JP6544737B2 - Test method and test kit for depression using biomarker - Google Patents

Description

  The present invention relates to a test method for diagnosing depression and a test kit for diagnosing depression.

  According to DSM-V, which is a standard of the American Psychiatric Association, depression is a disease in which symptoms such as depressed mood, marked decline in interest or pleasure, fatigue, power decline, etc. exist for almost two days according to DSM-V, a standard of the American Psychiatric Association It is supposed to be. The depressive symptoms as described above can fall into anyone who is stressed in daily life. And when you have such symptoms, you are anxious about depression. However, such symptoms often end with a temporary one, as the cause of the stress is eliminated, the time of the hobby is enjoyed, or forgotten due to the lapse of time. If the symptoms do not continue for as long as 2 weeks, a diagnosis of depression can not be made. Thus, depression and depressive symptoms tend to be confused, but depressive symptoms are not necessarily depression.

  In fact, there are several diseases that cause depressive symptoms other than depression. Schizophrenia, dementia, eating disorders, cerebral infarction and the like are mentioned as examples. Therefore, even a patient with severe depressive symptoms may not be depression. Therefore, properly diagnosing the cause of depressive symptoms is important for early treatment of the underlying disease. Also, patients with relatively mild depressive symptoms are difficult to diagnose. The diagnosis of depression is made based on the patient's subjective information obtained from interviews, and is easily influenced by the severity of depressive symptoms at the time of consultation. Therefore, there is a need for a method that can objectively examine the onset of depression regardless of the severity of depressive symptoms.

  In search of an objective indicator of depression, the present inventors have so far determined an indicator that can objectively evaluate the presence or severity of depressive symptoms by quantifying the expression of markers such as CIDEC in a subject It discovered (Japanese Patent Application 2014-025568).

  In addition, as an objective index of depression, diagnostic use of brain imaging by PET, NIRS, etc. has begun to be used supplementary. However, the medical facilities that can use them are limited, and there is a bias between facilities, so it has not been possible to establish a uniform evaluation method.

  In addition, as a method for objectively evaluating depression, Patent Document 1 discloses, from messenger RNA derived from peripheral blood of a subject, an apoptosis related gene, an ATPase related gene, a cell cycle related gene, a cytokine related gene, heat shock The expression levels of at least one or more genes selected from a protein related gene, a polymerase related gene, a GTP binding protein related gene, a protein kinase C related gene, and a mitochondrial cytochrome C oxidase related gene are analyzed by a DNA chip, and the analysis result is analyzed A method of evaluating depression is described, which is based on the evaluation of a subject's medical condition. In addition, Patent Document 2 includes FASLG, CX3CR1, TBX21, ID2, SLAMF7, PRSS23, YWHAQ, TARDBP, ADRB2, PPPA8, MMAA, SQLE, PDHA1, HAVCR2, RACGAP1, AHNAK, EDG8, and DUSP5 18 gene expression amounts A method of testing for depression is described, which comprises: determining a subject and determining whether the subject suffers from depression based on the measurement result. However, none of these have been clinically applied.

Unexamined-Japanese-Patent No. 2004-208547 JP 2008-253258 A

  An object of the present invention is to provide a test method for objective depression diagnosis and a test kit used for the test using a sample such as patient's blood, in view of the above problems. .

  As a result of intensive investigations to solve the above-mentioned problems, the present inventors searched for genes whose expression was significantly changed in the depression patient group and the depression remission group as compared with the healthy subject group, and The present invention has been accomplished by finding that it is a useful marker that can objectively determine the onset of depression regardless of the severity of depressive symptoms.

