WO2023143327A1 - 缺氧诱导因子1α作为标志物在抑郁症复发诊断中的应用 - Google Patents

缺氧诱导因子1α作为标志物在抑郁症复发诊断中的应用 Download PDF

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WO2023143327A1
WO2023143327A1 PCT/CN2023/072984 CN2023072984W WO2023143327A1 WO 2023143327 A1 WO2023143327 A1 WO 2023143327A1 CN 2023072984 W CN2023072984 W CN 2023072984W WO 2023143327 A1 WO2023143327 A1 WO 2023143327A1
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depression
risk
protein
hif
recurrence
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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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    • 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
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • 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
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16H—HEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
    • G16H50/00—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics
    • G16H50/30—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics for calculating health indices; for individual health risk assessment
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16H—HEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
    • G16H50/00—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics
    • G16H50/70—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics for mining of medical data, e.g. analysing previous cases of other patients
    • 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
    • G01N2333/4701—Details
    • G01N2333/4703—Regulators; Modulating activity
    • 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
    • G01N2333/4701—Details
    • G01N2333/4703—Regulators; Modulating activity
    • G01N2333/4706—Regulators; Modulating activity stimulating, promoting or activating activity
    • 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 field of biological sciences, and relates to fast, convenient and specific early screening or differential diagnosis of depression recurrence, in particular to the application of hypoxia-inducible factor 1 ⁇ protein as a marker in early screening or differential diagnosis of depression recurrence.
  • MDD Major depressive disorder
  • the main clinical features are significant and persistent depression and loss of interest. In severe cases, there may even be suicidal tendencies and important disability risks.
  • the 12-month prevalence rate of depression in China is 3.6%, while the lifetime prevalence rate reaches 6.9%.
  • the prevention and treatment of the disease is urgent.
  • 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. Therefore, in order to make the diagnosis of depression relapse more objective, reduce human factors, and improve the consistency and accuracy of diagnosis, so as to achieve early diagnosis, early detection and early intervention of depression relapse, develop effective objective biomarkers and establish effective detection methods Significant for people with depression. Therefore, how to specifically diagnose depression recurrence and provide effective treatment has become a very urgent matter in the current clinical depression diagnosis and treatment.
  • the 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. This marker has good sensitivity and specificity.
  • the purpose of the present invention is to provide a highly sensitive and highly specific specific marker hypoxia-inducible factor 1 ⁇ for predicting and/or diagnosing the recurrence risk of depression and its application.
  • 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.
  • genes 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 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 detection reagents include reagents for detecting HIF-1 ⁇ protein content or activity by enzyme-linked immunosorbent assay.
  • the reagent for detecting HIF-1 ⁇ protein content by ELISA is: ELISA kit for HIF-1 ⁇ protein content.
  • the reagent for detecting HIF-1 ⁇ protein content by enzyme-linked immunosorbent assay is ELISA kit.
  • the ELISA kit includes: HIF1A (Human) ELISA Kit (RAB0507, Abnova), human HIF-1 alpha ELISA kit (ab171577, Abcam).
  • the depression recurrence risk markers further include genes, transcripts, or proteins of one or more markers selected from group B: (B1) HSP90; (B2) BICC1.
  • the diagnostic reagent or kit is used to detect: the protein level of HIF-1 ⁇ and the transcript level or protein quantity or activity of HSP90 in the test sample.
  • the diagnostic reagent or kit is used to detect: the protein level of HIF-1 ⁇ and the transcript level or protein quantity or activity of HSP90 and BICC1 in the test sample.
  • the expression level C1 of the risk marker HIF-1 ⁇ protein is significantly higher than the control reference value C0, it indicates that the subject has a high risk of depression recurrence.
  • the diagnostic reagent or kit is also used to detect: the mRNA level of HIF-1 ⁇ in the sample to be tested.
  • HIF-1 ⁇ increases but the mRNA level of HIF-1 ⁇ has no significant change (or does not change)
  • HSP90 protein quantity or activity or mRNA level increases, it prompts the test object Increased risk of relapse of depression (a type of depression associated with reduced degradation of HIF-1 ⁇ protein).
  • 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 HIF-1 ⁇ protein level of 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 concentration C1 of the risk marker (such as HIF-1 ⁇ ) of the detected subject is significantly higher than the control reference value C0, the probability of relapse of the subject is greater than that of 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 of HIF-1 ⁇ increases, and the protein quantity or activity or mRNA level of HSP90 increases, it indicates that the risk of depression relapse is increased for the test subject.
