JP2007306883A - Method for evaluating chronic stress - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new method for readily, objectively and highly accurately evaluating the condition of chronic stress of a testee. <P>SOLUTION: The condition of the chronic stress of the testee is evaluated by using a messenger-RNA derived from peripheral blood of the testee, based on the result of the expression analysis of a specific marker gene selected as the marker gene of the chromic stress. Further, the gene expression data of the testee are compared and analyzed with the previously obtained gene expression data of a healthy subject and a person in the chronic stress condition. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for evaluating chronic stress. More specifically, evaluation of chronic stress characterized by evaluating the chronic stress state of the subject based on the expression analysis result of the selected marker gene using messenger RNA derived from the peripheral blood of the subject. Regarding the method.

  At present, lifestyle changes and changes in social structure that people are facing have had various effects on human mental and physical health. The increase in stress is closely related not only to mental disorders and psychosomatic disorders in the narrow sense, but also to lifestyle-related diseases and intractable chronic diseases.

  Non-specific reactions that occur when a living body is exposed to harmful environments and factors are defined as "stress". To date, factors induced by stress in various diseases have been identified and related to the disease state, but a method for accurately evaluating the degree of stress has not yet been established. The reason for this is that stress is a complex system reaction and requires various evaluations such as psychology, sociology, physiology, and clinical medicine, and each evaluation method lacks certain indicators.

  For example, as an individual stress evaluation method, evaluation of stressors such as life events and daily irritation, and evaluation of individual biological characteristics such as personality determination tests have been performed, and certain results have been achieved. However, these evaluations require an enormous amount of time and labor, and problems remain in quantitativeness and objectivity. Also, methods for measuring limited factors such as stress hormones, for example, methods for quantifying the concentration of chromogramin in body fluids (Patent Document 1), methods for measuring the activity of nitric oxide synthase in the adrenal gland or pituitary gland (Patent Document 2), a method using an adrenal sex hormone or its metabolite concentration in saliva as an index (Patent Document 3), a method of measuring the amount of glutathione-added hemoglobin (Patent Document 4), and measuring the cortisol concentration in saliva Method (Patent Document 5), method of measuring concentration change rate of monoamines (Patent Document 6), method of measuring V-1 gene expression level or V-1 protein mass (Patent Document 7), KYN in blood Many methods have been proposed, such as a method for measuring the rate of change in concentration (Patent Document 8) and a method using the expression level of the perl gene and perl2 gene as an index (Patent Document 9). , It is difficult to evaluate the reaction of complex systems by endocrine and nervous, immune and (stress reaction).

  The present inventors have comprehensively analyzed biological reactions appearing in peripheral leukocytes using a microarray (DNA chip), and have found a new methodology for objectively evaluating a stress response that is a complex system reaction. It has been applied to the diagnosis of related diseases.

  The inventors have already provided an oligonucleotide array and a test method for evaluating stress and stress-related diseases (Patent Document 10), and using this, an evaluation method for depression (Patent Document 11) and healthy Provided a method (Non-patent Document 1) for graduate students to evaluate acute mental stress that appears at the time of the open examination of a thesis.

  This time, we provide a stress assessment method for mental stress that lasts for a long time, so-called chronic stress. When exposed to intense stress, post-traumatic stress disorder (PTSD) may occur, but most acute stress returns to its original state with no underlying stressors. On the other hand, chronic stress that is continuously exposed to stressors for a long time causes the onset of diseases caused by stress, such as mental illness such as depression, cancer, allergic diseases, etc. It is closely linked to the exacerbation of many diseases such as illness. If this chronic stress state can be easily and accurately evaluated, it will be useful information that can be fed back to the individual's daily life and treatment. It can also be used at medical checkup centers, workplace health management rooms, disaster evacuation sites, etc., and can contribute to improving national life and reducing medical expenses in the field of preventive medicine.