That is, the present invention provides the following.
[1] A step of measuring the expression level of one or more markers selected from the group consisting of IGHA1, THEM4 and TNFRSF25 in a sample collected from a test animal, wherein the expression level of the marker is a reference value A test method for diagnosing depression in an animal, wherein the presence or absence of onset of depression in the subject animal is determined by comparing with.
[2] The test method according to [1], wherein the markers are IGHA1, THEM4 and TNFRSF25.
[3] Furthermore, one species selected from the group consisting of ANKRD35, CLEC3B, FAM26F, NRG1, UTS2, CCDC64, GPR15, GSTM2, IGLV6-57, PDGFB, PRKAR1B, SLC34A1, AIG1, TMX1, ATN1, CD27, CD3E, LZTS2 Or the test method as described in [1] or [2] including the process of measuring the expression level of 2 or more types of markers.
[4] A step of further measuring the expression levels of ANKRD35, CLEC3B, FAM26F, NRG1, UTS2, CCDC64, GPR15, GSTM2, IGLV6-57, PDGFB, PRKAR1B, SLC34A1, AIG1, TMX1, ATN1, CD27, CD3E, and LZTS2 The inspection method according to [1] or [2], including
[5] The test method according to any one of [1] to [4], wherein the sample is blood.
[6] One or more selected from the group consisting of microarray, real-time PCR, ELISA, Western blotting, FACS analysis, spectrophotometry, fluorometry, mass spectrometry, and chromatography The inspection method according to any one of [1] to [5], which is performed by
[7] A test kit for diagnosing depression, comprising a reagent capable of measuring the expression level of one or more markers selected from the group consisting of IGHA1, THEM4 and TNFRSF25.
[8] The test kit according to [7], wherein the markers are IGHA1, THEM4 and TNFRSF25.
[9] Furthermore, one species selected from the group consisting of ANKRD35, CLEC3B, FAM26F, NRG1, UTS2, CCDC64, GPR15, GSTM2, IGLV6-57, PDGFB, PRKAR1B, SLC34A1, AIG1, TMX1, ATN1, CD27, CD3E, LZTS2 Or the test kit as described in [7] or [8] which comprises the reagent which can measure the expression level of 2 or more types of markers.
[10] Further, reagents capable of measuring the expression levels of ANKRD35, CLEC3B, FAM26F, NRG1, UTS2, CCDC64, GPR15, GSTM2IGLV6-57, PDGFB, PRKAR1B, SLC34A1, AIG1, TMX1, ATN1, CD27, CD3E, and LZTS2 The test kit according to [7] or [8], which comprises.

  According to the present invention, IGHA1, THEM4 and TNFRSF25 are provided as biomarkers for depression test. In addition, the method of the present invention makes it possible to diagnose depression based on an objective index regardless of the severity of depressive symptoms.

The results of a forced swimming test and an open field test performed on four groups of Sham + NS, OVX + NS, Sham + CUMS, and OVX + CUMS, including model animals, are shown. The microarray analysis result by the RNA obtained from a test subject and a mouse is shown. The quantitative analysis result of real-time quantitative PCR by RNA obtained from a subject is shown.

Hereinafter, embodiments of the present invention will be described in detail.
(1) Test Method for Diagnosis of Depression The first of the present invention is the expression of one or more markers selected from the group consisting of IGHA1, THEM4 and TNFRSF25 in a sample collected from a subject animal A test method for diagnosing depression in an animal, comprising the step of measuring the amount, wherein the presence or absence of onset of depression in the subject animal is determined by comparing the expression level of the marker with a reference value. .

  In the present specification, “depression” is a depressive disorder according to the diagnostic criteria of WHO “ICD-10” or “Manual for diagnosis and statistics of mental diseases, DSM-V)” of the American Psychiatric Association. , Its severity is SIGH-D (Hamilton Depression Rating Scale: Structured Interview Guide for the Hamilton Depression Rating Scale), HAM-D (Hamilton Depression Rating Scale: Hamilton Rating Scale for Depression), MADRS (Montgomery There is no particular limitation on depression as long as it can be determined by conventional diagnostic methods such as Asberg Depression Rating Scale: Montgomery Asberg Depression Rating Scale or BDI (Beck Depression Inventory). As long as being diagnosed with depression, it also includes the case where other diseases such as anxiety disorder, sleep disorder and eating disorder are coexisted.

  In the present specification, IGHA1 means immunoglobulin heavy constant alpha 1. The human gene sequence is available from GenBank under the accession number S55735.

  In the present specification, THEM 4 means thioesterase superfamily member 4. Human gene sequences are available from GenBank under the accession number NM-053055.

  As used herein, TNFRSF 25 means tumor necrosis factor receptor superfamily, member 25. The human gene sequence is available from GenBank under the accession number NM_001039664.

  In the test method of the present invention, the amount of expression of the marker in the sample collected from the subject animal can be measured, and the presence or absence of onset of depression can be inspected using this as an index. In addition, the expression levels of IGHA1, THEM4 and TNFRSF25 may all be measured. In addition, the expression levels of other markers for depression test may be combined. Furthermore, more accurate examination can be performed by using a composite index associated with observation of clinical symptoms and results of brain image diagnosis by PET, NIRS and the like.