  • the protein quantity or activity or mRNA level of BICC1 is increased, and the protein quantity or activity of HIF-1 ⁇ is increased, it indicates that the risk of depression relapse is increased for the test subject.
  • the protein quantity or activity of HSP90, HIF-1 ⁇ and BICC1 is increased, and/or the mRNA level of HSP90 and BICC1 is increased, it indicates that the risk of depression relapse is increased in the test subject.
  • the diagnostic reagent or kit is used to detect the level of the risk marker in the sample to be tested.
  • the diagnostic reagent or kit comprises: a monoclonal antibody or a polyclonal antibody that binds to the HIF-1 ⁇ protein.
  • 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.
  • 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 markers include HIF-1 ⁇ protein
  • 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 expression level C1 of the input marker and the control reference 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
  • the risk markers also include genes, transcripts, or proteins of one or more markers selected from group B: (B1) HSP90; (B2) BICC1
  • the device further includes a detection module, which is used to detect the protein level or protein activity of the risk marker.
  • the detection module is selected from the group consisting of ELISA analyzer, PCR machine, sequencer, or a combination thereof.
  • the depression recurrence risk marker data includes HIF-1 ⁇ protein quantity or activity data, and HIF-1 ⁇ mRNA (or transcript) level.
  • the data processing module simultaneously evaluates the protein quantity and mRNA level of HIF-1 ⁇ .
  • the treatment includes: when the protein quantity of HIF-1 ⁇ is significantly increased, but the mRNA level of HIF-1 ⁇ is not significantly increased (such as unchanged or substantially unchanged), it is suggested that the subject is depressed High risk of recurrence.
  • HIF-1 ⁇ protein quantity or activity of HIF-1 ⁇ increases but the mRNA level of HIF-1 ⁇ has no significant change (or does not change)
  • HSP90 protein quantity or activity or mRNA level increases, it prompts the test object An increased risk of depression relapse was associated with reduced degradation of the HIF-1 ⁇ protein.
  • Figure 1 shows the changes in the peripheral blood HIF-1 ⁇ protein content in the peripheral blood of patients with first-episode major depression in Example 1 of the present invention and the relapsed and non-relapsed cases after drug treatment.
  • Fig. 2 shows the correlation analysis between the HAMD-17 score and peripheral blood HIF-1 ⁇ expression of healthy and depressed subjects in Example 1 of the present invention.
  • Fig. 3 shows the correlation analysis between the HAMD-17 score and peripheral blood HIF-1 ⁇ expression of healthy, relapsed after treatment, and non-relapsed depression subjects after treatment in Example 1 of the present invention.
  • Fig. 4 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 During the baseline period, the criteria for animal inclusion were: sugar water preference ranged from 60% to 90%, And the range of threading times in autonomous activities is 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%. 2Non-depressed/normal control animals: the sugar water preference range is still 60%-90% and the decrease or increase is ⁇ 30%.
  • Figure 5 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 risk markers for depression recurrence for the first time, including HIF-1 ⁇ , and developed a corresponding method for predicting the risk of depression recurrence and/or judged methods and kits.
  • HIF-1 ⁇ alone, or in combination with other depression recurrence risk markers (such as HSP90, BICC1, etc.)
  • HSP90, BICC1, etc. can predict and/or diagnose depression recurrence risk with high sensitivity and high specificity.
  • the present invention has been accomplished on this basis.
  • the depression recurrence risk markers of the present invention or their combination can effectively warn or diagnose the potential risk of depression patients' recurrence, and use the depression recurrence risk markers to predict and/or detect the possibility of depression patients' recurrence or Disease status, with high accuracy and specificity, provides a reference standard for early warning and/or diagnosis of depression recurrence in clinical practice.
  • a relatively objective technical means is provided for evaluation of depression recurrence, which can effectively avoid the problem of diagnostic bias caused by scale analysis and subjective experience evaluation, and contribute to improving depression.
  • the objectivity and accuracy of symptom recurrence assessment provide a new detection method.
  • 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
  • Marker HIF-1 ⁇ all refer to those with the risk of depression recurrence of the present invention.
  • HSP90 HSP90, BICC1.
