDNA synthesis reagent.
  • RNA extraction reagent Manton (registered trademark) II 1st strand cDNA Synthesis Kit (Takara Bio Co., Ltd./model number: 6210A) and nucleic acid amplification reagent (PowerUp SYBR (registered trademark) Green Master Mix (Thermo Fisher Scientific) model number: Thermo Fisher Scientific) Combined to form an aging degree determination kit.
  • the aging degree determination kit can be used by the following procedure. Peripheral blood is obtained from multiple mice of known age and some of them are used as body fluid samples for standard preparation. Total RNA is extracted from the analysis sample using the RNA extraction reagent constituting the aging degree determination kit. Using the obtained total RNA as a template, cDNA is synthesized using the cDNA synthesis reagent constituting the aging degree determination kit.
  • a real-time PCR reaction is carried out using a primer pair solution for Glut3 gene amplification, a primer pair solution for 18S ribosomal RNA gene amplification, and a nucleic acid amplification reagent that constitute the aging degree determination kit.
  • the real-time PCR system uses the Agilent AriaMx Real-Time PCR System (Agilent).
  • the real-time PCR reaction conditions are 50 ° C. 3 minutes for 1 cycle, 95 ° C. for 3 minutes for 1 cycle, 95 ° C. for 5 seconds and 60 ° C. for 30 seconds for 40 cycles, 95 ° C. for 30 seconds for 1 cycle, and 65 ° C. for 30 seconds for 1 cycle.
  • the relative expression level of the Glut3 gene with respect to the 18S ribosomal RNA gene is analyzed by the Pfaffl method.
  • the relational expression between the obtained relative expression level and the age of the week is obtained.
  • peripheral blood is collected from the mouse as the subject, and a part of it is used as a body fluid sample to be analyzed.
  • the relative expression level of the Glut3 gene with respect to the 18S ribosomal RNA gene is measured by the same method.
  • the relative expression level of the Glut3 gene in the body fluid sample to be analyzed as an index is determined based on the above relational expression.
  • Comparative Example 2 ⁇ Method> In Comparative Example 2, an index for determining dementia and brain function in a subject was examined using a body fluid sample derived from a human (volunteer donor).
  • MMSE Mini-Mental State Examination
  • the MMSE is a cognitive function test consisting of a total of 11 questions in the form of questions. Time orientation, location orientation, immediate recall, attention and calculation ability, delayed reproduction (short-term memory), linguistic ability, and graphic. Cognitive functions such as ability (spatial cognition) can be evaluated.
  • the maximum score of the MMSE score was 30 points, and a case of 27 points or less was judged to be mild cognitive impairment (MCI), and a case of 23 points or less was judged to be dementia.
  • MCI mild cognitive impairment
  • Peripheral blood was collected from volunteer donors and the proportion of mononuclear cells in the peripheral blood sample was measured using an automatic blood cell counter.
  • RNA isolation PAXgene (registered trademark) RNA collection tube
  • Becton Dickinson The blood collection tube was gently inverted and mixed, and then allowed to stand at room temperature to obtain total RNA derived from the peripheral blood (whole blood) of the volunteer donor. Total RNA was frozen and stored at ⁇ 20 to ⁇ 80 ° C.
  • RNA was obtained as a template, 0.3 ⁇ g of total RNA using a cDNA synthesis kit (PrimeScript (registered trademark) II 1st reverse cDNA Synthesis Kit (Takara Bio Inc./model number: 6210A)) according to the manufacturer's protocol. CDNA was synthesized (reverse transcription) from.
  • a real-time PCR reaction was carried out using a nucleic acid amplification reagent (PowerUp SYBR (registered trademark) Green Master Mix (Thermo Fisher Scientific / model number: A25777)).
  • Agilent AliaMx Real-Time PCR System Agilent AliaMx Real-Time PCR System (Agilent) was used.
  • the real-time PCR reaction conditions are 50 ° C. 3 minutes for 1 cycle, 95 ° C. for 3 minutes for 1 cycle, 95 ° C. for 5 seconds and 60 ° C. for 30 seconds for 40 cycles, 95 ° C. for 30 seconds for 1 cycle, and 65 ° C. for 30 seconds for 1 cycle.
  • And 95 ° C. for 30 seconds was defined as one cycle.
  • the 18S ribosomal RNA gene which is a housekeeping gene, was used as the endogenous control, and the relative expression level of each target gene with respect to the endogenous control was analyzed by the Pfaffl method.