Other markers for depression test to be used in combination are ANKRD35, CLEC3B, FAM26F, NRG1, UTS2, CCDC64, GPR15, GSTM2, IGLV6-57, PDGFB, PRKAR1B, SLC34A1, AIG1, TMX1, ATN1, C
It is preferable to use one or more markers selected from the group consisting of D27, CD3E and LZTS2. Also, all the markers may be measured.

  As used herein, ANKRD35 means ankyrin repeat domain 35. The human gene sequence is available from GenBank under the accession number NM_144698.

  In the present specification, CLEC3B means C-type lectin domain family 3, member B. Human gene sequences are available from GenBank under the accession number NM_003278.

As used herein, FAM26F refers to family with sequence similarity 26, member
Means F. The human gene sequence is available from GenBank under the accession number NM_001010919.

  As used herein, NRG1 refers to neuregulin 1. Human gene sequences are available from GenBank under the accession number NM_004495.

  In the present specification, UTS2 means urotensin 2. Human gene sequences are available from GenBank under the accession number NM_021995.

  As used herein, CCDC 64 means coiled-coil domain containing 64. Human gene sequences are available from GenBank under the accession number NM_207311.

  As used herein, GPR15 means G protein-coupled receptor 15. Human gene sequences are available from GenBank under the accession number NM_005290.

  As used herein, GSTM2 means glutathione S-transferase mu 2 (muscle). The human gene sequence is available from GenBank under the accession number NM_000848.

  As used herein, IGLV6-57 refers to immunoglobulin globulin variable 6-57. Human gene sequences are available from GenBank under the accession number BC023973.

  In the present specification, PDGFB means platelet-derived growth factor beta polypeptide. Human gene sequences are available from GenBank under the accession number NM_002608.

  In the present specification, PRKAR1B means protein kinase, cAMP-dependent, regulatory, type I, beta. The human gene sequence is available from GenBank under the accession number NM_001164761.

  In the present specification, SLC34A1 refers to solute carrier family 34 (sodium phosphate), member 1. Human gene sequences are available from GenBank under the accession number NM_003052.

  As used herein, AIG1 means androgen-induced 1. Human gene sequences are available from GenBank under the accession number NM_ 016108.

In the present specification, TMX1 means thioredoxin-related transmembrane protein 1 Human gene sequences are available from GenBank under the accession number NM_030755.

  As used herein, ATN1 refers to atrophin 1. The human gene sequence is available from GenBank under the accession number NM_001007026.

  As used herein, CD27 means CD27 molecule. Human gene sequences are available from GenBank under the accession number NM_001242.

  As used herein, CD3E means CD3 molecule, epsilon (CD3-TCR complex). The human gene sequence is available from GenBank under the accession number NM_000733.

  As used herein, LZTS2 means leucine zipper, putative tumor suppressor 2. The human gene sequence is available from GenBank under the accession number NM_032429.

  With regard to the gene sequence information, when the test animal is different from human, the homolog derived from the animal is to be measured. As the measurement of the expression level of the above marker, mRNA may be measured or protein may be measured. Furthermore, the above markers also include fragments, derivatives and variants having the same function as the above markers.

  In the above test method, the “test animal” may be any animal as long as it may cause depression, and specifically, it may be a primate such as a human or a monkey, a mouse, a rat or the like. Examples thereof include rodents, mammals such as dogs, cats, pigs, ferrets and the like. Among the above, the method for testing depression of the present invention is particularly preferably performed in a human suspected of having depression.

  Moreover, as a "sample" extract | collected from the said test animal, it will not be restrict | limited in particular, if it contains mRNA or protein of the said marker and can measure the density | concentration. Specifically, blood, plasma, serum, urine, saliva and the like can be mentioned. Among these, blood, plasma and serum are preferably used because they can be collected simply, are easy to store, and have a large collection amount. The blood or the like is preferably subjected to heparin treatment immediately after collection. In addition, it is preferable to use mRNA or protein extracted from nucleated cells (mainly white blood cells) in blood.