  • the risk markers of the present invention include genes (DNA), 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.
  • depression recurrence risk markers A protein includes its entire amino acid sequence, its secreted protein, its mutants, and its functionally active fragments.
  • 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 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 the depression recurrence risk marker HIF-1 ⁇ in blood, plasma or serum, the present invention also provides a corresponding method for diagnosing depression recurrence.
  • the invention relates to a diagnostic test method for quantitatively and positionally detecting the gene or protein level of HIF-1 ⁇ , a risk marker for human depression relapse.
  • HIF-1 ⁇ a risk marker for human depression relapse.
  • the human depression relapse risk marker HIF-1 ⁇ gene and protein levels detected in the test can be used to diagnose (including auxiliary diagnosis) the risk of depression relapse.
  • 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: contacting the sample with a protein-specific antibody of the depression recurrence risk marker HIF-1 ⁇ ; observing whether the antibody complex is formed The formation of antibody complexes indicates the presence of the protein of depression relapse risk marker HIF-1 ⁇ 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.
  • HIF-1 ⁇ the protein level of HIF-1 ⁇ is significantly increased in patients with relapsed depression. Therefore, HIF-1 ⁇ can be used as a marker for detection or diagnosis (especially auxiliary diagnosis and/or early diagnosis) of depression relapse risk.
  • the ratio (C1/C0) of the expression level C1 of the marker gene (ie HIF-1 ⁇ ) to the corresponding expression level C0 in the normal population is ⁇ 1.5, preferably ⁇ 2 and more preferably ⁇ 3, then all May be seen as an increased risk of depression relapse.
  • the inventors also unexpectedly found that in subjects with high risk of depression recurrence, although the test sample The HIF-1 ⁇ protein level increases (or significantly increases), but the mRNA level of HIF-1 ⁇ is basically unchanged or there is no significant difference, which suggests that after the expression of the HIF-1 ⁇ protein of the present invention, due to its degradation rate Lower or inhibited degradation, resulting in increased levels of final HIF-1 ⁇ protein content.
  • the research of the present inventors shows that the increase of HIF-1 ⁇ protein but not the increase of mRNA is often accompanied by the increase of HSP90 protein level, which suggests that the decrease of HIF-1 ⁇ degradation is related to HSP90 protein.
  • HIF-1 ⁇ protein quantity or activity of HIF-1 ⁇ increases but the mRNA level of HIF-1 ⁇ has no significant change (or does not change), while the HSP90 protein quantity or activity or mRNA level increases, it indicates that the subject is depressed An increased risk of disease recurrence was associated with reduced degradation of the HIF-1 ⁇ protein.
  • the depression recurrence risk marker HIF-1 ⁇ 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 for detecting HIF-1 ⁇ gene, protein, or a combination thereof, which is a risk marker for depression recurrence.
  • the kit contains the anti-depression relapse risk marker HIF-1 ⁇ antibody or immunoconjugate of the present invention, or an active fragment thereof; or specifically amplifies the depression relapse risk marker HIF-1 ⁇ , Primers or primer pairs, probes or chips for cDNA.
  • 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.
  • 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; severe or chronic physical diseases and brain organic diseases (neurological degenerative diseases, brain trauma or cerebrovascular diseases); users of psychoactive substances; pregnancy and breastfeeding Women in the late stage; communication difficulties; non-cooperative persons; medical records including multiple depressive episodes without complete records due to
  • 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.
  • HIF-1 ⁇ protein was constructed for the detection and analysis of HIF-1 ⁇ protein content in peripheral blood of first-episode depression patients, recurrent depression patients and healthy controls.
  • the serum HIF-1 ⁇ protein concentration was detected using an ELISA kit, which provided the necessary but not all reagents or materials needed to detect the HIF-1 ⁇ protein concentration: 1 piece of 96-well microtiter plate (coated with recombinant human HIF- 1 ⁇ protein specific antibody), 2 tubes of human HIF-1 ⁇ protein standard, 1 vial of 12ml assay diluent RD1 38, 1 vial of 21ml human HIF-1 ⁇ protein conjugated substrate solution, 1 vial of 21ml calibration diluent RD5P , 1 bottle of 21ml washing solution (25-fold dilution), 1 bottle of 12ml color developing solution A, 1 bottle of 12ml color developing solution A and 1 bottle of 6ml stop solution.