Japanese Patent Laid-Open No. 11-23572 JP-A-8-262025 Japanese Patent Laid-Open No. 11-38004 JP 2000-74923 A JP 2000-275248 A JP 2002-156378 A JP 2003-61654 A JP 2004-198325 A JP 2005-143420 A Japanese Patent Laid-Open No. 2002-34017 JP 2004-208547 A Neuroscience Letters, 381 (2005) 57-62

  An object of the present invention is to provide a novel method for diagnosing a chronic stress state easily, objectively and with high accuracy.

  In order to objectively evaluate the state of chronic stress, the present inventors focused on peripheral blood leukocytes that are easily obtained as specimens and that express many receptors for stress-related factors. Then, by comprehensively analyzing and patterning the expression pattern of messenger RNA of about 1500 genes related to stress response, a method capable of evaluating the state of chronic stress was found, and the present invention was completed.

  That is, the present invention evaluates the subject's chronic stress state based on the expression analysis result of the marker gene selected from Table 1, using messenger RNA derived from the peripheral blood of the subject.

  The genes listed in Table 1 are useful marker genes for chronic stress. According to the doferro-corrected doitall.pair significant difference test, the error rate is 0.05 or less, and 14 genes whose expression is increased 1.5 times or more in the chronic stress state compared with the normal one and 1. It consists of 10 genes whose expression is reduced 5 times or more. Table 1 shows the gene symbol of each marker gene, the average value of the expression ratio, and the p value.

  In the present invention, the expression of a marker gene selected from Table 1 can be examined in a subject's sample to determine whether or not the subject is in a chronic stress state.

  Regarding the marker gene of chronic stress, as described below, the 6th year medical student (chronic Blood was collected from 18 persons and 13 months of medical science students (healthy persons) who were not yet stressed 10 months before taking the doctor's national examination at 16: 00 to 18:00 RNA was extracted from whole blood, gene expression analysis was performed using a DNA chip, and the result was determined. The subject is a person who gave written explanation and consent to participate in the research for developing this evaluation method. In addition to blood sampling, the state-traffic anxiety inventory (STAI) questionnaire surveys state anxiety and characteristic anxiety and assesses the anxiety that the subject feels, thereby applying stress. It was carried out by the method of indirectly evaluating the gap.

  In the examples of the present specification, RNA was extracted from whole blood obtained from a chronically stressed person and a healthy person, and gene expression analysis was performed on the marker genes shown in Table 1 using a DNA chip. A DNA chip is obtained by immobilizing a DNA fragment having a base sequence corresponding to a large number of genes on a support substrate such as glass, and detects RNA in a sample by hybridization. As long as the gene expression can be comprehensively analyzed, other DNA-immobilized samples (DNA arrays, beads, membrane filters, etc.) and quantitative methods may be used instead of the DNA chip.

  The comparative control was a mixture of equal amounts of RNA samples from 13 healthy subjects (universal RNA) for each chronically stressed person. Then, the expression level ratio of each gene between the chronically stressed person RNA and the healthy person universal RNA was obtained, and the gene group having the fluorescence intensity of 300 or more in all the chronically stressed person / healthy person comparison data was used as the analysis target gene. . From this analysis target gene, genes whose expression level was significantly increased or decreased in the chronically stressed / healthy person comparison data were extracted by a significant difference test. Then, compared with healthy subjects, the genes whose expression level is increased 1.5 times or more (14 genes) and those whose gene expression is decreased 1.5 times or more (10 genes) in those who are chronically stressed are those of chronic stress. It was selected as an index for evaluating the state, that is, “a marker gene for chronic stress”.

  The evaluation of the state of chronic stress of the subject can be performed by comparatively analyzing the gene expression profiles of the subject, the chronic stressed state, and the healthy subject with respect to the marker genes described in Table 1.