  The method for measuring the expression level of the above marker is not particularly limited as long as it is a method capable of measuring the content of the mRNA or protein thereof. For example, as the measurement of mRNA, PCR method, microarray method, Northern blot method, Spectrophotometry, fluorometry and the like can be mentioned. In particular, real-time PCR by PCR method is preferred in terms of simplicity and speed of measurement. The primers used for PCR can be designed by known methods based on the gene sequences of the above markers, and for example, the primers described in Tables 3 and 4 can be used.

  The method of measuring the protein of the above marker includes FACS analysis, spectrophotometry, fluorometry, mass spectrometry, chromatography, as well as existing immunoassay using an antibody against the protein, chromatography technology and flight. Combined mass spectrometry (TOF-MS), mass of all components captured under fixed conditions on chromatography carrier (eg cation exchanger, anion exchanger, hydrophobic chromatography carrier, metal ion etc) Measurement method, two-dimensional gel electrophoresis, etc. are used.

As the immunoassay, a Western blot method, a method of quantitative detection in accordance with an enzyme immunoassay, a method of measurement by a fluorescence immunoassay, a chemiluminescence immunoassay, etc. are preferable. Enzyme-linked immunosorbent assay is a detection method using an enzyme as a labeling substance among labeled immunoassay methods. Among them, it is particularly preferable to select an enzyme-linked immunosorbent assay (ELISA) method using an immunosorbent. Further, among ELISAs, the sandwich method is mentioned as one of particularly preferable measurement modes in consideration of the easiness of operation, the economic convenience, especially the versatility in clinical examination. These measuring methods are described, for example, in the Laboratory for Neo-Chemistry of Chemistry (edited by the Japan Biochemical Society; Tokyo Kagaku Dojin), Molecular Cloning, A Laboratory Manual (T. Maniatis et al., Cold Spring Harbor Laboratory (2001)), Antibodies-A Laboratory It can carry out in accordance with the method as described in general experiment books, such as Manual (E.Harlow, et al., Cold Spring Harbor Laboratory (1988)), or it.

  An antibody or the like (including an antibody fragment) used when performing the above-mentioned measurement can be obtained by a method known as a target marker protein as an antigen. However, the protein of the target marker does not have to be produced as an antigen, and it may be any one that exhibits at least cross-reactivity with the protein and whose content can be measured.

  As the sample used in the method of the present invention, it is preferable to use the sample immediately after collection from the subject animal for the above measurement, but it is also possible to use a preserved sample. The blood sample storage method is not particularly limited as long as the marker amount for depression test does not change, but in the case of mRNA, for example, a reagent for RNA extraction is added and it is carried out at -80 ° C or liquid nitrogen It is preferable to cryopreserve. In the case of protein, low temperature conditions such as 0 to 10 ° C. without freezing, dark conditions and vibrationless conditions are preferable, and in the case of constant freezing, decomposition or oxidation reaction of marker molecules such as deep freezer etc. A method that can avoid

  By comparing the expression level of the obtained marker with a reference value, the presence or absence of onset of depression can be determined. As a reference value, the expression level of the marker in a healthy person or a subject who does not develop depression is exemplified. This reference value may be measured together at the time of the marker measurement of the subject animal, or the expression level of the marker is previously measured in a healthy person or a subject who does not develop depression, etc. You may set it. When the expression level of the obtained marker and the reference value are compared, and the expression level in the test animal is significantly different from the reference value, it is determined that depression is developed. The “significantly different from the reference value” is not a difference in the error range, but, for example, a significant difference that can be statistically indicated by a P value or the like. The expression level in the test animal may be significantly different from the reference value, and the expression level in the test animal may be higher or lower than the reference value. For example, IGHA1, THEM4 and TNFRSF25 determine that there is a onset of depression if they are significantly lower than the reference value.

(2) Test Kit for Diagnosis of Depression The second of the present invention comprises a reagent capable of measuring the expression level of one or more selected from the group consisting of IGHA1, THEM4 and TNFRSF25. The present invention relates to a test kit for diagnosing depression. All IGHA1, THEM4 and TNFRSF25 may be included. The content of the kit is configured by a combination of devices or reagents, but the composition or form is as long as it contains a substance which is essentially the same as each component described below, or essentially the same as a part thereof Even if different, they are included in the kit of the present invention.

