CN116008565A - Biomarker for auxiliary diagnosis of depression and application thereof - Google Patents

Abstract

The invention relates to a method for detecting a depression (Major depressive disorder) plasma biomarker kit, which is used for detecting a plasma biomarker of depression. The invention provides an application method of detecting one or more proteins in ErbB3, MIF, NSE and HGF R in blood plasma as biomarkers of Depression, which is used for assisting in diagnosing Depression and improving sensitivity and specificity of screening Depression (Depression) of common people.

Description

Biomarker for auxiliary diagnosis of depression and application thereof

Technical Field

The invention relates to the technical field of clinical medicine psychiatry, in particular to the technical field of depression, in particular to an auxiliary diagnosis and detection technology for judging depression.

Background

Depression (Major depressive disorder) is a severe chronic mental disorder characterized by core symptoms such as low mood, slow thinking, reduced cognitive function, and motor inhibition, and has the characteristics of high prevalence, high recurrence rate, high suicide rate, and high disabling performance. The prevalence of depression is about 6% overall for 12 months, while the risk of developing a lifetime is increased by about 3-fold (15-18%), meaning that almost one fifth of people experience at least one depressive episode at a time during their lifetime. Currently, about 3.5 million depressive patients worldwide, about 9500 ten thousand depressive patients in our country, and about 28 ten thousand suicides are suicidal for depression each year. Depression severely affects the physical health, daily life and psychosocial functions of the patient, causing a serious socioeconomic burden. In 2010, depression was listed by the World Health Organization (WHO) as the second greatest disease burden worldwide, and it is expected that 2030 will be the first to be the most urgent mental health problem to be solved worldwide

Unfortunately, current diagnosis of depression relies mainly on clinical phenomenology, lacking in practical and effective objective biological indicators. The research results show that the consistency of diagnosis in ten years of depression is only 45.5%, which severely limits the correct diagnosis of the disease by clinicians in the early stage of depression onset, so that the clinical diagnosis and treatment of depression are trapped. In fact, at present, on the one hand, diagnosis of patients can only be determined by trial and error strategies and each patient is administered with antidepressants that the doctor deems to be most effective: this means prolonged remission, patient suffering, poor compliance, occurrence of adverse therapeutic reactions, worsening suicidal ideation and high social costs. On the other hand, because the emotion of a person typically fluctuates with inheritance, context, event or socioeconomic environment, a trial-and-error strategy may misdiagnose the depression state of the general population as mild depression. Therefore, there is an urgent need to explore biomarkers that may be diagnostic for depression and the general population.

At present, most research on development of biological markers for exploring differential diagnosis of depression is mainly focused on basic research such as hematology, genetics, imaging and the like, but research results are inconsistent and even contradictory. Leading to serious difficulties in these studies under current medical conditions, and actual clinical application is quite visible. Even if good biological markers are found, effective popularization cannot be achieved because of the limitations of medical staff in awareness, medical insurance, material equipment and the like. At the same time, clinical transformations remain limited due to the strong subjectivity in the clinical evaluation process, or the complexity of the specific experimental design involved. Of all possible biomarkers, peripheral blood biomarkers are the easiest to obtain because of their applicability and availability in clinical practice, and are not susceptible to material equipment and clinical subjective judgment.

Therefore, it is highly desirable to establish a method for rapidly determining depression using plasma biological indicators.

Disclosure of Invention

The invention aims to provide an application method for accurately screening and assisting in diagnosing depression, which aims at the defects of early identification and accurate diagnosis of depression in the prior art: namely, measuring the differential expression level of one or more of 4 proteins such as plasma HER3 (Human epidermal growth factor receptor, erbB 3), macrophage migration inhibitory factor (macrophage migration inhibitory factor, MIF), human brain Neuron Specific Enolase (NSE) and hepatocyte growth factor receptor (Hepatocyte growth factor receptor, HGF R) becomes an objective index for clinical diagnosis.

In order to achieve the above object, the present invention provides an application of a biomarker in preparing a product for assisting diagnosis of depression, which is mainly characterized in that the biomarker is one or more of ErbB3, MIF, NSE and HGF R proteins.

The invention also provides application of the reagent for detecting the biomarker content in preparing a kit for assisting in diagnosis of depression, which is mainly characterized in that the biomarker is one or more of ErbB3, MIF, NSE and HGF R proteins.

The invention also provides application of the kit in auxiliary diagnosis of depression, which is mainly characterized in that the kit is used for detecting the content of biomarkers, wherein the biomarkers are one or more of ErbB3, MIF, NSE and HGF R proteins.

