WO2023143328A1 - 用于预测或诊断抑郁症复发的标志物组合及其应用 - Google Patents

用于预测或诊断抑郁症复发的标志物组合及其应用 Download PDF

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WO2023143328A1
WO2023143328A1 PCT/CN2023/072988 CN2023072988W WO2023143328A1 WO 2023143328 A1 WO2023143328 A1 WO 2023143328A1 CN 2023072988 W CN2023072988 W CN 2023072988W WO 2023143328 A1 WO2023143328 A1 WO 2023143328A1
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depression
risk
recurrence
hsp90
bicc1
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French (fr)
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王闯
徐佳
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宁波大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/30—Psychoses; Psychiatry
    • G01N2800/304—Mood disorders, e.g. bipolar, depression
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/54—Determining the risk of relapse

Definitions

  • the invention belongs to the technical fields of biology and medicine, and in particular relates to a combination of biomarkers for predicting and/or diagnosing recurrent depression patients and related applications thereof.
  • MDD Major depressive disorder
  • the main difficulties in the diagnosis and treatment of depression are: 1slow onset of antidepressant drugs; 2low medication compliance; 3slow development of new drugs; 4high relapse rate. Especially prone to relapse has become the focus and difficulty of increasing the severity of depressive symptoms and prevention and treatment of depression. Studies have shown that 34-83% of patients with major depression experience a new depressive episode within 6 months. It is worth noting that 60% of patients with depression have the risk of developing new depressive episodes after the first episode, and the probability of relapse is as high as 70% to 90% in patients with depression who have had two or more episodes. The long-term recurrence of chronic depression causes depression to become a chronic major mental disease, which brings difficulties to the prevention and treatment of depression.
  • the diagnosis of depression recurrence is mainly based on the patient's detailed medical history and mental symptoms, and comprehensive judgments are made through subjective scores such as the Hamilton Depression Scale (HAMD) and the Montgomery Depression Scale (MADRS).
  • the subjective experience of patients is different, and the diagnostic results are also different.
  • HAMD Hamilton Depression Scale
  • MADRS Montgomery Depression Scale
  • researchers from all over the world have been working on finding biomarkers of depression recurrence and establishing effective detection methods in recent years.
  • Peripheral blood of patients with depression is easy to obtain, less traumatic, and cheap, and is easy to automate detection. It can effectively assist uncertain human factors in the current diagnosis and reduce the rate of misdiagnosis.
  • the markers have good sensitivity and specificity.
  • the purpose of the present invention is to provide a highly sensitive and highly specific specific marker for predicting and/or diagnosing the risk of depression recurrence and its application.
  • a use of a gene, transcript, protein, or detection reagent thereof for a depression recurrence risk marker is provided for the preparation of a diagnostic reagent or kit, and the diagnostic reagent or kit To assess the risk of relapse of depression;
  • depression recurrence risk marker is selected from the following group:
  • M Genes, transcripts, or proteins of one or more markers selected from M1-M3: (M1) HSP90; (M2) HIF-1 ⁇ ; (M3) BICC1.
  • the depression recurrence risk markers include genes, transcripts, or proteins of M1 markers (HSP90).
  • the depression recurrence risk markers include genes, transcripts, or proteins of M2 markers (HIF-1 ⁇ ), preferably, proteins of M2 markers (HIF-1 ⁇ ).
  • the depression recurrence risk markers further include the gene, transcript, or protein of the M3 marker (BICC1).
  • the diagnostic reagent or kit is used to detect the level of the risk marker in the sample to be tested.
  • the sample to be tested is selected from the group consisting of blood, plasma, serum, or a combination thereof.
  • the expression levels of the risk markers include the expression levels in blood, plasma or serum.
  • the assessment includes early diagnosis, auxiliary diagnosis, or a combination thereof.
  • the genes, mRNA, cDNA, or proteins of the risk markers are derived from humans.
  • the detection reagent is coupled with or bears a detectable label.
  • the detectable label is selected from the group consisting of chromophores, chemiluminescent groups, fluorophores, isotopes or enzymes.
  • the antibody is a monoclonal antibody or a polyclonal antibody.
  • the diagnostic reagents include antibodies, primers, probes, sequencing libraries, nucleic acid chips (such as DNA chips) or protein chips.
  • the nucleic acid chip includes a substrate and specific oligonucleotide probes spotted on the substrate, and the specific oligonucleotide probes include Probes that specifically bind to polynucleotides (mRNA or cDNA).
  • the sample to be tested is from a subject selected from the group consisting of subjects without depression, subjects susceptible to depression, first-episode patients with depression, patients with recurrent depression, or a combination thereof.
  • the kit further includes a label or an instruction, and the label or instruction indicates that the kit is used for (a) diagnosing the recurrence risk of depression, and/or (b) evaluating depression relapse treatment effect.
  • the label or instructions indicate the following content: if the transcription levels of HSP90 and BICC1 in the risk markers of the test subject are significantly higher than the reference value of the control, it indicates that the test subject has depression. The risk of relapse is higher than that of general depressed patients.
  • the transcription levels of HSP90 and BICC1 among the risk markers of the test subject are not higher than the reference value of the control, it indicates that the test subject has a low risk of depression recurrence.
  • the label or instructions indicate the following content: If the translation level of the risk marker of the test subject is significantly higher than the control reference value, it indicates that the test subject has a higher risk of depression recurrence than normal of depressed patients.
  • the translation level of the risk marker in the test subject is not higher than the control reference value, it indicates that the test subject has a low risk of depression recurrence.
  • the label or instructions indicate the following:
  • the subject has a higher probability of relapse of depression than general depressed patients.
  • control reference value C0 is the concentration of the risk marker in the same sample in the normal population or patients without relapse of depression.
  • the "significantly higher than” refers to the ratio of C1/C0 ⁇ 1.5, preferably ⁇ 2, more preferably ⁇ 3.
  • the protein quantity or activity of HSP90 or the mRNA level increases, it indicates that the risk of depression relapse is increased in the test subject.
  • the protein quantity or activity or mRNA level of BICC1 and the protein quantity or activity of HIF-1 ⁇ are increased, it indicates that the risk of depression relapse is increased in the test subject.
  • HSP90 protein or mRNA level and the protein amount or activity of HIF-1 ⁇ increase, it indicates that the risk of relapse of depression in the test subject is increased.
  • HSP90, HIF-1 ⁇ and BICC1 or the mRNA level of HSP90 and BICC1 increase, it indicates that the risk of relapse of depression is increased in the test subject.
  • the expression level C1 of two or three risk marker genes selected from the following group or their combination is significantly higher than the control reference value C0, it indicates that the subject has a high risk of depression recurrence: (M1) HSP90 ; (M2) HIF-1 ⁇ ; (M3) BICC1.
  • a marker set for predicting the risk of depression recurrence includes 2-3 markers selected from M1 to M3:
  • M3 A gene, transcript, or protein of BICC1.
  • a depression recurrence risk assessment device comprising:
  • an input module which is used to input the data of depression recurrence risk markers in the blood, plasma or serum of a test subject
  • the risk marker gene is selected from the following group:
  • M genes, transcripts, or proteins selected from one or more markers of M1-M3: (M1) HSP90; (M2) HIF-1 ⁇ ; (M3) BICC1;
  • a data processing module the data processing module is used to process the depression recurrence risk marker data, and give the recurrence risk assessment value, wherein, the processing includes: for the transcription or expression level C1 of the input marker and Compared with the reference value C0, wherein, when C1 is significantly higher than C0, it indicates that the subject has a high risk of depression recurrence; when C1 is not significantly higher than C0, it indicates that the subject has a low risk of depression recurrence; and (c) An output module, the output module is used to output the evaluation result.
  • the "significantly higher than” refers to the ratio of C1/C0 ⁇ 1.5, preferably ⁇ 2, more preferably ⁇ 3.
  • the device further includes a detection module, which is used to detect the transcript level, protein level, or protein activity of the risk marker.
  • the detection module is selected from the group consisting of a PCR detector, a sequencer, or a combination thereof.
  • the input module is also used for inputting quantitative scoring data of depression based on the Hamilton Depression Scale.
  • the processing module further performs comprehensive data processing on the depression quantification score data and relapse risk assessment value, so as to provide a treatment plan for adjuvant treatment or intervention treatment.
  • the treatment plan is used to reduce the risk of depression relapse of the test subject, or to prevent the test subject from relapse of depression.
  • Fig. 1 shows the peripheral blood of patients with first-episode major depression in Example 1 of the present invention and the peripheral blood HSP90 of patients with relapse and non-relapse after drug treatment, HIF-1 ⁇ and BICC1 protein content changes (Fig. 1A) and their relationship with depression Correlation analysis of symptom recurrence (Fig. 1B,C).