  The kit of the present invention further comprises the group consisting of ANKRD35, CLEC3B, FAM26F, NRG1, UTS2, CCDC64, GPR15, GSTM2, IGLV6-57, PDGFB, PRKAR1B, SLC34A1, AIG1, TMX1, ATN1, CD27, CD3E, LZTS2 A reagent capable of measuring one or more selected expression levels may be included. All reagents capable of measuring the expression level of the above marker may be included in the kit.

As the reagent, for example, in the case of measuring the expression level of the above marker by the PCR method,
It contains a primer capable of specifically amplifying the marker. Primers can be prepared by known methods based on the gene sequences of the markers described above. In addition, reverse transcriptase, Taq polymerase, reaction buffer and the like may be included, as necessary. In the case of real time PCR, fluorescent reagents, fluorometers, thermal cyclers and the like are also included.

  In the case of measuring the expression level of a marker by microarray, the marker gene fragment is spotted on a support such as glass, plastic, silicon or membrane. In addition, if necessary, a labeling reagent, a buffer for hybridization, a buffer for fragmentation, and the like may be included.

  When the expression level of the above marker is measured by immunoassay, it contains an antibody against the marker. In addition, as necessary, a dilution liquid of a biological sample, an antibody-immobilized solid phase, a reaction buffer, a washing solution, a labeled secondary antibody or an antibody fragment thereof, a reagent for detecting a labeled form, a standard substance and the like are included. As a dilution liquid of a biological sample, an aqueous solution containing a protein such as BSA or casein as a surfactant or a buffer may be mentioned.

  As the antibody-immobilized solid phase, one obtained by immobilizing an anti-marker antibody or an antibody fragment thereof on a material obtained by shaping various polymer materials to suit the application is used. Shapes are tubes, beads, plates, fine particles such as latex, sticks etc., and materials are polystyrene, polycarbonate, polyvinyl toluene, polypropylene, polyethylene, polyvinyl chloride, nylon, polymethacrylate, gelatin, agarose, cellulose, polyethylene terephthalate etc. Polymer materials, glass, ceramics, metals and the like. Examples of methods for immobilizing antibodies include known methods such as physical methods and chemical methods, or a combination of these methods. For example, a 96-well polystyrene microtiter plate for immunoassays in which an antibody or antibody fragment or the like is immobilized on a hydrophobic surface can be mentioned.

  Any reaction buffer may be used as long as it provides a solvent environment for the binding reaction between the antibody on the antibody-immobilized solid phase and the antigen in the biological sample, but a surfactant, a buffer, BSA, or the like may be used. Examples include reaction buffers containing proteins such as casein, preservatives, stabilizers, reaction accelerators and the like.

  As a labeled secondary antibody or antibody fragment thereof, an antibody or antibody fragment used in the present invention labeled with an enzyme for labeling such as horseradish peroxidase (HRP), calf intestinal alkaline phosphatase, β-galactosidase, etc., buffer A mixture of proteins such as BSA and casein and preservatives is used.

  As a reagent for detecting a labeled form, depending on the enzyme for labeling described above, for example, in the case of horseradish peroxidase, a substrate for absorbance measurement such as tetramethylbenzidine or orthophenylenediamine, fluorescence such as hydroxyphenylpropionic acid or hydroxyphenylacetic acid If the substrate and the luminescent substrate such as luminol are alkaline phosphatase, substrates for absorbance measurement such as 4-nitrophenyl phosphate, fluorescent substrates such as 4-methyl umbelliferyl phosphate and the like can be mentioned.

  The invention will be further described with reference to the following non-limiting examples.

<Subject recruitment>
Among patients with major depression (MDD) who are being treated at Gunma University Hospital, their first age is 50 years or older, they exhibit melancholic form of major depression in the DSM-IV classification, and the merger of other mental illness The subjects were those who did not approve. The severity of MDD was scored by the Structured Interview Guide for the Hamilton Depression Rating Scale (SIGH-D), and remission was scored less than 8 points.
We recruited age- and gender-matched healthy individuals as controls. We confirmed that there was no history of mental illness or family history in recruited healthy people. Cognitive function tests (Mini Mental State Examination; MMSE) were performed on recruited subjects to confirm that their cognitive function was normal.
Table 1 below shows the recruited subjects.