Preferably, the test sample is plasma.

The invention provides an application method for detecting one or more differential expression levels in ErbB3, MIF, NSE and HGF R proteins as melancholy type depression blood plasma biomarkers.

Drawings

FIG. 1 is a comparison of 4 protein differential expression levels (in pg/ml) for distinguishing between depressed patients and healthy controls, specifically NSE (A), erbB3 (B), MIF (C) and HGF R (D) proteins of depressed patients and healthy controls, with the bar graph representing mean and error bars being Standard Errors (SE). In the figure "×" represents the difference between the two groups P <0.001 after correction using Bonferroni method.

Fig. 2 shows ROC curve results for 4 proteins for distinguishing depression from healthy controls, specifically NSE (a), erbB3 (B), MIF (C) and HGF R (D) proteins, respectively. The abscissa is 1-specificity, the ordinate is sensitivity, and AUC (Area Under Curve) is the area enclosed by the coordinate axis under the ROC curve.

Detailed Description

For a further understanding of the present invention, preferred embodiments of the invention are described below in conjunction with the examples, but it should be understood that these descriptions are merely intended to illustrate further features and advantages of the invention and are not limiting of the invention claims.

Considering depression as a disease with extremely high clinical heterogeneity, the clinical symptoms, treatment strategies, treatment effects and prognosis, and even biological phenotypes of the disease have extremely high heterogeneity, the invention provides a method for assisting in diagnosis of depression by detecting the differential expression level of one or more of 4 proteins such as ErbB3, MIF, NSE and HGF R in blood plasma, and the method can be used as objective biological indexes of depression and applied to patients with mental examination considered as depression onset or depression state. And calculating the protein differential expression level of the 4 plasma proteins according to the detection result of one or more of the 4 plasma proteins to obtain a reference value for auxiliary diagnosis of depression, further diagnosing the patient, avoiding misdiagnosis and misdiagnosis, improving the diagnosis accuracy and treatment pertinence of a clinician and improving the prognosis of the disease.

The protein chip technology can be used for detecting one or more expression levels of ErbB3, MIF, NSE and HGF R proteins of patients, and can effectively distinguish depression patients and healthy control groups, so that the protein chip technology can be used as objective indexes for accurate screening and auxiliary diagnosis of depression. In addition, the patients with depressed emotion can be further diagnosed by combining with comprehensive mental examination, so that false positives are avoided.

Therefore, by detecting the differential expression level of one or more of ErbB3, MIF, NSE and HGF R proteins in plasma, the patients with depression can be identified early and diagnosed in an assisted manner, and the patients with depression can be clearly distinguished from healthy controls, and can be directly provided for a clinician to refer to, so that the accurate treatment scheme can be determined in a short time, the diagnosis and treatment time can be shortened, and the working efficiency and the accuracy of the diagnosis and treatment scheme can be improved.

In the invention, the biomarker is one or more of ErbB3, MIF, NSE and HGF R proteins, and is suitable for screening and auxiliary diagnosis of patients suffering from depression or depression onset.

In the present invention, terms such as "depression", "depression symptom", "depression onset" and the like and their meanings are as follows:

depression (Major depressive disorder) is a general term for diseases mainly represented by mood or mood depression, accompanied by various degrees of cognitive and behavioral changes, and possibly psychotic symptoms such as hallucinations, delusions, etc., and has the characteristics of high prevalence, high recurrence rate, high suicide rate and high disability. Currently, the depression is diagnosed clinically mainly by using an equivalent diagnosis system such as International Classification of diseases (ICD-10), manual of mental disease diagnosis and statistics (DSM-5) and the like.

Depressive episodes (major depressive episode) are disease states characterized by depression and are characterized by reduced mood, reduced interest, and loss of pleasure, often accompanied by other cognitive, somatic, and behavioral manifestations such as inattention, hypo-responsive, sleep disorders, reduced behavioral activity, and fatigue. Single depressive episodes last for at least 2 weeks, frequently repeated episodes, most of which can be alleviated each time, and part of which may have residual symptoms or be converted to chronic, may cause serious social function impairment.

Depression symptoms (depressive symptoms) refer to the clinical manifestations of patients in the event of depression, and can be largely divided into core symptoms, psychological symptoms and somatic symptoms. Wherein the core symptom group includes depressed mood, reduced interest, and loss of pleasure; psychological symptom groups include anxiety, mental retardation, cognitive symptoms, self-criminal responsibilities, suicidal and behavioral disorders, psychomotor retardation or agitation, psychotic symptoms, self-awareness, and the like; somatic symptom groups include sleep disorders, eating and weight disorders, loss of energy, depressed mood, light day and night, sexual dysfunction, other non-specific somatic symptoms, and the like.