  • Fig. 2 shows the scheme roadmap of Chronic Unpredictable Mild Stress (CUMS)-induced depression relapse model in Example 2 of the present invention.
  • SPT sugar water preference test
  • OFT field test
  • NSFT novel environment feeding suppression test
  • FST forced swimming test
  • the criteria for behavioral judgment and evaluation were as follows: (1) Experimental In the baseline period, the criteria for animal inclusion were: sugar water preference ranged from 60% to 90%, and the range of threading times in voluntary activities was 150-200 times/5 minutes. (2) After the first 5-week CUMS and the second 5-week CUMS modeling, the screening criteria for depression-like and non-depression-like animals: 1Depressed-like mice: sugar water preference ⁇ 60% and its decrease >30%. 2 For non-depressed/normal control animals: the sugar water preference range is still 60%-90% and the decrease or increase is ⁇ 30%.
  • Fig. 3 shows the initial sugar water preference test (SPT) evaluation in Example 2 of the present invention.
  • SPT sugar water preference test
  • Fig. 4 shows the depressive behavior test after the first stress in Example 2 of the present invention.
  • 129 mice screened by SPT were subjected to SPT, OFT, NSFT and FST depression-like behavior test results.
  • Round 1-SPT screening and state scoring Figure 3
  • 129 mice were included for 5-week CUMS modeling, and the initial chronic Unpredictable mild stress induces depression-like behaviors.
  • Fig. 5 shows the evaluation of depressive behavior after Fluoxetine drug treatment in Example 2 of the present invention.
  • Fig. 6 shows the SPT depressive behavior evaluation of mice after being washed with Fluoxetine in Example 2 of the present invention.
  • Fig. 7 shows the evaluation of depression relapse behavior after secondary stress in Example 2 of the present invention.
  • SPT, OFT, NSFT and FST were tested and analyzed after the secondary stress (RE-CUMS), combined with SPT and state scoring criteria to screen 17 NCs into the group, and 18 rats to the secondary stress depression group (RE-CUMS-DE) , 15 rats in the secondary stress non-depressed group (RE-CUMS-w/o DE).
  • Figure 8 shows the expression and transcription evaluation of HSP90, HIF-1 ⁇ and BICC1 in the prefrontal cortex after depression relapse in Example 2 of the present invention.
  • RE-CUMS significantly induced HSP90, HIF-1 ⁇ and BICC1 protein expression significantly increased.
  • the transcriptional changes of hsp90 and bicc1 were significantly increased, but hif-1 ⁇ was not significantly increased.
  • the results of immunohistochemical studies showed that RE-CUMS significantly induced HSP90 and BICC1 positive neurons markedly increased.
  • the present inventors unexpectedly discovered new depression recurrence risk markers for the first time, including (M1) HSP90, (M2) HIF-1 ⁇ , and/or (M3 ) BICC1, and correspondingly developed methods and kits for predicting and/or judging the risk of depression recurrence.
  • the present invention has been accomplished on this basis.
  • the depression recurrence risk markers or combinations thereof of the present invention can effectively warn or diagnose the potential risk of recurrence in patients with depression, and use the depression recurrence risk marker group to predict and/or detect the possibility of recurrence in depression patients or disease state, with high accuracy and specificity, and provides a reference standard for early warning and/or diagnosis of depression recurrence in clinical practice.
  • the application detection system based on the markers of depression recurrence or its combination of the present invention provides a relatively objective technical means for evaluating depression recurrence, which can effectively avoid the problem of diagnostic bias caused by scale analysis and subjective experience evaluation, To provide a new detection method for improving the objectivity and accuracy of depression relapse assessment.
  • depression recurrence risk marker of the present invention relapse risk marker of the present invention
  • risk marker of the present invention relapse risk marker of the present invention
  • disease Any one or more of the recurrence risk markers (M1)HSP90, (M2)HIF-1 ⁇ , (M3)BICC1.
  • the risk markers of the present invention include genes (DNA), transcripts (mRNA), cDNA, proteins or combinations thereof.
  • the proteins of the markers of the present invention may or may not contain an initial methionine.
  • the term also includes full-length depression relapse risk marker protein and fragments thereof.
  • the depression recurrence risk marker protein includes its complete amino acid sequence, its secreted protein, its mutant and its functionally active fragment.
  • HIF-1 ⁇ is hypoxia-inducible factor 1-alpha (Hypoxia-inducible factor 1-alpha), gene name: HIF1AN (Homo sapiens), UniProtKB ID: Q9NWT6, NCBI Gene: 55662. Functions as a master transcriptional regulator of the adaptive response to hypoxia. Under hypoxic conditions, activates the transcription of more than 40 genes, including erythropoietin, glucose transporters, glycolytic enzymes, vascular endothelial growth factor, HILPDA, and other genes whose protein products increase oxygen delivery or promote metabolic adaptation to hypoxia . Plays an important role in embryonic vascularization, tumor angiogenesis, and the pathophysiology of ischemic disease.
  • HSP90 is heat shock protein 90 (Heat shock protein HSP 90), including alpha subunit (gene name: HSP90AA1-Human, UniProtKB ID: P07900, NCBI Gene: 3326) and beta subunit (gene name: HSP90AB1-Human, UniProtKB ID : P08238, NCBI Gene: 3326).
  • HSP90 is a molecular chaperone that promotes maturation, structure maintenance, and proper regulation of specific target proteins, for example involved in cell cycle control and signal transduction. Goes through a functional cycle associated with its ATPase activity, which is critical for its chaperone activity. Involved in regulating substrate recognition, ATPase cycle and chaperone function.
  • BICC1 double-tailed C homolog 1 (Protein bicaudal C homolog 1), gene name: BICC1-Human, UniProtKB ID: Q9H694, NCBI Gene: 80114.
  • BICC1 as a potential RNA-binding protein, acts as a negative regulator of Wnt signaling and may be involved in regulating gene expression during embryonic development.
  • the inventors unexpectedly discovered that HSP90 is involved in the regulation of initiation of stress-induced depression relapse.
  • HSP90 can bind to the PAS region of HIF-1 ⁇ in an oxygen-dependent manner, preventing the ubiquitination and degradation of HIF-1 ⁇ . Therefore, the present invention proposes that the recurrence of depression is significantly related to the upregulation of BICC1, and the overexpression of HSP90 is induced by stress conditions, thereby initiating its regulation of HIF-1 ⁇ , rapidly increasing the level in the brain, and participating in various pathological, physiological stress and tissue stress in the brain. Growth and development, and then induce depression recurrence.
  • BICC1 was significantly increased in the peripheral blood, prefrontal cortex and hippocampus of patients with depression, further clarifying that BICC1 can be used as a key molecule in the pathogenesis of a new type of depression.
  • the inventors provided clinical detection evidence for research prediction, and found that HSP90, HIF- The expression levels of 1 ⁇ and BICC1 were significantly higher than those in the control group, and the expression levels of HSP90, HIF-1 ⁇ and BICC1 were significantly higher in the relapsed group after drug treatment than in the non-relapsed group, indicating that peripheral blood HSP90 in patients with relapsed depression, High expression of HIF-1 ⁇ and BICC1.
  • HSP90, HIF-1 ⁇ and BICC1 were correlated with depression recurrence. Utilizing these data, the present invention has developed and verified a detection method/system for predicting the risk of depression recurrence, which is used to predict the recurrence risk of depression.
  • the invention also provides the construction of chronic unpredictable mild stress (CUMS) induced depression relapse model and its screening scheme.
  • CUMS chronic unpredictable mild stress
  • a preferred construction model and screening scheme may include the following steps:
  • the present invention Based on the high expression of depression recurrence risk markers HSP90, HIF-1 ⁇ , BICC1, or their combination in blood, plasma or serum, the present invention also provides a corresponding method for diagnosing depression recurrence.
  • the present invention relates to a diagnostic test method for quantitative and localized detection of human depression relapse risk markers HSP90, HIF-1 ⁇ , BICC1, or a combination thereof at the gene, transcript level or protein level.
  • HSP90, HIF-1 ⁇ , BICC1, or a combination thereof at the gene, transcript level or protein level.
  • These assays are well known in the art.
  • the genes, transcript levels and protein levels of human depression recurrence risk markers HSP90, HIF-1 ⁇ , BICC1, or their combinations detected in the test can be used to diagnose (including auxiliary diagnosis) the risk of depression recurrence.
  • a preferred method is to perform quantitative detection of mRNA or cDNA by PCR.
  • a preferred method is quantitative detection of mRNA or cDNA, sequencing.
  • a preferred method is to quantitatively detect the proteins of depression recurrence risk markers.