DP:中高年発症うつ病患者（病相期）
RM:中高年発症うつ病患者（寛解期）
HC:健常者
SIGH-D：ハミルトンうつ病評価尺度構造化面接法
MMSE: 認知機能検査

DP: Middle-aged and elderly onset depression patients (disease phase)
RM: Middle-aged and elderly onset depression patients (remitting phase)
HC: Healthy people
SIGH-D: Hamilton Depression Rating Scale Structured Interview Method
MMSE: cognitive function test

  Two patients with late-onset depression declined MMSE. Data are expressed as mean ± standard error (mean ± SE). The asterisk (*) in the table indicates that the p value for the corresponding healthy group is p <0.05 (Bonferroni test).

<Extraction of RNA from subject's blood>
10 mL of blood was collected from the subject, and RNA was extracted from nucleated cells (mainly white blood cells) in the blood using QIAmp RNA Blood Mini Kit (QIAGEN KK). The extraction procedure was performed with some modifications to the description of the instruction manual attached to the kit. The operation is summarized as follows. The collected 10 ml of blood was divided in half into 50 ml tubes, and 25 ml of buffer EL was added to each tube and thoroughly mixed by inversion. After placing each tube in ice for 20 minutes, it was centrifuged at 480 × g for 15 minutes at 4 ° C., and the supernatant was discarded. The pellet in the tube was resuspended in 10 ml Buffer EL, vortexed and resuspended. The resuspended sample was centrifuged at 480 × g for 15 minutes at 4 ° C., and the supernatant was discarded. 3 ml of buffer RLT containing 2-mercaptoethanol was added to the pellet and dissolved by vortexing. The lysate was homogenized by centrifugation for 2 minutes at room temperature on QIAshredder spin columns. An equal volume of 70% ethanol was added to the homogenized lysate and mixed well by vortexing. The mixed sample was added to the QIAamp spin column and centrifuged for 15 seconds. After washing the column, it was digested with DNaseI. The elution step was performed according to the instructions attached to the kit. The eluted sample was processed by standard ethanol precipitation. The RNA pellet was dissolved in RNase free water.
The amount of RNA was measured by Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific Inc.).

<Preparation of model animals>
Female C57BL / 6J mice (8 weeks old) were purchased from Charles River Laboratories Japan, and after 1 week of acclimatization, the ovaries were removed under pentobarbital anesthesia (Ovariectomy; OVX). Similarly, pentobarbital anesthesia was performed, and a ovary exposed but sutured without excision was used as a control animal (Sham-operation; Sham).
From 2 weeks after the above treatment, loading of chronic ultra-mild stress (CUMS) was started. The method of CUMS applied the load for six weeks according to the previous report (Uchida et al., Neuron, 69, 359-372, 2011). In addition, the group which did not perform the stress load during the same period (Nonstress:
Provided an NS group). That is, this test was performed with four groups of Sham + NS, Sham + CUMS, OVX + NS, and OVX + CUMS.

The forced swimming test and the open field test were conducted on the first day after stopping the load of the CUMS.
The forced swimming test was conducted as follows. The N number of this test was Sham + NS (n = 24), Sham + CUMS (n = 24), OVX + NS (n = 24), and OVX + CUMS (n = 24). Room temperature water was poured to an acrylic cylinder (height 22 cm, diameter 11.5 cm) to a height of 15 cm, and the mouse was placed therein. The behavior of the mouse was recorded for 5 minutes by a CCD camera connected to a personal computer, and analyzed by ImageJ PS1 (manufactured by Obara Medical Industries Co., Ltd.). The recorded immobility time was used as an indicator of depression-like behavior.

  The open field test was conducted as follows. The N number of this test was Sham + NS (n = 9), Sham + CUMS (n = 8), OVX + NS (n = 8), and OVX + CUMS (n = 9). The open field device (50 × 50 × 40 cm) was illuminated with light emitting diodes (30 lux in the middle of the field) and the mouse was placed at the center of the field and allowed to move freely for 5 minutes. The total distance traveled within the central area (36% of the field) and the time spent in the central area were taken as indicators. The collected data was analyzed using ImageJ OF4 (manufactured by Ohara Medical Industry Co., Ltd.).

  The results of animal behavior analysis are shown in FIG. OVX + CUMS mice exhibited depression-like behavior in the forced swim test and anxiety-like behavior in the open field test. On the other hand, no behavioral change was observed in Sham + CUMS mice and OVX + NS mice. From these behavioral analysis results, OVX + CUMS mice were defined as a model of depression, and Sham + Nonstress mice were used as a control group.