In the present invention, the 4 proteins involved (NSE, erbB3, MIF and HGF R) are all human proteins. The basic information and functions of these 4 major proteins are presented one by one.

Human brain neuron-specific enolase (NSE, also known as ENO 2) is an enolase, a "metal-activated metalloenzyme," which catalyzes the dehydration of 2-phospho-D-glycerate to phosphoenolpyruvate in the glycolytic pathway and the reverse reaction in gluconeogenesis. Anaerobic conversion of glucose to a metabolite suitable for oxidation is necessary. In vertebrate organisms, there are three enolase isozymes expressed from different genes. Enolase alpha is ubiquitous; enolase β is muscle-specific, enolase γ is neuronal-specific, and all known eukaryotic enolases are dimers.

Human epidermal growth factor receptor 3 (HER 3/ErbB 3) is a membrane-bound protein encoded by the human ErbB3 gene, erbB3 protein is a member of the Epidermal Growth Factor Receptor (EGFR) family of receptor tyrosine kinases, and like other EGFR family members, consists of three parts, the extracellular ligand binding domain, the transmembrane domain, and the intracellular domain. ErbB3 is present in blood, peripheral organs and brain. Functionally, erbB3 plays an important role as a member of the EGFR family, while tyrosine protein kinases are cell surface receptors that function as neuromodulators. It binds to and is activated by neuregulin-1 (NRG 1); ligand binding increases phosphorylation of tyrosine residues and promotes binding to the p85 subunit of phosphatidylinositol 3-kinase. Can also bind to Adenosine Triphosphate (ATP) and nucleotides, mediating intracellular signal transduction. In addition, related pathways for cell proliferation or differentiation may also be initiated.

Macrophage Migration Inhibitory Factor (MIF) is a class of pleiotropic immunomodulatory cytokines with unique structures, has chemokine-like functions, and is a pro-inflammatory cytokine. It was first discovered in 1966 that it is primarily involved in the natural immune response to bacterial pathogens (innate immunity) and is also involved in cell-mediated immunity, immunomodulation and inflammatory processes. It plays a role in regulating macrophage function in host defense, mainly by inhibiting the anti-inflammatory action of glucocorticoids. Mainly distributed in the blood and brain.

Hepatocyte growth factor receptor (HGF R) is a glycosylated receptor tyrosine kinase, a product encoded by the MET gene of the proto-oncogene receptor tyrosine kinase (receptor tyrosine kinase), the encoded preprotein being proteolytically processed to produce 50kDa extracellular alpha chain alpha and 145kDa transmembrane beta chains, these subunits being linked by disulfide bonds to form mature dimers, which dimerize and activate to play a role in cell survival, embryogenesis and cell migration and invasion. In addition, the β subunit of the receptor HGF R can be further processed to form an M10 peptide.

The term "ROC curve" (receiver operator characteristic curve) as used herein, working characteristics curve, refers to a graphical curve showing the performance of a binary classification system as its discrimination threshold varies, which curve is created based on plotting true-to-false-positive rates under conditions of different threshold settings. Here, the true positive rate is referred to as sensitivity; the false positive rate was then counted as 1-specificity. Therefore, the ROC curve is based on a graphical display of true positive rate (sensitivity) versus false positive rate (1-specificity) over a range of cut-offs, and further a clinical use of the best cut-offs is selected with accuracy expressed as area under ROC curve (AUC), thereby providing a viable parameter for comparing test performance. In the ROC curve, the closer the AUC value is to 1, the more sensitive and highly specific the test is, while the closer the AUC value is to 0.5, the test is neither sensitive nor specific.

When referring to the difference between a test sample and a control sample or reference sample, the term "statistical difference" or "statistical significance" refers to the case where each group has less than 5% of the same probability (e.g., P < 0.05) when using an appropriate statistical analysis method, i.e., the probability of obtaining the same result on a completely random basis is less than 5 out of 100 attempts. Similarly, if the probability of each group being identical is less than 1%o (e.g., P < 0.001), the probability of achieving identical results on a completely random basis is less than 1 out of 1000 attempts. In the present invention we have chosen an appropriate statistical analysis method, for example, based on whether the study variable is normally distributed (using the Kolmogorov-Smirnov test), using a parametric model test such as the t-test or ANOVA test (normally distributed), or a non-parametric model such as the MannWhitney U test or the Kruskal-Wallis test (non-normally distributed). Meanwhile, the antibody chip detection data for 4 proteins were also log-transformed (base 2) so that the protein detection data after log2 transformation fit into a normal distribution (determined by Kolmogorov-Smirnov test).