  • additional reagents and materials are required as follows: enzyme label detector, pipette, pipette, graduated cylinder, absorbent paper, distilled or deionized water, data analysis and drawing software, etc.
  • Washing liquid configuration Take 20mL washing liquid and add deionized water, dilute to 500mL washing liquid for use;
  • 4HIF-1 ⁇ protein standard configuration Add the diluted calibration diluent RD5P into 1 tube of HIF-1 ⁇ protein standard to construct a concentration gradient standard.
  • the standard concentration is: 100ng/mL, 50ng/mL, 25ng/mL , 6.25 ng/mL.
  • kits that can be detected for hypoxia-inducible factor 1 ⁇ are consistent with the kits used in the above examples, such as HIF1A (Human) ELISA Kit (RAB0507, Abnova), human HIF-1alpha ELISA kit (ab171577, Abcam) and the like.
  • Fig. 2 is the correlation analysis of HAMD-17 score and peripheral blood HIF-1 ⁇ expression in healthy and depressed subjects in Example 1 of the present invention
  • Example 3 is a correlation analysis of HAMD-17 scores and peripheral blood HIF-1 ⁇ expression in healthy, relapsed after treatment, and non-relapsed depression subjects after treatment in Example 1 of the present invention
  • HDRS-17 Hamilton Depression Scale
  • HDRS-17 Hamilton Depression Scale
  • mice 150 C57BL/6 male adult mice were selected for model construction, and the CUMS model was used for 5 week stress. After the mice acclimated to the environment for one week, the SPT behavioral evaluation was carried out, and healthy male mice with normal behavioral performance were screened. 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. Then, the depression-like mice were divided into two groups: one group was given Fluoxetine (20mg/kg/day), and the other group and the control group were given the same dose of normal saline.
  • Fluoxetine 20mg/kg/day
  • 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 (Fig. 4).
  • 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.
  • Behavioral evaluation of animals before the start of the experiment, after the first 4 weeks of CUMS, after the first CUMS-induced depression-like mice recovered, and after the second exposure to CUMS for 4 weeks, corresponding behavioral evaluations were carried out, including: (1) Sucrose Preference Test (SPT); (2) Open-field test (OFT); (3) Novelty Suppressed Feeding Test (NSFT); (4) Forced swimming Experiment (Forced swimming Test, FST), the experimental method refers to the standard operating procedure.
  • SPT Sucrose Preference Test
  • OFT Open-field test
  • NSFT Novelty Suppressed Feeding Test
  • FST Forced swimming Experiment
  • Hypoxia inducible factor-1 is a heterodimeric molecule, in which the functionally active subunit is hypoxia inducible factor 1 ⁇ (HIF-1 ⁇ ), which is involved in the adaptation of host cells to hypoxia. Regulatory core factor. When activated, HIF-1 ⁇ is involved in: (1) tissue recovery after hypoxia; (2) pro-inflammatory and antimicrobial effects; (3) promotion of tumor growth; (4) specific pro-apoptotic effects; (5) regulation of embryonic development and many other physiological processes.
  • hypoxia-inducible factor 1 ⁇ (HIF-1 ⁇ ) has a positive effect on the recurrence of depression. There is a significant correlation. Due to the low compliance and serious damage of HIF-1 ⁇ detection in the brain or cerebrospinal fluid of patients with depression, it is difficult to be applied clinically.
  • peripheral blood HIF-1 ⁇ (and combined with HSP90, BICC1, etc.) as a biomarker to diagnose the condition of patients with recurrence of depression
  • an objective diagnosis method for recurrence of depression can be realized.
  • the diagnosis has high sensitivity and specificity.
  • peripheral blood HIF-1 ⁇ protein can be used as an early warning or diagnosis of depression relapse.
  • the key markers can be used to judge the potential risk of depression relapse or the status of depression relapse process through gene screening and protein expression level detection.
  • the method of the present invention uses peripheral blood or serum samples as detection samples, it is easy to obtain and has high patient compliance, which helps to solve the problems of early warning and relapse diagnosis of depression.