<RNA extraction from mouse blood>
After collecting 300 μL of blood from the vena cava of the mouse under pentobarbital anesthesia, the cells were immediately heparinized and centrifuged at 1000 × g for 2 minutes. The resulting pellet was dissolved in a cooled lysis buffer containing 2-mercaptoethanol. From the lysate, total RNA was extracted using the GeneJet Whole Blood RNA Purification Mini Kit (Thermo Fisher Scientific Inc.) according to the instructions attached to the kit.
The amount of RNA was measured by Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific Inc.).

<Microarray>
The RNA obtained from the subject and the mouse was labeled with Cyanine 3 according to the instruction attached to the kit using Agilent Low Input Quick Amp Labeling Kit, one-color (Agilent Technologies). The labeled RNA was purified by RNeasy mini spin column (QIAGEN). The resulting labeled RNA is fragmented and hybridized for 17 hours at 65 ° C. with Agilent SurePrint G3 Human GE 8 × 60 K v2 Microarray (Design ID: 039494) and Agilent SurePrint G3 Mouse GE 8 × 60 K Microarray (Design ID: 028005) After this, the microarray was washed and measured by Agilent DNA microarray scanner. The luminescence intensity was quantified by Agilent feature extraction software version 10.7.3.1 and standardized by Agilent GeneSpring GX version 11.0.2.

  Statistical analysis is performed on the basis of the obtained data, and among the genes whose expression fluctuates in the stage of depression and remission, patients with 1,130 genes, which have particularly remarkable fluctuation compared to healthy people, and depression We found 637 kinds of genes whose expression changes specifically in model mice (FIG. 2). As a result of collating the gene lists, seven matching genes were found (Table 2).

<Quantitative analysis by real-time quantitative PCR in subjects>
After reverse transcription of the RNA obtained from the subject using the PrimeScript RT reagent Kit (Takara Bio Inc.) to obtain a cDNA sample, the cDNA sample and the specificity against the candidate biomarkers obtained from the results of the subject or mouse microarray Real-time quantitative PCR was performed by mixing the primer pairs (Table 3 and Table 4) with SYBR Premix Ex Taq II (Takara Bio Inc.) or Premix Ex Taq (Takara Bio Inc.). Real-time quantitative PCR was performed with 50 cycles of 95 ° C. for 3 seconds and 60 ° C. for 30 seconds by StepOnePlus real-time PCR system (Applied Biosystems). When primer pairs in Table 3 and SYBR Premix Ex Taq II are used as internal standard substances, RPS 29 is used. When primer pairs in Table 4 and Premix Ex Taq are used, 18S-rRNA, GAPDH, GUSB. , HPRT1 was selected. The amount of amplification of the target gene was quantitatively analyzed according to the ΔΔCt method, and compared with that of a healthy person.

  Based on the results of microarray analysis, among RNA expression profiles that are fluctuating in expression during depression and in remission, patients with depression have particularly remarkable fluctuation compared to healthy people, and real-time quantitative PCR The 14 genes that could be detected by analysis are shown in Table 5 below.

  From the results of real-time PCR analysis of a total of 21 marker candidate genes described in Tables 2 and 5 above, IGHA1, THEM4 and TNFRSF25 were regarded as the most important measurement items, and the remaining 18 genes were regarded as supplementary measurement items. The results of real-time PCR analysis of IGHA1, THEM4 and TNFRSF25 are shown in FIG. FIG. 3 shows that the expression of IGHA1, THEM4 and TNFRSF25 was significantly reduced in the disease phase and remission phase of depression as compared to healthy subjects.

  From the above results, it was shown that IGHA1, THEM4 and TNFRSF25 are markers excellent in depression reflection, whose expression fluctuates in real-time quantitative PCR. Furthermore, depression can be tested with high accuracy by using other 18 markers in combination.