  • a method for detecting whether there is a marker protein in a sample is to use a specific antibody for detection, which includes: combining the sample with the protein specificity of HSP90, HIF-1 ⁇ , BICC1, or a combination of depression risk markers Antibody contact: observe whether an antibody complex is formed, and the formation of an antibody complex indicates the presence of depression recurrence risk markers HSP90, HIF-1 ⁇ , BICC1, or a combination of proteins in the sample.
  • the depression recurrence risk marker protein or its polynucleotide can be used for the diagnosis of depression recurrence.
  • a part or all of the polynucleotides of the present invention can be fixed on microarrays or DNA chips as probes for analysis of differential expression of genes in mononuclear cells and gene diagnosis.
  • the anti-depression relapse risk marker antibody can be immobilized on the protein chip to detect the depression relapse risk marker protein in the sample.
  • HSP90, HIF-1 ⁇ , and BICC1 can be used alone or in combination as markers for detecting or diagnosing (especially auxiliary diagnosis and/or early diagnosis) the risk of depression recurrence.
  • the ratio (C1/C0) of the expression level C1 of the marker gene (ie HSP90, HIF-1 ⁇ , BICC1) to the corresponding expression level C0 in the normal population is ⁇ 1.5, preferably ⁇ 2, more preferably ⁇ 3, it can be regarded as an increased risk of depression recurrence.
  • the expression levels of 2 or 3 markers are comprehensively detected, more accurate detection results can be obtained.
  • depression recurrence risk markers HSP90, HIF-1 ⁇ , BICC1, or a combination thereof can be used as a diagnostic marker for depression recurrence risk.
  • the present invention also provides a kit for diagnosing the risk of depression recurrence, the kit contains a detection reagent, and the detection reagent is used to detect the depression recurrence risk markers HSP90, HIF-1 ⁇ , BICC1 gene, transcript , protein, or a combination thereof.
  • the kit contains the anti-depression relapse risk markers HSP90, HIF-1 ⁇ , BICC1 antibodies or immunoconjugates of the present invention, or active fragments thereof; or specifically amplifies depression relapse risk markers Primers or primer pairs, probes or chips for mRNA or cDNA of HSP90, HIF-1 ⁇ , BICC1.
  • the kit further includes a label or an instruction, which indicates that the kit is used for diagnosing the risk of depression recurrence and/or evaluating the therapeutic effect of depression recurrence.
  • the risk markers of the present invention can efficiently and accurately predict the risk of depression recurrence
  • the detection system of the present invention can provide accurate early warning and assess the risk of depression recurrence.
  • Real-time fluorescent quantitative PCR is an experimental method that applies fluorescent energy technology to polymerase chain reaction.
  • a fluorescent dye called SYBR Green I is used in this experiment.
  • SYBR Green I In the PCR reaction system, after SYBR Green I is specifically incorporated into the DNA double strand, it will emit a fluorescent signal; the SYBR dye molecules that are not incorporated into the strand will not emit any fluorescent signal. Because this method keeps the increase of the fluorescent signal synchronized with the increase of the PCR product, that is, the intensity of the fluorescent signal emitted by the fluorescent dye is directly proportional to the DNA yield. Therefore, the initial concentration of the target sequence can be obtained by detecting the fluorescence signal intensity during the PCR process, thereby achieving the purpose of quantification.
  • the subjects were Ningbo Kangning Hospital affiliated to Ningbo University and Ningbo Mental Health Center uniformly included 20 first-episode depression patients and recurrent depression patients, and recruited 20 age- and gender-matched healthy volunteers. For control. All physicians participating in the diagnosis work have the qualifications of psychiatrists and have more than 10 years of experience in psychiatry. They are proficient in using the SCID-1 checklist, proficient in the ICD-10 and DSM-V diagnostic criteria, and use the Hamilton Depression Scale ( HAMD-17) evaluates the psychopathological state of the patient, the operation standard is unified, the consistency test meets the requirements (Kappa 0.68-0.82), and the depressed patients with comorbidities are excluded. This study was approved by the Ethics Committee of Ningbo University, and all research subjects signed the informed consent form. The next morning after enrollment, 20ml of venous blood was drawn on an empty stomach for scientific research.
  • First-episode depression patients group (1) Inclusion criteria: All cases meet the diagnostic criteria for depressive episodes (F32) in the International Classification of Mental and Behavioral Disorders ICD-10, and this episode is the first episode; (2) Statistics: Han nationality, aged 18-60 years, primary school education or above, gender, duration, medication, etc.; (3) Hamilton Depression Scale (17 items, HAMD-17) score greater than or equal to 17; (4) the patient himself or their legal guardians signed the informed consent; (5) were included in the Ningbo Kangning Hospital affiliated to Ningbo University and Ningbo Mental Health Center. (6) Exclusion criteria: the same as the depression recurrence group mentioned above.
  • Recurrent depressive disorder group (1) Inclusion criteria: all cases meet the diagnostic criteria for recurrent depressive disorder (F33) in the International Classification of Mental and Behavioral Disorders ICD-10, and have had at least one depressive episode in the past; (2) Statistics : Han nationality, age 18-60 years old, primary school education or above, gender split, course of disease, medication, etc.; (3) Hamilton Depression Scale (17 items, HAMD-17) score greater than or equal to 17 points; (4) Patient I or my legal guardian signed the informed consent; (5) All were included in the Ningbo Kangning Hospital affiliated to Ningbo University and Ningbo Mental Health Center, with complete medical records such as "first visit record” and "discharge record”; (6) ) Exclusion criteria: other mental disorders currently or in the past; combined with severe or chronic physical diseases and brain organic diseases (neurological degenerative diseases, brain trauma or cerebrovascular diseases); users of psychoactive substances; pregnant and lactating women Those who have communication difficulties; those who do not cooperate; those who have multiple depressive episodes
  • Healthy control group (1) Inclusion criteria: no previous and current mental illnesses and definite physical illnesses; no family history of mental illnesses; (2) Statistics: Han nationality, age 18-60 years, primary education and above, gender Half, course of disease, medication, etc.; (3) Hamilton Depression Scale (17 items, HAMD-17) score less than 7 points; (4) Exclusion criteria: the same as the above-mentioned depression recurrence group and first-episode group.
  • HDRS-17 Hamilton Depression Scale
  • HDRS-17 Hamilton Depression Scale
  • HSP90, HIF-1 ⁇ and BICC1 in the peripheral blood of patients with first-episode major depression and the peripheral blood of patients with relapse and non-relapse after drug treatment found that HSP90, HIF-1 ⁇ and BICC1 in major depression were significantly higher than those in the control group , HSP90, HIF-1 ⁇ and BICC1 were significantly increased after drug treatment compared with the non-relapse group (Fig. 1A).
  • HSP90, HIF-1 ⁇ and BICC1 were correlated with depression recurrence
  • HSP90, HIF-1 ⁇ and BICC1 were initially found to be biomarkers of depression recurrence in clinical cases (Fig. 1B, C).
  • CUMS chronic unpredictable mild stress
  • Figure 2 Construction of chronic unpredictable mild stress (CUMS)-induced depression relapse model ( Figure 2) and evaluation of related depression-like behaviors and brain pathological changes: 150 C57BL/6 male adult mice were selected for model construction (Fig. 3), the CUMS model uses each 5-week stress. After the mice acclimated to the environment for one week, the SPT behavioral evaluation was performed, and healthy male mice with normal behavioral performance were screened (Fig. 4A-D). First, the mice were randomly divided into the control group and the experimental group. The mice in the control group were fed normally, and the mice in the experimental group were stimulated by CUMS for 5 weeks. mouse.
  • CUMS chronic unpredictable mild stress
  • mice were divided into two groups: one group was given Fluoxetine (20 mg/kg/day), and the other group and the control group were given the same dose of normal saline (Figure 5).
  • the mice recovered from fluoxetine treatment were given CUMS again for 5 weeks, and after another 5 weeks of slow stress, the depressive mice with CUMS again, the non-depressed mice after CUMS again and the control mice were screened out. , and three groups of mice were studied.
  • the CUMS methods used in this experiment include: swimming in cold water (4°C, 5min), swimming in hot water (42°C, 5min), tilting the mouse cage (tilting 45°C, 24h), feeding in pairs (24h), fasting (24h) , water deprivation (24h), day and night reversal (24h), continuous lighting (36h), wet mouse cage (24h, adding appropriate water to the mouse cage), a total of nine stimuli were randomly arranged to complete within 4 weeks, and the same stimulus could not Appears consecutively, making animals unable to predict the occurrence of stimuli.
  • the experimental group was reared in a single cage, and the control group was reared in pairs.
  • Administration method before each administration, the fluoxetine hydrochloride capsule is dissolved in the normal saline, is mixed with the fluoxetine solution of 10mg/ml, carries out intragastric gavage administration to mice every day during administration and gives fluoxetine ( 10mg/kg) or the same dose of normal saline for 3 weeks.