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Abstract

本发明涉及缺氧诱导因子1α作为标志物在抑郁症复发诊断中的应用。具体地，本发明提供了缺氧诱导因子1α蛋白或其检测试剂在抑郁症复发诊断中的用途。本发明的研究证实，缺氧诱导因子1α蛋白在干预治疗后抑郁症复发患者血清中特异性升高，因此可作为外周血液生物标记物用于抑郁症复发的早期筛查或鉴别诊断，以及抑郁症患者干预治疗后疗效评估。本发明检测方法简便快捷，患者依从性高，具有高灵敏度和高特异性，从而有助于进行抑郁症复发的诊断和预测。

Description

缺氧诱导因子1α作为标志物在抑郁症复发诊断中的应用 技术领域
本发明属于生物科学领域，涉及快速方便特异性早期筛查或鉴别诊断抑郁症复发，具体地，涉及缺氧诱导因子1α蛋白作为标志物在抑郁症复发早期筛查或鉴别诊断中的应用。
背景技术
抑郁症(Major depressive disorder，MDD)是一种基因与环境交互影响的重大精神疾病，以显著而持久的心境低落与兴趣减少为主要临床特征，严重者甚至有自杀倾向和重要的致残风险。据2019年世界卫生组织统计的流行病学资料显示，全球有超过3.5亿人罹患抑郁症，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。因此，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，从而实现抑郁症复发早诊断早发现早干预，发展有效的客观生物标记物并建立有效的检测方法对抑郁症患者来说意义重大。因此，如何特异性诊断抑郁症复发，进而提供有效的治疗成为当前临床抑郁症诊疗中十分紧迫的事情。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，该标志物具有良好的具备良好的灵敏度和特异度。
采用分子生物学检测的方法科学检测抑郁症患者复发的潜在风险，具有重要的临床意义和社会价值。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缺氧诱导因子1α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包括HIF-1α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，所述检测试剂包括利用酶联免疫方法检测HIF-1α蛋白含量或活性的试剂。
在另一优选例中，所述利用酶联免疫方法检测HIF-1α蛋白含量的试剂为：HIF-1α蛋白含量酶联免疫吸附测定试剂盒。
在另一优选例中，所述利用酶联免疫方法检测HIF-1α蛋白含量的试剂为 ELISA试剂盒。
在另一优选例中，所述的ELISA试剂盒包括：HIF1A(Human)ELISA Kit(RAB0507,Abnova)、人HIF-1 alpha ELISA试剂盒(ab171577,Abcam)。
在另一优选例中，所述抑郁症复发风险标志物还包括选自下组B的一种或多种标志物的基因、转录本、或蛋白：(B1)HSP90；(B2)BICC1。
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HIF-1α的蛋白水平以及HSP90的转录本水平或蛋白数量或活性。
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HIF-1α的蛋白水平以及HSP90和BICC1的转录本水平或蛋白数量或活性。
在另一优选例中，当风险标志物HIF-1α蛋白的表达水平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。
在另一优选例中，所述的诊断试剂或试剂盒还用于检测：待测样品中HIF-1α的mRNA水平。
在另一优选例中，如果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)，而HSP90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(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，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HIF-1α蛋白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(如HIF-1α)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，并且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水平升高，且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、HIF-1α和BICC1的蛋白数量或活性升高，和/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，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。
在另一优选例中，所述诊断试剂或试剂盒包含：与HIF-1α蛋白结合的单克隆抗体或多克隆抗体。
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。
在本发明的第二方面，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，所述的风险标志物包括HIF-1α蛋白；
(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，所述风险标志物还包括选自下组B的一种或多种标志物的基因、转录本、或蛋白：(B1)HSP90；(B2)BICC1
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：ELISA分析仪、PCR仪、测序仪、或其组合。
在另一优选例中，所述抑郁症复发风险标志物数据包括HIF-1α的蛋白数量或活性数据，和HIF-1α的mRNA(或转录本)水平。
在另一优选例中，所述的数据处理模块同时评估HIF-1α的蛋白数量和mRNA水平。
在另一优选例中，所述处理包括：当HIF-1α的蛋白数量显著升高，而HIF-1α的mRNA水平未显著升高(如不变或基本不变)，则提示该对象抑郁症复发风险高。
在另一优选例中，如果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)，而HSP90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，并且与HIF-1α蛋白的降解减少相关联。