  For example, a subject-derived RNA sample and a healthy subject universal RNA sample are labeled with fluorescent dyes having different emission wavelengths, and then competitive high on the same chronic stress evaluation DNA chip carrying the genes shown in Table 1. Perform hybridization. The fluorescence intensity of each probe on the chip indicates the expression intensity ratio of each gene of the subject and the healthy subject, and the expression profile of the genes shown in Table 1 above was compared with the expression of the chronic stress state / healthy subject. By subjecting the gene expression profile source data to comparative analysis, the state of chronic stress of the subject can be evaluated. In addition, instead of the healthy subject universal RNA sample, the subject, chronic stress, etc., for the genes listed in Table 1, using the RNA samples of healthy individuals whose sex and age matched that of the chronically stressed subjects. You may perform comparative analysis between a state person and a healthy person. Furthermore, instead of the fluorescence intensity of the universal RNA sample, a so-called 1 that uses a data set obtained by normalizing fluorescence intensity of one color of each of the subject, chronic stressed person, and healthy person between chips and between genes. It can also be carried out by a color method.

  The gene expression analysis method is not limited to a DNA chip, but a nucleic acid hybridization method using other DNA-immobilized sample such as a DNA array or a membrane filter, a quantitative PCR method such as RT-PCR or real-time PCR, Northern Any analysis method known in the art can be used, such as a blot method, a subtraction method, a differential display method, and a differential hybridization method. In particular, a DNA solid-phase sample such as a DNA chip, a DNA array, a membrane filter, and beads is preferable because a large number of genes can be comprehensively analyzed at once.

A probe for detecting a gene can be designed as a sequence complementary to a highly specific region (for example, 3′UTR portion) of a marker gene selected from the genes listed in Table 1 according to a well-known method. A synthetic oligo probe having a length of 25-100 bases or a PCR product having a length of 300-1000 bases can be used. The method for immobilizing the probe on the solid phase is not particularly limited, and the synthesized probe may be spotted on the solid phase or the probe may be synthesized on the solid phase according to a known method. An example of an inspection system using a DNA chip is shown in FIG.
In an actual clinical laboratory, a system having a means for comparing and analyzing gene expression data of a subject and gene expression data of a healthy person and a chronic stressed person acquired in advance is prepared. In this system, in addition to the gene expression data of the subject, the healthy person, and the chronically stressed person, it is further preferable to add and compare the clinical data such as age and sex. For example, if you store the expression data of chronically stressed and healthy individuals as a database divided by age and gender, you can simply retrieve the data that matches the age, sex and the subject from the database. A comparative analysis of the state person and the healthy person can be performed. The allowable age difference is preferably within 5 years. In addition, by letting a computer learn in advance the expression data of a chronically stressed person and a healthy person, and letting the computer determine whether the expression data of the subject is close to the expression pattern of a chronically stressed person or a healthy person The state of chronic stress of the subject can also be evaluated.

  The data analysis method is not limited to cluster analysis, and any analysis method known in the technical field such as a machine learning algorithm such as a support vector machine can be used. An overview of the evaluation system is shown in FIG.

  According to the present invention, the state of chronic stress can be easily and objectively and accurately diagnosed by gene expression analysis of RNA obtained from only a few cc of peripheral blood of a subject. Chronic stress that has been difficult can be easily evaluated. Since the method of the present invention grasps biological functions from a large number of RNA expression levels from a number of RNA expression levels as compared with conventional methods for measuring limited factors, a method for evaluating a complex state such as chronic stress As such, it is appropriate in principle and has great utility.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, this invention is not limited to these.

[Example 1]
1. Chronic stressed and healthy subjects The subjects were those of the Tokushima University School of Medicine 6th year students who gave written explanation and consent to participate in the research for developing this evaluation method. This study has been approved by Tokushima University Hospital Ethics Committee.

  Chronic stressed individuals were selected from 18 sixth-year medical students who had been in a chronic stress state for more than half a year after taking the National Examination for Physicians. 18 people are 10 males and 8 females, and the average age is 25.4 years old and 23 to 32 years old. About these 18 people, the blood sample of each person was collected on the day before the test, and the state anxiety and characteristic anxiety were scored by the STAI questionnaire and evaluated.