  • Drug washout period It is known that the metabolic elimination half-life of fluoxetine in the human body is 4 to 6 days, while in mice, the elimination half-life of fluoxetine is 9 hours. While other references have reported that the elimination half-life of fluoxetine in mice varies, the drug elimination period in this experiment was one week.
  • Sucrose Preference Test Before the start of the experiment, the mice underwent 1 week of sugar water adaptation training, and two drinking bottles were placed on each mouse cage, containing 1% sucrose water and normal water respectively. Tap water and sucrose water were freshly prepared daily.
  • SPT Sucrose Preference Test
  • first deprive water for 23 hours, then place two bottles of water (1% sucrose water and ordinary tap water) weighed in the same mouse cage, after drinking for 1 hour, remove the two bottles of water and weigh them , calculating the animal's preference for sugar water: the animal's preference for sugar water (%) sugar water consumption (g)/[sugar water consumption (g)+tap water consumption (g)].
  • the experiment time is arranged between 8:00-10:30.
  • Open-field test The JLBehv animal behavior analysis system purchased from Shanghai Geely Company was used to track and analyze the activity of mice.
  • the system includes an open box for field activities and a correspondingly configured computer. system.
  • the open box is made of opaque material, with an inner diameter of 75cm long x 75cm wide x 40cm high. Soundproof enclosure with video recording and lighting around the perimeter.
  • the mice are placed in the central area of the open box, and the video equipment can automatically record the data of the mouse's activity distance, number of activities, rest time, activity distance in the central area, and average speed of activity within 5 minutes. After each mouse finished a field experiment, the excrement in the open box should be completely removed before the next mouse test.
  • the experimental time is arranged between 8:00-14:00.
  • Novelty Suppressed Feeding Test Use a test box with a side length of 50cm and a height of 40cm, and a 2cm-high sawdust on the bottom of the box. Place sugar pills or small pieces of food on white paper in the center of the bottom of the box. Before the experiment, the animals were fasted for 24 hours. During the experiment, the mice were put into the box from any corner of the box, observed for 5 minutes through the camera system, and the momentum and time of the mice were recorded to reflect the degree of anxiety and depression of the mice. Put it back into the cage and record the consumption of affairs within 5 minutes to exclude the influence of appetite difference on the time of eating.
  • novel depression relapse risk markers including (M1) HSP90, (M2) HIF-1 ⁇ , and/or (M3) BICC1 .
  • peripheral blood HSP90, HIF-1 ⁇ and BICC1 as biomarkers to diagnose the condition of patients with recurrence of depression
  • an objective diagnosis method for recurrence of depression can be realized, and the diagnosis of depression has a higher Sensitivity and specificity.
  • HSP90, HIF-1 ⁇ , BICC1 can be expressed at the protein and gene levels
  • gene screening and protein expression level detection can be used to judge the potential risk of depression relapse or the status of depression relapse process.

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Abstract

本发明提供了用于预测或诊断抑郁症复发的标志物组合及其应用，具体地，本发明提供了检测来自检测对象的样品中HSP90，HIF-1α，BICC1基因和蛋白水平的试剂材料和/或仪器设备在制备用于诊断抑郁症复发风险的检测设备中的应用。单独或联合使用HSP90、HIF-1α、BICC1蛋白，HSP90、BICC1的转录本作为外周血液生物标记物检测其含量用于预测和诊断抑郁症复发，可提高抑郁症首发与复发的鉴别诊断准确性，检测方法更方便快速，患者依从性高，有助于解决抑郁症早期预警和复发诊断困难的问题。本发明的抑郁症复发风险标志物可作为诊断抑郁症复发和评价抑郁症复发治疗效果的标记物，具有高灵敏度和特异性等优点。

Description

用于预测或诊断抑郁症复发的标志物组合及其应用 技术领域
本发明属于生物学和医学技术领域，具体地，涉及用于预测和/或诊断复发型抑郁患者的生物标志物组合及其相关应用。
背景技术