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HIF-1α蛋白含量变化图。
图2显示了本发明实施例1中健康与抑郁症受试者HAMD-17评分及外周血HIF-1α表达相关性分析。
图3显示了本发明实施例1中健康、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HIF-1α表达相关性分析。
图4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5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HIF-1α，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。单独使用HIF-1α，或将其与其他抑郁症复发风险标志物(如HSP90、BICC1等)联用，可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。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”、“本发明风险标志物”可互换使用，都指具有本发明的抑郁症复发风险标志物HIF-1α。
此外，可与本发明的风险标志物HIF-1α联用的额外标志物包括(但并不限于)：HSP90、BICC1。
在本发明中，本发明的风险标志物包括基因(DNA)、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。
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HIF-1α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HIF-1α基因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HIF-1α基因和蛋白水平，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HIF-1α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HIF-1α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，HIF-1α的蛋白水平，在抑郁症复发病人中存在显著上升。因此，HIF-1α可用作检测或诊断(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HIF-1α)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。
此外，本发明人还意外地发现，在抑郁症复发高风险的对象中，虽然检测样本 中HIF-1α蛋白水平升高(或显著升高)，但HIF-1α的mRNA水平居然基本不变或未存在显著差异，这提示：在本发明HIF-1α蛋白在表达后，由于其降解速度较低或降解受抑制，从而导致最终HIF-1α蛋白含量的水平升高。本发明人的研究表明，这种HIF-1α蛋白升高而mRNA不升高的情况，往往伴随着HSP90蛋白水平的升高，这提示HIF-1α降解下降与HSP90蛋白是相关的。
因此，在本发明中，还可对HIF-1α的mRNA水平进行定量检测(如PCR或测序)，以便鉴别出这种特定的抑郁症复发高风险亚型。
在本发明中，如果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)，而HSP90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，并且与HIF-1α蛋白的降解减少相关联。
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HIF-1α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HIF-1α基因、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HIF-1α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HIF-1α、的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HIF-1α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HIF-1α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HIF-1α蛋白含量检测分析。
血清HIF-1α蛋白浓度使用ELISA试剂盒检测，所用试剂盒提供了检测HIF-1α蛋白浓度所需的必要但非全部试剂或者材料：1块96孔酶标板(已包被重组人的HIF-1α蛋白特异性抗体)、2管人的HIF-1α蛋白标准品、1瓶12ml试验稀释液RD1 38、1瓶21ml的人HIF-1α蛋白共轭结合底物液、1瓶21ml校准稀释液RD5P、1瓶21ml洗涤液(25倍稀释)、1瓶12ml显色液A、1瓶12ml显色液A和1瓶6ml终止液。此外，还需要的额外试剂与材料如下：酶标检测仪、移液器、移液管、量筒、吸水纸、蒸馏水或去离子水、数据分析与绘图软件等。
在检测开始之前准备好所有需要的物品：
①洗涤液配置：取20mL洗涤液加入去离子水，稀释至500mL洗涤液待用；
②底物液：于使用开始前15分钟，将等体积显色液A和B混合；
③校准稀释液RD5P稀释，将20mL的RD5P原液加入80ml去离子水中，混合成为100mL稀释的校准稀释液RD5P，待用；
④HIF-1α蛋白标准品配置：使用稀释的校准稀释液RD5P加入1管HIF-1α蛋白标准品中，构建浓度梯度的标准品，标准品浓度依次为：100ng/mL、50ng/mL、25ng/mL、6.25ng/mL。
实验操作如下：
①使用试剂盒中提供的96孔酶标板(已包被重组人的HIF-1α蛋白特异性 抗体)，确定本次检测所需酶标板孔数目，每个样品、标准品和空白应当设置复孔；此外，也可以根据自身需要，采用常规免疫抗体制备方法由纯化的HIF-1α蛋白或它的抗原片段注射入动物体内以产生特异性抗体或者由表达人HIF-1α蛋白或它的抗原片段的细胞用来对动物免疫而产生抗体，包被于干净的酶标板中自制新的试剂盒。
②每孔先加入50μl试验稀释液RD1-38；
③添加样品：倍比稀释HIF-1α蛋白标准品后将100ng/mL、50ng/mL、25ng/mL、6.25ng/mL的标准品各50μL依次加入酶标板孔中，并设置1孔空白比较(空白比较孔不添加样品和ELISA试剂，其他各步操作相同)。其余各孔加入使用校准稀释液RD5P稀释后的待测血清样本50μL(需确保稀释后的血清HIF-1α蛋白浓度处于标准品所示浓度区间的中间，并记录每一样本稀释倍数，便于实验结束后计算，以确保试剂盒检测的准确性)；室温下，摇床上孵育2小时；摇床速度设定为500±50rpm；