SEQ ID NO: 1: Sequence of IGHA1.
Sequence number 2: Sequence of THEM4.
SEQ ID NO: 3: Sequence of TNFRSF25.
SEQ ID NO: 4: Sequence of ANKRD 35.
SEQ ID NO: 5: Sequence of CLEC 3B.
SEQ ID NO: 6: Sequence of FAM26F.
SEQ ID NO 7: Sequence of NRG1.
SEQ ID NO: 8: sequence of UTS2.
Sequence number 9: arrangement | sequence of CCDC64.
SEQ ID NO: 10: Sequence of GPR15.
SEQ ID NO: 11: Sequence of GSTM2.
SEQ ID NO: 12: Sequence of IGLV 6-57.
SEQ ID NO: 13: Sequence of PDGFB.
SEQ ID NO: 14: Sequence of PRKAR1B.
SEQ ID NO: 15: Sequence of SLC34A1.
SEQ ID NO: 16: Sequence of AIG1.
Sequence number 17: sequence of TMX1.
SEQ ID NO: 18: Sequence of ATN1.
SEQ ID NO: 19: Sequence of CD27.
SEQ ID NO: 20: Sequence of CD3E.
SEQ ID NO: 21: Sequence of LZTS2.
SEQ ID NO: 22: AIG forward primer.
SEQ ID NO: 23: AIG reverse primer.
SEQ ID NO: 24: ATN1 forward primer.
SEQ ID NO: 25: ATN1 reverse primer.
SEQ ID NO: 26: CD27 forward primer.
SEQ ID NO: 27: CD27 reverse primer.
SEQ ID NO: 28: CD3 Eforward primer.
SEQ ID NO: 29: CD3 Reverse primer.
SEQ ID NO: 30: LZTS2 forward primer.
SEQ ID NO: 31: LZTS2 reverse primer.
SEQ ID NO: 32: TMX1 forward primer.
SEQ ID NO: 33: TMX1 reverse primer.
SEQ ID NO: 34: TNFRSF 25 forward primer.
SEQ ID NO: 35: TNFRSF 25 reverse primer.

  According to the method of the present invention, depression can be diagnosed based on an objective index regardless of the severity of depressive symptoms, which is useful in the field of medical diagnosis.

Claims (10)

  Measuring the expression level of one or more markers selected from the group consisting of IGHA1, THEM4 and TNFRSF25 in a sample collected from a test animal, and comparing the expression level of the marker with a reference value The test method for the diagnosis of the depression in this animal by which the presence or absence of the onset of the depression of a test animal is determined.   The test method according to claim 1, wherein the markers are IGHA1, THEM4 and TNFRSF25.   Furthermore, one or two species selected from the group consisting of ANKRD35, CLEC3B, FAM26F, NRG1, UTS2, CCDC64, GPR15, GSTM2, IGLV6-57, PDGFB, PRKAR1B, SLC34A1, AIG1, TMX1, ATN1, CD27, CD3E, LZTS2 The test method according to claim 1, comprising the step of measuring the expression level of the above marker.   Furthermore, a step of measuring the expression level of ANKRD35, CLEC3B, FAM26F, NRG1, UTS2, CCDC64, GPR15, GSTM2, IGLV6-57, PDGFB, PRKAR1B, SLC34A1, AIG1, TMX1, ATN1, CD27, CD3E, and LZTS2 is included. The inspection method according to claim 1 or 2.   The test method according to any one of claims 1 to 4, wherein the sample is blood.   The measurement is performed by one or more selected from the group consisting of microarray, real-time PCR, ELISA, Western blotting, FACS analysis, spectrophotometry, fluorometry, mass spectrometry, and chromatography. The inspection method according to any one of claims 1 to 5.   A test kit for diagnosing depression, comprising a reagent capable of measuring the expression level of one or more markers selected from the group consisting of IGHA1, THEM4 and TNFRSF25.   The test kit according to claim 7, wherein the markers are IGHA1, THEM4 and TNFRSF25.   Furthermore, one or two species selected from the group consisting of ANKRD35, CLEC3B, FAM26F, NRG1, UTS2, CCDC64, GPR15, GSTM2, IGLV6-57, PDGFB, PRKAR1B, SLC34A1, AIG1, TMX1, ATN1, CD27, CD3E, LZTS2 The test kit according to claim 7 or 8, comprising a reagent capable of measuring the expression level of the above marker.   Furthermore, it contains a reagent capable of measuring the expression level of ANKRD35, CLEC3B, FAM26F, NRG1, UTS2, CCDC64, GPR15, GSTM2, IGLV6-57, PDGFB, PRKAR1B, SLC34A1, AIG1, TMX1, ATN1, CD27, CD3E, and LZTS2 The test kit according to claim 7 or 8, which comprises

Images