抑郁症(Major depressive disorder，MDD)是由多种因素引起的以情绪持续低落为主要症状的情感障碍性精神疾病，其作为重大精神疾病之一，也被认为是最主要致残原因之一。2019年世界卫生组织统计显示全球有超过3.5亿人罹患抑郁症，我国组织完成的3万余人的流行病学调查显示，中国的抑郁症12个月的患病率为3.6％，而终身患病率则达到6.9％，抑郁症的防治刻不容缓。
目前，抑郁症的诊治面临的主要困难在于：①抗抑郁药物起效慢；②用药依从性低；③新药开发慢；④复发率高等。尤其易复发已经成为抑郁症状日益加重及抑郁防治的重点与难点。研究表明，34-83％的重症抑郁患者在6个月内出现新的抑郁发作。值得注意的是，60％的抑郁症患者在第一次发作后有发展新的抑郁发作的风险，而发作过二次或以上的抑郁症患者，其复发的几率高达70％～90％。长期的慢性抑郁复发，导致抑郁症成为一种慢性重大精神疾病，为抑郁症的防治带来困难。
临床上，对抑郁症复发的诊断主要是根据病人的详细病史与精神症状，通过汉密尔顿抑郁量表(Hamilton Depression Scale，HAMD)和蒙哥马利抑郁量表(MADRS)等主观评分来做综合判断，由于医师的主观经验不同，诊断结果也存在差异。为了使抑郁症复发的诊断更加客观，减少人为因素，提高诊断的一致性和准确度，近年来世界各地的科研工作者一直致力于寻找抑郁症复发的生物标志物，建立有效的检测方法。抑郁症患者外周血易于获取，创伤较小，且价格便宜，易于自动化检测，可以有效辅助当前诊断中不确定的人为因素，降低误诊率，标志物具有良好的具备良好的灵敏度和特异度。
然而，本领域当前尚未见抑郁症复发的生物标志物相关报道。因此，本领域迫切需要开发用于临床诊疗的高灵敏度和特异度的抑郁症复发的标志物，用于解决抑郁症复发诊断困难的问题，并且需要一种预测和/或诊断抑郁症复发风险的检测系统，来提高抑郁症复发评估的客观性和准确性。
发明内容
本发明的目的就是提供一种高灵敏和高特异性地对抑郁症复发风险进行预测和/或诊断的特异性标志物及其应用。
在本发明的第一方面，提供了一种抑郁症复发风险标志物的基因、转录本、蛋白、或其检测试剂的用途，用于制备一诊断试剂或试剂盒，所述诊断试剂或试剂盒用于评估抑郁症的复发风险；
其中，所述的抑郁症复发风险标志物选自下组：
(M)选自M1-M3的一种或多种标志物的基因、转录本、或蛋白：(M1)HSP90；(M2)HIF-1α；(M3)BICC1。
在另一优选例中，所述的抑郁症复发风险标志物包括M1标志物(HSP90)的基因、转录本、或蛋白。
在另一优选例中，所述的抑郁症复发风险标志物包括M2标志物(HIF-1α)的基因、转录本、或蛋白，优选地，包括M2标志物(HIF-1α)的蛋白。
在另一优选例中，所述的抑郁症复发风险标志物进一步包括M3标志物(BICC1)的基因、转录本、或蛋白。
在另一优选例中，所述的诊断试剂或试剂盒用于检测待测样品中所述风险标志物的水平。
在另一优选例中，所述的待测样品选自下组：血液、血浆、血清、或其组合。
在另一优选例中，所述风险标志物的表达水平包括血液、血浆或血清中的表达水平。
在另一优选例中，所述的评估包括早期诊断、辅助性诊断、或其组合。
在另一优选例中，所述风险标志物的基因、mRNA、cDNA、或蛋白来源于人。
在另一优选例中，所述的检测试剂偶联有或带有可检测标记。
在另一优选例中，所述可检测标记选自下组：生色团、化学发光基团、荧光团、同位素或酶。
在另一优选例中，所述的抗体是单克隆抗体或多克隆抗体。
在另一优选例中，所述诊断试剂包括抗体、引物、探针、测序文库、核酸芯片(如DNA芯片)或蛋白质芯片。
在另一优选例中，所述的核酸芯片包括基片和点样在基片上的特异性寡核苷酸探针，所述的特异性寡核苷酸探针包括与所述风险标志物的多核苷酸(mRNA或cDNA)特异性结合的探针。
在另一优选例中，所述待测样品来自选自下组的对象：无抑郁症的对象、抑郁易感对象、抑郁症的首发患者、抑郁症复发患者、或其组合。
在另一优选例中，所述的试剂盒还包括标签或说明书，所述标签或说明书注明所述试剂盒用于(a)诊断抑郁症的复发风险，和/或(b)评价抑郁症的复发治疗效果。
在另一优选例中，所述的标签或说明书中注明以下内容：如果检测对象的所述风险标志物中的HSP90和BICC1的转录水平显著高于对照参比值，则提示检测对象抑郁症的复发风险高于一般的抑郁症患者。
在另一优选例中，如果检测对象的所述风险标志物中的HSP90和BICC1的转录水平不高于对照参比值，则提示检测对象抑郁症的复发风险较低。
在另一优选例中，所述的标签或说明书中注明以下内容：如果检测对象的所述风险标志物的翻译水平显著高于对照参比值，则提示检测对象抑郁症的复发风险高于一般的抑郁症患者。
在另一优选例中，如果检测对象的所述风险标志物的翻译水平不高于对照参比值，则提示检测对象抑郁症的复发风险较低。
在另一优选例中，所述的标签或说明书中注明以下内容：
如果检测对象的所述风险标志物浓度C1显著高于对照参比值C0，则该对象复发抑郁症的几率大于一般抑郁症患者。
在另一优选例中，所述的对照参比值C0为正常人群或无抑郁症复发患者中相同样本中的所述风险标志物的浓度。
在另一优选例中，所述“显著高于”指C1/C0的比值≥1.5，较佳地≥2，更佳地≥3。
在另一优选例中，如果HSP90的蛋白数量或活性或mRNA水平升高，则提示检测对象抑郁症患病复发风险增高。
在另一优选例中，如果HIF-1α的蛋白数量或活性升高，则提示检测对象抑郁症患病复发风险增高。
在另一优选例中，如果BICC1和HSP90的蛋白数量或活性或mRNA水平升高，则提示检测对象抑郁症患病复发风险增高。
在另一优选例中，如果BICC1的蛋白数量或活性或mRNA水平和HIF-1α的蛋白数量或活性升高，则提示检测对象抑郁症患病复发风险增高。
在另一优选例中，如果HSP90蛋白数量或活性或mRNA水平和HIF-1α的蛋白数量或活性升高，则提示检测对象抑郁症患病复发风险增高。
在另一优选例中，如果HSP90蛋白数量或活性或mRNA水平升高，且HIF-1α的蛋白数量或活性升高但HIF-1α的mRNA水平无显著变化(或不变)，则提示检测 对象抑郁症患病复发风险增高(一种与HIF-1α蛋白的降解减少相关联的抑郁症患病复发风险增高)。
在另一优选例中，如果HSP90、HIF-1α和BICC1的蛋白数量或活性或HSP90和BICC1的mRNA水平升高，则提示检测对象抑郁症患病复发风险增高。
在另一优选例中，当选自下组的二个或三个风险标志物基因或其组合的表达水平C1显著高于对照参比值C0，则提示检测对象抑郁症复发风险高：(M1)HSP90；(M2)HIF-1α；(M3)BICC1。
在本发明的第二方面，提供了一种用于对抑郁症复发风险进行预测的标志物集合，所述的集合包括选自M1至M3的2-3种标志物：
(M1)HSP90的基因、转录本、或蛋白；
(M2)HIF-1α的基因、转录本、或蛋白；
(M3)BICC1的基因、转录本、或蛋白。
在本发明的第三方面，提供了一种抑郁症复发风险评估设备，所述设备包括：
(a)输入模块，所述输入模块用于输入某一检测对象血液、血浆或血清中的抑郁症复发风险标志物数据；
其中，所述的风险标志物基因选自下组：
(M)选自M1-M3的一种或多种标志物的基因、转录本、或蛋白：(M1)HSP90；(M2)HIF-1α；(M3)BICC1；
(b)数据处理模块，所述数据处理模块用于处理抑郁症复发风险标志物数据，并给出复发风险评估值，其中，所述处理包括：对于输入的标志物的转录或表达水平C1与对照参比值C0进行比较，其中，当C1显著高于C0时，则提示该对象抑郁症复发风险高；当C1不显著高于C0时，则提示该对象抑郁症复发风险低；和(c)输出模块，所述输出模块用于输出所述的评估结果。
在另一优选例中，所述“显著高于”指C1/C0的比值≥1.5，较佳地≥2，更佳地≥3。
在另一优选例中，所述设备还包括一检测模块，所述检测模块用于检测所述风险标志物的转录本水平、蛋白水平、或蛋白活性。
在另一优选例中，所述的检测模块选自下组：PCR检测仪、测序仪、或其组合。
在另一优选例中，所述输入模块还用于输入基于汉密尔顿抑郁量表的抑郁症量化评分数据。
在另一优选例中，所述的处理模块还对所述抑郁症量化评分数据和复发风险评估值进行综合性数据处理，从而给出辅助治疗或干预治疗的治疗方案。
在另一优选例中，所述治疗方案用于降低所述检测对象的抑郁症复发风险，或用于防止所述检测对象复发抑郁症。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了本发明实施例1中首发重症抑郁症患者外周血及其经过药物治疗后复发与未复发病例的外周血HSP90，HIF-1α及BICC1蛋白含量变化图(图1A)及其与抑郁症复发的相关性分析(图1B,C)。
图2显示了本发明实施例2中，慢性不可预测轻度应激(CUMS)诱导抑郁症复发模型方案路线图。其中，采用糖水偏好实验(SPT)，旷野实验(OFT)，新环境进食抑制实验(NSFT)及强迫游泳实验(FST)进行抑郁样行为测试评估，行为学判断评价的标准如下：(1)实验基线期，动物纳入的标准是：糖水偏好范围为 60％～90％，并且自主活动中穿线次数范围是150-200次/5分钟。(2)首次5周CUMS及再次5周CUMS造模后，抑郁样、未抑郁样动物筛选标准：①抑郁样小鼠为：糖水偏好<60％且其下降量>30％。②未抑郁样/正常对照动物是：糖水偏好范围仍是 60％～90％且其下降或上升量<30％。