  On the other hand, as healthy individuals, 13 students from the Tokushima University School of Medicine 6th grade students who were not yet in the chronic stress state due to preparation for the doctor's national exam were selected 10 months ago. Thirteen people are 5 males and 8 females, with an average age of 23.8 years and 23-27 years. For these 13 people, blood samples of each person were collected and the anxiety and characteristic anxiety were scored and evaluated using the STAI questionnaire as in the case of the chronic stress person.

  FIG. 3 shows the average values (control) of the state anxiety of 18 chronically stressed persons scored by the STAI scale and the state anxiety of 13 healthy persons.

2. Gene expression analysis 5 cc blood samples were collected from 18 persons with chronic stress using PAXgene Blood RNA System (Qiagen), and total RNA was extracted. Blood was collected on an empty stomach from 4 pm to 6 pm, and the doctor collected blood from the elbow vein while resting. Total RNA yield was 5-15 micrograms.

  Next, 5 micrograms of total RNA extracted from each chronically stressed person was taken out and annealed with oligo (dT) 24 primer to which a T7 promoter sequence was added. First strand DNA synthesis was performed. Next, Second strand DNA having a T7 promoter sequence was synthesized using this First strand DNA as a template. Finally, RNA was synthesized with T7 RNA polymerase using Second strand DNA as a template. 6 micrograms of synthesized RNA was annealed with random hexamers and subjected to reverse transcriptase reaction to synthesize fluorescently labeled cDNA by incorporating Cy5-dCTP into the chain.

  After collecting 5 cc of blood from 13 healthy controls, extract total RNA, mix the same amount to make a universal sample, and use Cy3-dCTP instead of Cy5-dCTP as a fluorescent label. cDNA was synthesized by the same procedure except that dCTP was used.

  After mixing equal amounts of two types of cDNAs for comparative analysis, hybridization was performed at 62 ° C. for 12 hours on a DNA chip (Hitachi, Ltd., pharmaceutical response analysis DNA chip). After washing, the fluorescence intensity of each spot was measured with a scanner (ScanArray 5000 manufactured by GSI-Lumonics), and the ratio of the expression level of each gene in the chronic stress state sample and the healthy subject universal sample was determined.

3. Data analysis (selection of marker genes for chronic stress)
The genes to be analyzed were a gene group (1235) having a fluorescence intensity of 300 or more in all 18 sets of data. First, in the universal comparison data of chronic stressed / healthy persons, genes whose expression levels are significantly increased or decreased are extracted by a significant difference test (with Bonferroni correction: doitall.pair: error rate <0.05), Furthermore, genes with a variation of 1.5 times or more were selected. Compared with healthy subjects, there were 14 genes whose expression levels were significantly increased and 10 genes that were significantly decreased in those with chronic stress. These genes are shown in Table 1. These genes are useful as marker genes for evaluating whether or not a subject is in a chronic stress state.

[Example 2] Evaluation of chronic stress by marker gene of subject 1. Subjects As in 1.1 of Example 1, 3 persons one month before the doctor's national exam (here, assumed to be in a chronic stress state) 3 persons 12 months before the doctor's national examination (here, assumed to be healthy) ) As the subject. The sample names were blinded and the subjects A, B, C, D, E, and F were used so that the six subjects did not know whether they were chronically stressed or healthy.

2. Gene expression analysis From the subject, 5 cc of blood was collected from each subject using PAXgene Blood RNA System (Qiagen), and total RNA was extracted. Total RNA yield was 5-10 micrograms. First, first strand DNA synthesis was performed on 5 micrograms of total RNA extracted from each subject by annealing an oligo (dT) 24 primer added with a T7 promoter sequence. Next, Second strand DNA having a T7 promoter sequence was synthesized using this First strand DNA as a template. Finally, RNA was synthesized with T7 RNA polymerase using Second strand DNA as a template. For 6 micrograms of RNA, random hexamers were annealed to carry out reverse transcriptase reaction, and Cy5-dCTP was incorporated into the chain to synthesize fluorescently labeled cDNA.