④完全倒去孔内液体，使用稀释好的洗涤液重复4次洗涤每孔，每孔约加洗涤液400μl，洗涤完全后，将板倒置吸水纸上拍干；
⑤每孔加入200μl人HIF-1α蛋白共轭结合底物液，室温下，摇床上孵育1小时；
⑥重复操作4洗涤；
⑦每孔加入200μl底物液，室温下，避光孵育30分钟；
⑧每孔加入50μl终止液，停止反应(颜色立即由蓝色变为黄色)；
⑨以空白孔为零，添加终止液后15分钟内用酶标检测仪在450nm处测定光密度(OD值)。通过HIF-1α的标准蛋白测出的OD值使用ELISACalc软件绘制出标准曲线及曲线的数学公式，随后将每个血清样本的OD值依次输入软件所得到的标准曲线公式中计算当前样品中HIF-1α蛋白的含量，基于实验开始前记录的每个样本稀释倍数，计算未稀释时的浓度，作为最终的血清HIF-1α蛋白的含量。
图1可见，HIF-1α蛋白的血清水平仅在抑郁症复发患者中显著增高，而健康受试者和药物治疗后未复发抑郁症受试者之间没有统计学上的显著性差异，说明本发明利用检测HIF-1α蛋白含量可以有效地特异性早期筛查或鉴别诊断抑郁症复发，还可用于抑郁症患者干预治疗后疗效评估的生物标记物。此外，一些其他可以检测的缺氧诱导因子1α的酶联免疫试剂盒效果与上述实施例中采用的试剂盒效果一致，如HIF1A(Human)ELISA Kit(RAB0507,Abnova)、人 HIF-1alpha ELISA试剂盒(ab171577,Abcam)等。
图2为本发明实施例1中健康与抑郁症受试者HAMD-17评分及外周血HIF-1α表达相关性分析；
图3为本发明实施例1中健康、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HIF-1α表达相关性分析；
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HIF-1α含量与汉密尔顿抑郁量表HAMD-17相关性分析，测试结果见表1。
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HIF-1α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HIF-1α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HIF-1α表达相关性分析
在受试者外周血中HIF-1α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HIF-1α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HIF-1α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HIF-1α的变化发现重症抑郁症中HIF-1α较对照组显著升高，经过药物治疗后复发较未复发组相比HIF-1α显著升高。通过相关性分析发现HIF-1α与抑郁症复发具有相关性，临床病例上初步发现HIF-1α作为抑郁症复发的生物标志物。
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，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。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。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(图4)。
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，包括：(1)糖水偏好实验(Sucrose Preference Test,SPT)；(2)旷野实验(Open-field test,OFT)；(3)新环境进食抑制实验(Novelty Suppressed Feeding Test,NSFT)；(4)强迫游泳实验(Forced Swimming Test,FST)，实验方法参照标准操作规程。
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，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，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(图5)。
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。这种HIF-1α蛋白升高而mRNA不升高的情况，往往伴随着HSP90蛋白水平的升高，这提示HIF-1α降解下降与HSP90蛋白是相关的。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。HIF-1α蛋白表达水平或活性升高而mRNA不升高的情况，可用于抑郁症复发评价，同时包括但不限HSP90和BICC1基因组、转录组和蛋白组水平的变化。
讨论
缺氧诱导因子1(Hypoxia inducible factor-1,HIF-1)是异二聚体分子，其中具有功能活性的亚基是缺氧诱导因子1α(HIF-1α)，是参与宿主细胞缺氧适应性调节的核心因子。当HIF-1α活化后，参与：(1)缺氧后的组织恢复；(2)促炎和抗微生物作用；(3)促进肿瘤生长；(4)特异促凋亡作用；(5)调节胚胎发育等多种生理过程。
本发明人通过研究意外地发现，缺氧诱导因子1α(HIF-1α)与抑郁症复发具 有显著相关性。由于抑郁症患者脑内或脑脊液中HIF-1α检测患者依从性低，损伤性严重，因此难以在临床上应用。
在本发明中，通过选择外周血HIF-1α(并与HSP90，BICC1等组合)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，确定外周血HIF-1α蛋白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。
由于本发明方法采用外周血或血清样本作为检测样本，因此易获得，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。
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包括HIF-1α蛋白。
  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，所述抑郁症复发风险标志物还包括选自下组B的一种或多种标志物的基因、转录本、或蛋白：(B1)HSP90；(B2)BICC1。