  • the Pfaffl method is a kind of relative quantification method of the comparative Ct method in which the PCR amplification efficiency of the housekeeping gene and the target gene is taken into consideration.
  • Table 5 shows the base sequences of primers for each gene.
  • Example 5 ⁇ Method> In Example 5, a body fluid sample derived from a human (volunteer donor) was used to examine an index for determining dementia and brain function in the subject.
  • MMSE Mini-Mental State Examination
  • Table 6 shows the relative expression level of each target gene with respect to the expression level of the 18S ribosomal RNA gene calculated by the Pfaffl method in the peripheral blood samples of each volunteer donor of Comparative Example 2 and Example 5.
  • age is the age of each volunteer donor
  • mononuclear cells is the ratio of mononuclear cells in the peripheral blood sample of each volunteer donor (relative to the number of all leukocytes contained in the peripheral blood sample).
  • MMSE indicates the MMSE score.
  • the relative expression level of the Glut1 gene was 0.33, the relative expression level of the Glut3 gene was 0.25, the relative expression level of the MCT4 gene was 0.09, and PHD3.
  • the relative expression level of the gene was 0.50, and the relative expression level of the PDK1 gene was 0.72.
  • the relative expression level of the Glut1 gene was 0.45, the relative expression level of the Glut3 gene was 0.26, the relative expression level of the MCT4 gene was 0.19, and the relative expression level of the PHD3 gene was 0.
  • the relative expression level of the PDK1 gene was 0.35.
  • the relative expression level of the Glut1 gene was 0.12
  • the relative expression level of the Glut3 gene was 0.17
  • the relative expression level of the MCT4 gene was 0.72
  • the relative expression level of the PHD3 gene was 0.
  • the relative expression level of the PDK1 gene was 0.73.
  • the relative expression level of the Glut1 gene is 0.55
  • the relative expression level of the Glut3 gene is 0.32
  • the relative expression level of the MCT4 gene is 1.00
  • the relative expression level of the PHD3 gene is 0.
  • the relative expression level of the PDK1 gene was 0.58.
  • the relative expression level of the Glut1 gene was 0.92
  • the relative expression level of the Glut3 gene was 0.41
  • the relative expression level of the MCT4 gene was 0.75
  • the relative expression level of the PHD3 gene was 0.
  • the relative expression level of the PDK1 gene was 0.88.
  • the relative expression level of the Glut1 gene was 1.14, the relative expression level of the Glut3 gene was 1.15, the relative expression level of the MCT4 gene was 1.35, and PHD3.
  • the relative expression level of the gene was 1.11 and the relative expression level of the PDK1 gene was 1.26.
  • the relative expression level of the Glut1 gene was 2.96, the relative expression level of the Glut3 gene was 0.97, the relative expression level of the MCT4 gene was 1.44, and the relative expression level of the PHD3 gene was 2.
  • the relative expression level of the PDK1 gene was 2.28.
  • the relative expression level of the Glut1 gene is 1.52, the relative expression level of the Glut3 gene is 1.56, the relative expression level of the MCT4 gene is 1.38, and the relative expression level of the PHD3 gene is 1.
  • the relative expression level of the PDK1 gene was 1.27.
  • the relative expression level of the Glut1 gene is 1.76, the relative expression level of the Glut3 gene is 0.62, the relative expression level of the MCT4 gene is 2.08, and the relative expression level of the PHD3 gene is 2.
  • the relative expression level of the PDK1 gene was 1.28.
  • the possibility of using the above gene and / or the protein encoded by the above gene as an index in determining the onset of dementia and / or the decrease in brain function of the subject was shown.
  • the possibility of using a gene for a protein involved in energy metabolism and / or a protein involved in energy metabolism as an index for determining the onset of dementia and / or a decrease in brain function of a subject was shown. ..
  • the ratios of the relative expression levels of the Glut1 gene, Glut3 gene, MCT4 gene, PHD3 gene and PDK1 gene to the above reference values were 1.1, 2.3, 1.2 and 1, respectively. It was .2 and 1.3.
  • the ratios of the relative expression levels of the Glut1 gene, Glut3 gene, MCT4 gene, PHD3 gene and PDK1 gene to the above reference values were 3.0, 1.9, 1.3 and 2, respectively. It was 9.9 and 2.3.
  • the ratios of the relative expression levels of the Glut1 gene, Glut3 gene, MCT4 gene, PHD3 gene and PDK1 gene to the above reference values were 1.5, 3.1, 1.3 and 1, respectively. It was .8 and 1.3.