  The same sample as in Example 1 was used as a universal control for healthy persons. 6 micrograms of Cy5-cDNA prepared from each subject sample and Cy3-cDNA prepared from the universal control sample are mixed in equal amounts, and then applied to a DNA chip (Hitachi's DNA chip for drug response analysis). Hybridization was performed at 62 ° C. for 12 hours. After washing, the fluorescence intensity of each spot is measured with a scanner (GSI-Lumonics ScanArray 5000), and using the digitization software (GSI-Lumonics QuantArray), the universal control sample and each subject sample for each gene The expression intensity ratio was determined.

3. Evaluation of subjects Cluster analysis by hierarchical clustering method using unweighted cosine coefficient distance between clusters together with 18 subjects in the universal comparison of chronically stressed / healthy subjects who analyzed the above 6 subjects. As a result, subjects B, D, and E were classified into the chronic stress state group, and subjects A, C, and F were classified into the healthy group. When comparing the blinded sample names with the results of clustering, subjects B, D, and E were chronically stressed one month before the doctor's national exam, and subjects A, C, and F were 12 months before the doctor's national exam. It was confirmed that he was a healthy person and perfectly matched the clustering results.

  From the above results, it was shown that the evaluation of the chronic stress state based on the expression analysis result of the specific gene group described in Table 1 has very high effectiveness and great utility.

  The method of the present invention is useful as an objective evaluation method for chronic stress in daily life such as workplaces and schools as well as clinical sites.

FIG. 1 is a conceptual diagram of a test system using a chronic stress DNA chip of the present invention. In the figure, F1 represents a DNA chip, F2 represents a probe DNA corresponding to the gene selected in the present invention, F3 represents an excitation light source and a fluorescence detector, and F4 represents a fluorescence detector control computer. FIG. 2 is a conceptual diagram of the chronic stress evaluation system of the present invention. In the figure, information management such as sex and age is stored in the personal information database. FIG. 3 shows the average value of the state anxiety of 18 chronically stressed persons and the state anxiety of 13 healthy persons scored by the STAI scale.

Claims (8)

  Using the messenger RNA derived from the peripheral blood of the subject, the chronic stress state of the subject is evaluated based on the expression analysis result of any marker gene selected from Table 1. Evaluation methods.   A method for evaluating chronic stress characterized by evaluating a chronic stress state of the subject based on the result of expression analysis of the marker gene described in Table 1 using messenger RNA derived from the peripheral blood of the subject .   The chronic expression according to claim 1 or 2, wherein the gene expression analysis is performed by comparing and analyzing a gene expression profile of a subject and a gene expression profile of a chronically stressed person and a healthy person. Stress evaluation method.   The chronic expression according to any one of claims 1 to 3, wherein the gene expression analysis is performed using any one of a DNA solid-phased sample including a chip, an array, a membrane filter, and beads. Stress evaluation method.   The gene expression analysis is performed using any method selected from RT-PCR, real-time PCR, Northern blot method, subtraction method, differential display method, and differential hybridization method. Item 4. The method for evaluating chronic stress according to any one of Items 1 to 3.   A solid phase-immobilized sample for evaluation of chronic stress, wherein each probe specifically hybridizes to the marker gene shown in Table 1 and each probe for detecting the gene is immobilized on the solid phase.   A system for evaluating chronic stress, comprising means for comparing and analyzing gene expression data of a subject and gene expression data of a healthy person and a chronic stress person who have been acquired in advance.   8. The system according to claim 7, further comprising means for comparing and analyzing the data of the age, sex, and the like in addition to the gene expression data of the subject, the healthy person, and the chronically stressed person.

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