  4. 如权利要求1所述的用途，其特征在于，当风险标志物HIF-1α蛋白的表达水平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。
  5. 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。
  6. 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。
  7. 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包含：与HIF-1α蛋白结合的单克隆抗体或多克隆抗体。
  8. 如权利要求3所述的用途，其特征在于，所述的诊断试剂或试剂盒用于检测：待测样品中HIF-1α的蛋白水平以及HSP90和BICC1的转录本水平或蛋白数量或活性。
  9. 如权利要求3所述的用途，其特征在于，如果HIF-1α的蛋白数量或活性升高但HIF-1α的mRNA水平无显著变化，而HSP90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。
  10. 如权利要求3所述的用途，其特征在于，如果HIF-1α的蛋白数量或活性升高，并且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。
  11. 如权利要求3所述的用途，其特征在于，如果BICC1的蛋白数量或活性或mRNA水平升高，且HIF-1α的蛋白数量或活性升高，则提示检测对象抑郁症患病复发风险增高。
  12. 如权利要求6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。
  13. 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，所述的风险标志物包括HIF-1α蛋白；
    (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。
  14. 如权利要求13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蛋白水平或蛋白活性。
  15. 如权利要求13所述的设备，其特征在于，所述检测模块选自下组：ELISA分析仪、PCR测序仪、测序仪、或其组合
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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040185474A1 (en) * 2002-12-27 2004-09-23 Kazuhito Rokutan Method of diagnosing depression
WO2014193999A2 (en) * 2013-05-28 2014-12-04 Caris Science, Inc. Biomarker methods and compositions
US20160319361A1 (en) * 2013-08-28 2016-11-03 Caris Life Sciences Switzerland Holdings Gmbh Oligonucleotide probes and uses thereof
CN107621545A (zh) * 2017-07-26 2018-01-23 东南大学 Bicc1蛋白对精神疾病诊断的新用途
CN111334567A (zh) * 2020-02-24 2020-06-26 新疆医科大学第二附属医院 HIF-1α基因启动子区甲基化检测引物及检测方法与应用
CN114438191A (zh) * 2022-01-27 2022-05-06 宁波大学 缺氧诱导因子1α作为标志物在抑郁症复发诊断中的应用

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2005312435A (ja) * 2004-03-29 2005-11-10 Kazuhito Rokutan うつ病の評価方法
EP3009839B1 (en) * 2013-06-14 2018-11-28 Seoul National University R&DB Foundation Method for detecting hypoxia or diagnosing hypoxia-related diseases
EP3327134A1 (en) * 2016-11-28 2018-05-30 Carsten Korth Method and biomarkers for in vitro diagnosis of mental disorders
CN111351945B (zh) * 2020-03-18 2021-04-23 东南大学 维生素d结合蛋白作为标志物在精神疾病抑郁症诊断中的应用
CN112553328B (zh) * 2020-12-30 2022-06-17 浙江大学 检测基因表达水平的产品及其在制备重度抑郁症诊断工具中的应用
CN113528643A (zh) * 2021-07-21 2021-10-22 中国人民解放军军事科学院军事医学研究院 抑郁症相关的生物标志物及其诊断产品和应用

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040185474A1 (en) * 2002-12-27 2004-09-23 Kazuhito Rokutan Method of diagnosing depression
WO2014193999A2 (en) * 2013-05-28 2014-12-04 Caris Science, Inc. Biomarker methods and compositions
US20160319361A1 (en) * 2013-08-28 2016-11-03 Caris Life Sciences Switzerland Holdings Gmbh Oligonucleotide probes and uses thereof
CN107621545A (zh) * 2017-07-26 2018-01-23 东南大学 Bicc1蛋白对精神疾病诊断的新用途
CN111334567A (zh) * 2020-02-24 2020-06-26 新疆医科大学第二附属医院 HIF-1α基因启动子区甲基化检测引物及检测方法与应用
CN114438191A (zh) * 2022-01-27 2022-05-06 宁波大学 缺氧诱导因子1α作为标志物在抑郁症复发诊断中的应用

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