  • the ratios of the relative expression levels of the Glut1 gene, Glut3 gene, MCT4 gene, PHD3 gene and PDK1 gene to the above reference values were 1.8, 1.2, 1.9 and 2, respectively. It was .4 and 1.3.
  • the relative expression level of one or more of the above genes as an index higher than the above reference value, it is possible to obtain an index for determining dementia or brain function in a subject. That is, by measuring the relative expression level of one or more of the above genes in the body fluid sample derived from the subject and comparing the relative expression level with the reference value, the determination of dementia in the subject or the brain function can be determined. An index for judgment can be obtained.
  • the gene expression or enzyme activity of one or more proteins involved in the energy metabolism reaction in the sample derived from the subject is measured, and the measured values of the gene expression or enzyme activity of the one or more proteins are used as reference values.
  • an index for determining dementia or determining brain function in a subject can be obtained.
  • Example 6 based on the indexes for determining dementia and brain function obtained in Comparative Example 2 and Example 5, the dementia and brain function of the human are determined from the body fluid sample derived from a human (volunteer donor). Judged. In Example 6, the Glut1 gene and the PDK1 gene were used as indexes for determining dementia and brain function.
  • Quantitative RT-PCR The relative expression levels of the Glut1 gene and the PDK1 gene in the peripheral blood samples of volunteer donors were analyzed in the same manner as in "6-4. Quantitative RT-PCR" of Comparative Example 2.
  • Table 7 shows the relative expression levels of the Glut1 gene and the PDK1 gene with respect to the expression level of the 18S ribosomal RNA gene calculated by the Pfaffl method in the peripheral blood samples of each volunteer donor of Example 6.
  • MMSE Mini-Mental State Examination
  • Example 7 Manufacturing of dementia / brain function judgment kit> A kit for determining dementia or determining brain function (dementia / brain function determination kit) is manufactured by the following procedure.
  • the primer pair for amplifying the PDK1 gene shown in Table 5 is dissolved in TE buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA) to prepare a primer pair solution for PDK1 gene amplification having a final concentration of 10 ⁇ M. do.
  • the primer pair for amplifying the 18S ribosomal RNA gene shown in Table 5 is dissolved in TE buffer to prepare a primer pair solution for amplifying the 18S ribosomal RNA gene at a final concentration of 10 ⁇ M.
  • the primer pair solution for PDK1 gene amplification is used as a reagent for measuring gene expression of PDK1, which is a protein involved in the TCA cycle.
  • the PDK1 gene amplification primer pair solution the 18S ribosomal RNA gene amplification primer pair solution, a mononuclear cell separation reagent (Filel-Paque PLUS (GE Healthcare Co., Ltd./model number: 17144002)), and RNA extraction.
  • a mononuclear cell separation reagent Filel-Paque PLUS (GE Healthcare Co., Ltd./model number: 17144002)
  • the dementia / brain function determination kit can be used by the following procedure.
  • RNA extraction reagent constituting the dementia / brain function determination kit Collect peripheral blood from the human subject.
  • Mononuclear cells are separated from peripheral blood using the mononuclear cell separation reagent constituting the dementia / brain function determination kit, and a part of the obtained mononuclear cells is used as a body fluid sample to be analyzed.
  • Total RNA is extracted from the body fluid sample using the RNA extraction reagent constituting the dementia / brain function determination kit.
  • cDNA is synthesized using the cDNA synthesis reagent constituting the dementia / brain function determination kit.
  • a real-time PCR reaction is performed using a primer pair solution for PDK1 gene amplification, a primer pair solution for 18S ribosomal RNA gene amplification, and a nucleic acid amplification reagent that constitute the dementia / brain function determination kit.
  • the real-time PCR system uses Agilent AliaMx Real-Time PCR System (Agilent).
  • the real-time PCR reaction conditions are 50 ° C. 3 minutes for 1 cycle, 95 ° C. for 3 minutes for 1 cycle, 95 ° C. for 5 seconds and 60 ° C. for 30 seconds for 40 cycles, 95 ° C. for 30 seconds for 1 cycle, and 65 ° C. for 30 seconds for 1 cycle.
  • the relative expression level of the PDK1 gene with respect to the 18S ribosomal RNA gene is analyzed by the Pfaffl method. By comparing the relative expression level of the PDK1 gene with respect to the expression level of the 18S ribosomal RNA gene with the reference value of the gene expression of the PDK1 gene, it is possible to determine the presence or absence of dementia in humans.

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