图3显示了本发明实施例2中，初次糖水偏爱实验(SPT)评价。150只雄性小鼠经基线检测后，分组为：NC组(n＝30)、CUMS组(n＝120)，经初次SPT筛选评估小鼠抑郁样行为。
图4显示了本发明实施例2中，首次应激后抑郁行为测试。129只SPT筛选后小鼠经过5周的CUMS后分别进行SPT，OFT，NSFT及FST抑郁样行为测试结果。经过Round 1-SPT筛选及状态评分(图3)，纳入129只小鼠用于5周的CUMS造模，经SPT，OFT，NSFT及FST抑郁样行为测试(图4A-D)，评估初次慢性不可预测轻度应激诱导抑郁样行为。
图5显示了本发明实施例2中，Fluoxetine药物治疗后抑郁行为评价。初次CUMS诱导小鼠(n＝107)施加持续3周Fluoxetine(FLX)治疗，其分组为：NC+Sal 组(n＝25)、DE+Sal组(n＝32)，DE+FLX组(n＝50)，通过SPT、OFT、NSFT、FST实验评价Fluoxetine药物治疗后小鼠抑郁行为。
图6显示了本发明实施例2中，Fluoxetine药物清洗后小鼠SPT抑郁行为评价。通过首次CUMS诱导联合Fluoxetine(FLX)治疗后，经SPT、OFT、NSFT、FST抑郁样行为评价，共获得纳入小鼠87只，其中NC+Sal组(n＝22)、DE+Sal组(n＝27)，DE+FLX组(n＝36)，通过SPT评价Fluoxetine药物清洗后小鼠抑郁样行为。
图7显示了本发明实施例2中，二次应激后抑郁复发行为评价。二次应激(RE-CUMS)后进行SPT、OFT、NSFT及FST测试并分析，结合SPT及状态评分标准筛选NC入组17只，二次应激抑郁组(RE-CUMS-DE)18只，二次应激未抑郁组(RE-CUMS-w/o DE)15只。
图8显示了本发明实施例2中，抑郁症复发后前额叶皮层HSP90，HIF-1α及BICC1表达与转录评价。RE-CUMS显著诱导HSP90，HIF-1α及BICC1蛋白表达显著升高。而hsp90及bicc1转录变化显著升高，而hif-1α未显示显著增高。免疫组化研究结果显示RE-CUMS显著诱导HSP90及BICC1标记的阳性神经元显著升高。
具体实施方式
本发明人通过广泛而深入的研究，首次意外地发现了新的抑郁症复发风险标志物，所述抑郁症复发风险标志物包括(M1)HSP90、(M2)HIF-1α、和/或(M3)BICC1，并相应开发了用于对抑郁症复发风险进行预测和/或评判的方法和试剂盒。在此基础上完成了本发明。
实验表明，本发明的抑郁症复发风险标志物或其组合，可以有效预警或诊断抑郁症患者复发的潜在风险，采用该抑郁症复发风险标志物组预测和/或检测抑郁症患者复发的可能性或疾病状态，具有较高的准确率和特异性，为临床中抑郁症复发的预警和/或诊断提供参考标准。
基于本发明的抑郁症复发的标志物或其组合的应用检测系统，为评测抑郁症复发提供了一种较为客观的技术手段，可有效避免量表分析和主观经验测评带来的诊断偏差问题，为提高抑郁症复发评估的客观性和准确性提供新的检测手段。
抑郁症复发风险标志物
在本发明中，术语“本发明的抑郁症复发风险标志物”、“本发明的复发风险标志物”、“本发明的风险标志物”可互换使用，都指具有本发明的三种抑郁症 复发风险标志物(M1)HSP90、(M2)HIF-1α、(M3)BICC1中任何一种或多种。
在本发明中，本发明风险标志物包括基因(DNA)、转录本(mRNA)、cDNA、蛋白或其组合。
本发明标志物的蛋白可以含有或不含起始甲硫氨酸。此外，所述术语还包括全长的抑郁症复发风险标志物蛋白及其片段。在本发明中，抑郁症复发风险标志物蛋白包括其完整的氨基酸序列、其分泌蛋白、其突变体以及其功能上活性的片段。
HIF-1α为缺氧诱导因子-1α(Hypoxia-inducible factor 1-alpha)，基因名：HIF1AN(Homo sapiens)，UniProtKB ID：Q9NWT6，NCBI Gene：55662。作为缺氧适应性反应的主要转录调节器发挥作用。在缺氧条件下，激活40多个基因的转录，包括促红细胞生成素、葡萄糖转运蛋白、糖酵解酶、血管内皮生长因子、HILPDA和其他蛋白质产物增加氧气输送或促进代谢适应缺氧的基因。在胚胎血管化、肿瘤血管生成和缺血性疾病的病理生理学中起重要作用。
HSP90为热休克蛋白90(Heat shock protein HSP 90)，包括alpha亚单位(基因名：HSP90AA1-Human，UniProtKB ID：P07900，NCBI Gene：3326)和beta亚单位(基因名：HSP90AB1-Human，UniProtKB ID：P08238，NCBI Gene：3326)。HSP90可促进特定靶蛋白成熟、结构维持和适当调节的分子伴侣，例如参与细胞周期控制和信号转导。经历一个与其ATP酶活性相关的功能循环，而ATP酶活性对其伴侣活性至关重要。参与调节底物识别、ATP酶周期和伴侣蛋白功能。
BICC1双尾C同源蛋白1(Protein bicaudal C homolog 1)，基因名：BICC1-Human，UniProtKB ID：Q9H694，NCBI Gene：80114。BICC1作为潜在的RNA结合蛋白，充当Wnt信号的负调节器，可能参与调节胚胎发育过程中的基因表达。
抑郁症复发风险标志物与抑郁症复发的相关性
在本发明中，本发明人意外地发现HSP90参与应激诱导抑郁症复发的启动调节。研究表明，HSP90可以氧依赖地结合在HIF-1α的PAS区，阻止HIF-1α的泛素化降解。因此，本发明提出抑郁症复发与BICC1上调显著相关，应激条件诱导HSP90过表达，从而启动其对HIF-1α的调控，快速提升脑内水平，参与脑部多种病理、生理应激以及组织生长发育，进而诱发抑郁症复发。
此外，在本发明中，基于临床队列和基础研究，发现BICC1在抑郁症患者外周血及前额叶皮层与海马区显著增高，进一步明确BICC1可作为一种新型抑郁症发病机制中的关键分子。
在本发明中，本发明人为研究预测提供了临床检测证据，通过对首发重症抑郁症患者外周血及其经过药物治疗后复发和未复发病例的外周血分析，发现重症抑郁症中HSP90，HIF-1α及BICC1表达水平较对照组差异显著升高，经过药物治疗后复发较未复发组相比HSP90，HIF-1α及BICC1表达水平较对照组差异显著升高，表明抑郁症复发患者外周血HSP90，HIF-1α及BICC1高表达。通过相关性分析发现HSP90，HIF-1α及BICC1与抑郁症复发具有相关性。利用这些数据，本发明开发并验证了一种用于抑郁症复发风险预测的检测方法/系统，用于预测抑郁症的复发风险。
CUMS诱导抑郁症复发模型
本发明还提供了构建慢性不可预测轻度应激(CUMS)诱导的抑郁症复发模型及其筛选方案。
一种优选的构建模型和筛选方案，可包括以下步骤：
步骤1：Basline分组(Round 0)，150只雄性小鼠经基线检测后，分组为：NC组(n＝30)、CUMS组(n＝120)；
步骤2：SPT筛选(Round 1)，入选动物150只，其分组为：NC组(n＝30)、CUMS组(n＝120)，经SPT筛选后纳入动物129只，用于初次应激建模及行为学筛选；
步骤3：首次应激后抑郁行为测试(Round 2)，入选动物129只，其分组为：NC组(n＝25)、CUMS组(n＝104)，经SPT、OFT、NSFT、FST筛选后纳入动物107只，用于Fluoxetine药物治疗；
步骤4：Fluoxetine药物治疗后抑郁行为评价(Round 3)，入选动物107只，其分组为为：NC+Sal组(n＝25)、DE+Sal组(n＝32)，DE+FLX组(n＝50)；经Fluoxetine药物治疗后通过SPT、OFT、NSFT、FST筛选获得治疗后仍具有抑郁样行为的小鼠(n＝107)，进行药物清洗，再进行药物清洗动物SPT筛选，纳入动物87只，用于二次应激(Re-exposed CUMS)；
步骤5：二次应激后抑郁复发行为评价(Round 4)，入选动物87只，其分组为为：NC+Sal组(n＝22)、DE+Sal组(n＝27)，DE+FLX组(n＝38)；经二次应激后，通过SPT、OFT、NSFT、FST筛选，纳入动物50只，用于后续抑郁症复发小鼠动物模型评价；
步骤6：抑郁复发后小鼠脑内HSP90、HIF-1α、BICC1、或其组合评价(Round5)，入选动物50只，其分组为：NC组(n＝17)、RE-CUMS-DE组(n＝18)，RE-CUMS-w/o DE组(n＝15)，用于抑郁症复发后前额叶皮层HSP90，HIF-1α及BICC1表达与转录 变化评价。
检测方法
基于抑郁症复发风险标志物HSP90、HIF-1α、BICC1、或其组合在血液、血浆或血清中高表达，本发明还提供了相应的诊断抑郁症复发的方法。
本发明涉及定量和定位检测人抑郁症复发风险标志物HSP90、HIF-1α、BICC1、或其组合的基因、转录本水平或蛋白水平的诊断试验方法。这些试验是本领域所熟知的。试验中所检测的人抑郁症复发风险标志物HSP90、HIF-1α、BICC1、或其组合的基因、转录本水平和蛋白水平，可以用于诊断(包括辅助诊断)抑郁症复发的风险。
一种优选的方法是对mRNA或cDNA，进行PCR进行定量检测。
一种优选的方法是对mRNA或cDNA，测序进行定量检测。
一种优选的方法是对抑郁症复发风险标志物的蛋白进行定量检测。
优选地，一种检测样本中是否存在标志物蛋白的方法是利用特异性抗体进行检测，它包括：将样本与抑郁症复发风险标志物HSP90、HIF-1α、BICC1、或其组合的蛋白特异性抗体接触；观察是否形成抗体复合物，形成了抗体复合物就表示样本中存在抑郁症复发风险标志物HSP90、HIF-1α、BICC1、或其组合的蛋白。
抑郁症复发风险标志物蛋白或其多核苷酸可用于抑郁症复发的诊断。本发明的多核苷酸的一部分或全部可作为探针固定在微阵列或DNA芯片上，用于分析单个核细胞中基因的差异表达分析和基因诊断。抗抑郁症复发风险标志物的抗体可以固定在蛋白质芯片上，用于检测样本中的抑郁症复发风险标志物蛋白。
基于本发明的研究，HSP90、BICC1基因的表达量(mRNA水平或蛋白水平)和HIF-1α的蛋白水平，在抑郁症复发病人中存在显著上升。因此，HSP90、HIF-1α、BICC1可以单独地或组合地用作检测或诊断(尤其是辅助性诊断和/或早期诊断)抑郁症复发风险的标志物。在检测时，如果标志物基因(即HSP90、HIF-1α、BICC1)的表达量C1与正常人群中的相应表达量C0的比值(C1/C0)≥1.5，较佳地≥2更佳地≥3，则均可视为抑郁症复发的风险上升。尤其是综合检测2个或3个标志物的表达量时，可以获得更准确的检测结果。
检测试剂盒
基于抑郁症复发风险标志物与抑郁症复发风险的相关性，因此抑郁症复发风险标志物HSP90、HIF-1α、BICC1、或其组合可以作为抑郁症复发风险的诊断标志物。
本发明还提供了一种诊断抑郁症复发风险的试剂盒，所述的试剂盒含有一检测试剂，所述检测试剂用于检测抑郁症复发风险标志物HSP90、HIF-1α、BICC1基因、转录本、蛋白、或其组合。优选地，所述试剂盒含有本发明的抗抑郁症复发风险标志物HSP90、HIF-1α、BICC1的抗体或免疫偶联物，或其活性片段；或者含有特异性扩增抑郁症复发风险标志物HSP90、HIF-1α、BICC1的mRNA或cDNA的引物或引物对、探针或芯片。
在另一优选例中，所述的试剂盒还包括标签或说明书，所述标签或说明书注明所述试剂盒用于诊断抑郁症复发风险和/或评价抑郁症复发的治疗效果。
本发明的主要优点包括：
(a)本发明的风险标志物可以高效准确地预测抑郁症复发风险；
(b)本发明的检测体系可以精准早期预警、评估抑郁症复发风险。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
通用方法
实时荧光定量PCR
实时荧光定量PCR是将荧光能量技术应用于多聚酶链式反应的实验方法。一种称为SYBR Green Ⅰ的荧光染料在这个实验中会被使用。在PCR反应体系中，SYBR Green Ⅰ特异性地掺入DNA双链后，会发射荧光信号；而不掺入链中的SYBR染料分子不会发射任何荧光信号。因为这种方法使得荧光信号的增加与PCR产物的增加保持同步，也就是说，荧光染料发射出的荧光信号强度与DNA产量成正比。所以，检测PCR过程的荧光信号强度便可得知靶序列初始浓度，从而可以达到定量的目的。
实施例1
受试者为宁波大学附属宁波市康宁医院、宁波市精神卫生中心统一纳入首发抑郁症患者、复发抑郁症患者20例，并招募20名年龄和性别匹配的健康志愿者作 为对照。所有参与诊断工作的医师均具有精神科执业医师资格并有10年以上的精神科从业经验，熟练使用SCID-1检查表，熟练掌握ICD-10和DSM-V诊断标准，采用汉密尔顿抑郁量表(HAMD-17)对病人的精神病理状态进行评估，操作规范统一，一致性检测达到要求(Kappa＝0.68～0.82)，排除患有合并症的抑郁患者。本研究通过宁波大学伦理委员会批准，所有研究对象签署知情同意书，入组后的次日清晨空腹抽取20ml静脉血用于科研研究。
复发抑郁症患者、首发抑郁症患者及健康对照诊断、纳入及排除标准：
首发抑郁症患者组：(1)入组标准：所有病例符合《国际精神与行为障碍分类标准》ICD-10中抑郁发作的(F32)诊断标准，本次发作为首次发作；(2)统计：汉族、年龄18-60岁、小学及以上文化程度、性别各半、病程、用药情况等；(3)汉密尔顿抑郁量表(17项，HAMD-17)评分大于等于17分；(4)患者本人或其法定监护人签署知情同意书；(5)均在宁波大学附属宁波市康宁医院、宁波市精神卫生中心统一纳入。(6)排除标准：同上述抑郁复发组。
复发抑郁症患者组：(1)入组标准：所有病例符合《国际精神与行为障碍分类标准》ICD-10中复发性抑郁障碍(F33)诊断标准，既往至少有一次抑郁发作；(2)统计：汉族、年龄18-60岁、小学及以上文化程度、性别各半、病程、用药情况等；(3)汉密尔顿抑郁量表(17项，HAMD-17)评分大于等于17分；(4)患者本人或其法定监护人签署知情同意书；(5)均在宁波大学附属宁波市康宁医院、宁波市精神卫生中心统一纳入，有“首诊记录”和“出院记录”等完整的病历资料；(6)排除标准：当前或既往有其他精神障碍；合并严重或慢性躯体疾病和脑器质性疾病(神经系统变性疾病、脑外伤或脑血管病)；具有精神活性物质使用者；妊娠及哺乳期妇女；沟通困难者；不合作者；包括多次抑郁发作因在其他普通医院门诊或住院而无完整记录的病历，都给予排除。
健康对照组：(1)入组标准：既往及当前无精神疾病和明确的躯体疾病；无精神疾病家族史；(2)统计：汉族、年龄18-60岁、小学及以上文化程度、性别各半、病程、用药情况等；(3)汉密尔顿抑郁量表(17项，HAMD-17)评分小于7分；(4)排除标准：同上述抑郁复发组及首发组。
1.首发抑郁症患者、复发抑郁症患者及健康对照患者外周血中BICC1、HIF-1α、HSP90含量分析。
从每位受试者收集约4ml外周血，并使其在室温下凝结1小时，然后将样品在3000×g下离心10分钟以获得血清。然后将血清存放于在-80℃低温冰箱内或直接分析。构建BICC1、HIF-1α、HSP90蛋白ELISA检测试剂盒，用于首发抑郁症 患者、复发抑郁症患者及健康对照患者外周血中BICC1、HIF-1α、HSP90蛋白含量检测分析(附加专利申报内容)。
2.首发抑郁症患者、复发抑郁症患者及健康对照患者外周血中BICC1、HIF-1α、HSP90含量与汉密尔顿抑郁量表相关性分析。
表1受试者基本临床信息及HAMD-17检测结果
在受试者外周血中BICC1、HIF-1α、HSP90含量分析的基础上，收集患者汉密尔顿抑郁量表(HDRS-17)，结合HDRS-17打分情况与健康(Control，n＝20)和抑郁症(MDD，n＝20)患者外周血BICC1、HIF-1α、HSP90表达水平进行相关性分析。
表2健康与抑郁症受试者HAMD-17评分及外周血BICC1、HIF-1α、HSP90表达相关性分析
在受试者外周血中BICC1、HIF-1α、HSP90含量分析的基础上，收集患者汉密尔顿抑郁量表(HDRS-17)，结合HDRS-17打分情况与健康(Control，n＝20)、首发重症抑郁症患者药物治疗后未复发(No recurrence，n＝8)、首发重症抑郁症患者药物治疗后复发(Recurrence，n＝12)患者外周血BICC1、HIF-1α、HSP90表达水平进行相关性分析。
表3健康、治疗后复发、治疗后未复发抑郁症受试者HAMD-17评分及外周血BICC1、HIF-1α、HSP90表达相关性分析
结论：首发重症抑郁症患者外周血及其经过药物治疗后复发与未复发病例的外周血HSP90，HIF-1α及BICC1的变化发现重症抑郁症中HSP90，HIF-1α及BICC1较对照组显著升高，经过药物治疗后复发较未复发组相比HSP90，HIF-1α及BICC1显著升高(图1A)。通过相关性分析发现HSP90，HIF-1α及BICC1与抑郁症复发具有相关性，临床病例上初步发现HSP90，HIF-1α及BICC1作为抑郁症复发的生物标志物(图1B,C)。
实施例2
构建慢性不可预测轻度应激(CUMS)诱导的抑郁症复发模型(图2)及相关抑郁样行为和脑部病理变化评价：选取150只C57BL/6雄性成年小鼠用于模型构建 (图3)，CUMS模型采用每次5周应激。小鼠适应环境一周后，进行SPT行为学评定，筛选行为学表现正常的健康雄性小鼠(图4A-D)。首先，小鼠随机分为对照组和实验组，对照组小鼠正常饲养，实验组小鼠给予5周CUMS刺激，5周慢性应激结束后筛选出CUMS刺激后抑郁样小鼠和对照组小鼠。然后，抑郁样小鼠被分为两组：一组给Fluoxetine(20mg/kg/day)，另一组和对照组小鼠均给予同剂量生理盐水(图5)。一周药物清洗后,氟西汀治疗恢复的小鼠再次给予5周CUMS，再次5周慢应激结束后，筛选出再次CUMS抑郁样小鼠和再次CUMS后未抑郁样小鼠及对照组小鼠，并对三组小鼠进行研究。
本实验选用的CUMS方法包括：冷水游泳(4℃，5min)，热水游泳(42℃，5min)，倾斜鼠笼(倾斜45℃，24h)，成对饲养(24h)，禁食(24h)，禁水(24h)，昼夜颠倒(24h)，持续照明(36h)，潮湿鼠笼(24h，鼠笼中加入适当水)，共九种刺激随机安排在4周内完成，并且同种刺激不能连续出现，使动物不能预测刺激的发生。实验组单笼饲养，对照组成对正常饲养。
给药方法：每次给药前，把盐酸氟西汀胶囊溶于生理盐水中，配制成10mg/ml的氟西汀溶液，在给药期间每日对小鼠进行灌胃给予氟西汀(10mg/kg)或相同剂量生理盐水，共3周。
药物清洗期：已知氟西汀在人体内的代谢清除半衰期4～6天，而在小鼠体内，氟西汀的清除半衰期为9小时。然而其他一些参考文献报道小鼠体内氟西汀的清除半衰期各不一样，本实验的药物清除周期为一周。
动物行为学评定：在实验开始前，第一次4周CUMS后，第一次CUMS诱导的抑郁样小鼠恢复后及第二次接触4周CUMS后都分别进行对应的行为学评定。
(1)糖水偏好实验(Sucrose Preference Test,SPT)：实验开始前，小鼠进行1周的糖水适应性训练，每个鼠笼上放置两个饮用瓶，分别装有1％的蔗糖水和普通自来水,并且蔗糖水为每日新鲜配制。进行糖水偏好实验测试时，首先禁水23h，然后同一个鼠笼上放置事先称量好的两瓶水(1％的蔗糖水和普通自来水)，饮用1h后，撤下两瓶水并称重，计算动物对糖水的偏好度：动物对糖水的偏好度(％)＝糖水消耗量(g)/[糖水消耗量(g)+自来水消耗量(g)]。实验时间安排在8:00―10:30之间进行。
(2)旷野实验(Open-field test,OFT)：采用购自于上海吉利公司的JLBehv动物行为分析系统进行小鼠活动能力的跟踪分析，该系统包括进行旷野活动的敞箱和对应配置的计算机系统。敞箱由不透明材料制成，内径长75cm×宽75cm×高40cm， 外周带有视屏录像和照明的隔音箱。小鼠放置于敞箱中央区域，录像设备能自动记录小鼠在5min内的活动的路程、活动次数、休息时间、中央区域活动路程及活动平均速度等数据。每只小鼠结束一次旷野实验后，需先彻底清除敞箱内的排泄物后再进行下一只小鼠的测试。实验时间安排在8:00―14:00之间。
(3)新环境进食抑制实验(Novelty Suppressed Feeding Test,NSFT)：使用边长50cm、高40cm箱底铺匀2cm高木屑的试验箱，箱底中央放置糖丸或小块食物于白纸上。实验前动物禁食24h，实验时从任意的箱角放小鼠入箱内，通过摄像系统观察5min，记录小鼠动量和仅是时间的长短，反应小鼠的焦虑、抑郁程度，随后将动物放回笼中记录5min内事务消耗量，以排除食欲差异对进食时间的影响。
(4)强迫游泳实验(Forced Swimming Test,FST)：经典强迫游泳实验分2天进行。第一天让小鼠在25℃的深水中强迫游泳5min，取出后在室温烘干，归笼；第二天，在同样条件下进行强迫游泳5min，观察并记录不动时间图，以判断小鼠抑郁样行为，其中，不动时间越长，抑郁程度越强。
经糖水偏好实验(SPT)，旷野实验(OFT)，新环境进食抑制实验(NSFT)及强迫游泳实验(FST)进行抑郁样行为测试评估及小鼠筛选后(图6)，最终纳入对照组(NC)入组17只，二次应激抑郁组(RE-CUMS-DE)18只，二次应激未抑郁组(RE-CUMS-w/o DE)15只(图7)，通过Western Blot实验、Realtime PCR实验及免疫组化实验评价抑郁症复发后小鼠前额叶皮层HSP90，HIF-1α及BICC1表达与转录变化。
结论：Western Blot结果表明RE-CUMS显著诱导HSP90，HIF-1α及BICC1蛋白表达显著升高。Realtime PCR结果表明RE-CUMS显著诱hsp90及bicc1转录水平升高，而hif-1α未显示显著增高(图8)。免疫组化研究结果显示RE-CUMS显著诱导HSP90及BICC1标记的阳性神经元显著升高。以上结果表明本发明筛选出来基因及其对应蛋白包括以下一种或多种：BICC1，HSP90，HIF-1α，bicc1，hsp90，hif-1α，可作为预测或诊断抑郁症复发患者的特异性生物标志物。
讨论
在本发明中，本发明人首次意外地发现了新的抑郁症复发风险标志物，所述抑郁症复发风险标志物包括(M1)HSP90、(M2)HIF-1α、和/或(M3)BICC1。
在本发明中，通过选择外周血HSP90，HIF-1α及BICC1作为生物标记物对抑郁症复发患者的病情进行诊断，可实现了对于抑郁症复发的客观诊断方法，对抑郁症诊断有较高的灵敏度和特异性。
另一方面，通过对慢性不可预测轻度应激诱导的抑郁症复发小鼠模型构建，及相关抑郁样行为和脑部病理变化评价，确定HSP90，HIF-1α，BICC1，在蛋白和基因水平可作为预警或诊断抑郁症复发的关键标志物，可通过基因筛查、蛋白表达水平检测，判断抑郁症复发潜在风险或抑郁症复发进程状态。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (15)

  1. 一种抑郁症复发风险标志物的基因、转录本、蛋白、或其检测试剂的用途，其特征在于，用于制备一诊断试剂或试剂盒，所述诊断试剂或试剂盒用于评估抑郁症的复发风险；
    其中，所述的抑郁症复发风险标志物选自下组：
    (M)选自M1-M3的一种或多种标志物的基因、转录本、或蛋白：(M1)HSP90；(M2)HIF-1α；(M3)BICC1。
  2. 如权利要求1所述的用途，其特征在于，所述的待测样品选自下组：血液、血浆、血清、或其组合。
  3. 如权利要求1所述的用途，其特征在于，当选自下组的二个或三个风险标志物基因或其组合的表达水平C1显著高于对照参比值C0，则提示检测对象抑郁症复发风险高：(M1)HSP90；(M2)HIF-1α；(M3)BICC1。
  4. 如权利要求1所述的用途，其特征在于，所述的标签或说明书中注明以下内容：如果检测对象的所述风险标志物中的HSP90和BICC1的转录水平显著高于对照参比值，则提示检测对象抑郁症的复发风险高于一般的抑郁症患者。
  5. 如权利要求1所述的用途，其特征在于，如果HIF-1α的蛋白数量或活性升高，则提示检测对象抑郁症患病复发风险增高。
  6. 如权利要求1所述的用途，其特征在于，如果HSP90蛋白数量或活性或mRNA水平升高，且HIF-1α的蛋白数量或活性升高但HIF-1α的mRNA水平无显著变化或不变，则提示检测对象抑郁症患病复发风险增高。
  7. 如权利要求1所述的用途，其特征在于，如果HSP90、HIF-1α和BICC1的蛋白数量或活性或HSP90和BICC1的mRNA水平升高，则提示检测对象抑郁症患病复发风险增高。
  8. 如权利要求1所述的用途，其特征在于，所述的试剂盒还包括标签或说明书，所述标签或说明书注明所述试剂盒用于(a)诊断抑郁症的复发风险，和/或(b)评价抑郁症的复发治疗效果。
  9. 如权利要求1所述的用途，其特征在于，所述诊断试剂或试剂盒用于检测待测样品中所述风险标志物的水平。
  10. 如权利要求9所述的用途，其特征在于，所述待测样品来自选自下组的对象：无抑郁症的对象、抑郁易感对象、抑郁症的首发患者、抑郁症复发患者、或其组合。
  11. 一种用于对抑郁症复发风险进行预测的标志物集合，其特征在于，所述 的集合包括选自M1至M3的2-3种标志物：
    (M1)HSP90的基因、转录本、或蛋白；
    (M2)HIF-1α的基因、转录本、或蛋白；
    (M3)BICC1的基因、转录本、或蛋白。
  12. 一种抑郁症复发风险评估设备，其特征在于，所述设备包括：
    (a)输入模块，所述输入模块用于输入某一检测对象血液、血浆或血清中的抑郁症复发风险标志物数据；
    其中，所述的风险标志物基因选自下组：
    (M)选自M1-M3的一种或多种标志物的基因、转录本、或蛋白：(M1)HSP90；(M2)HIF-1α；(M3)BICC1；
    (b)数据处理模块，所述数据处理模块用于处理抑郁症复发风险标志物数据，并给出复发风险评估值，其中，所述处理包括：对于输入的标志物的转录或表达水平C1与对照参比值C0进行比较，其中，当C1显著高于C0时，则提示该对象抑郁症复发风险高；当C1不显著高于C0时，则提示该对象抑郁症复发风险低；和
    (c)输出模块，所述输出模块用于输出所述的评估结果。
  13. 如权利要求12所述的设备，其特征在于，所述输入模块还用于输入基于汉密尔顿抑郁量表的抑郁症量化评分数据。
  14. 如权利要求12所述的设备，其特征在于，所述设备还包括一检测模块，所述检测模块用于检测所述风险标志物的转录本水平、蛋白水平、或蛋白活性。
  15. 如权利要求12所述的设备，其特征在于，所述的检测模块选自下组：PCR检测仪、测序仪、或其组合。
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