The technical solutions in the embodiments of the present application are clearly and completely described below with reference to the accompanying drawings. The following examples are only illustrative of the present invention and are not intended to limit the scope of the invention. The experimental procedure, in which specific conditions are not specified in the examples, is generally carried out according to conventional conditions or according to conditions recommended by the manufacturer.

Examples

S1 clinicians first included acute stage patients who met the criteria for diagnosis of depression in ICD-10/DSM-5, who were first or recrudesces during the current episode of depression, and included healthy controls for mental and clinical evaluation, as shown in FIG. 1.

In this example, 120 subjects were included, and the number of healthy controls was 30 and the number of depressed patients was 90.

In this example, the inclusion and exclusion criteria for healthy control population and depression patients were:

healthy controls

Group entry criteria:

(1) Age 18-55 years;

(2) There is no family history of mental disease;

(3) The total score of the HAMD-17 scale is less than or equal to 5 points;

(4) No history of neurological degenerative diseases, brain trauma or cerebrovascular diseases;

(5) No substance abuse or substance addiction;

(6) Unstable angina, myocardial infarction, congestive heart failure, severe liver cirrhosis, acute and chronic renal failure, severe diabetes, aplastic anemia, epilepsy, and other somatic diseases such as severe barycenter, liver, kidney, endocrine, blood system, or diseases that may interfere with experimental evaluation (laboratory abnormal index is more than 2 times higher than normal);

(7) Voluntarily participate in the study, and sign informed consent.

Exclusion criteria:

(1) Excluding the history of mental illness, severe somatic illness, cerebrovascular disease or brain trauma;

(2) Excluding those who have developed severe allergic reactions or who have suffered from diseases of the immune system;

(3) Anti-inflammatory drugs or immunosuppressants were used within 1 month;

(4) Patients suffering from brain trauma or associated with severe somatic diseases;

(5) Pregnant, lactating women or those who are scheduled to become pregnant.

Patients suffering from depression

Group entry criteria:

(1) Meets the diagnosis standard of depressive episode in ICD-10/DSM-5, and patients in the first or recurrent acute phase of the depressive episode are present;

(2) Age 18-55 years;

(3) 17 Hamiltonian depression scale (HAMD-17) score total score is greater than or equal to 17 score;

(4) No antidepressant was taken within the first half of the group, no physical or psychological treatment was received;

(5) The method has a certain cultural degree and can understand the related content of research;

(6) Voluntarily attending the study, and signing a written informed consent.

Exclusion criteria:

(1) Secondary depression caused by other organic diseases;

(2) Drug-induced secondary depression;

(3) Alcohol or other substance abuse and dependence;

(4) Pregnant, lactating women or those who plan pregnancy;

(5) Severe suicidal attempts (HAMD-17 item 3 "suicidal" score > 3, or suicidal behavior during the current depressive episode);

(6) Co-morbid other mental disorders;

(7) Patients with severe somatic diseases.

S2, taking a plasma sample, and measuring plasma ErbB3, MIF, NSE and HGF R protein levels of the patient by adopting a protein chip technology.

In this example, the ErbB3, MIF, NSE and HGF R protein expression levels of plasma samples were detected using a quantitative protein chip kit.

The kit comprises the following items: quantitative protein glass chip (QAH-CUSTOM), sample dilutions, 20 Xwash I, 20 Xwash II, standard mixtures, immunoglobulin G antibodies to ErbB3, MIF, NSE and HGF R proteins, cy 3-streptavidin, slide washes and dryers, sealing strips, and the like.

The specific detection implementation method is as follows:

(1) Drying quantitative protein chips:

taking out the quantitative protein chip, balancing for 20-30 min at room temperature, and drying the chip in a vacuum drier for 1-2 hr to obtain the final product.

(2) And (3) preparing a standard:

(1) add 500. Mu.L of sample dilution to the vial of cytokine standard mixture and redissolve the standard. Before opening the small tube, the small tube is rapidly centrifuged, dissolved powder is gently blown up and down, and the small tube is marked as Std 1.

(2) 6 clean centrifuge tubes Std2, std3 through Std7, respectively, were labeled, and 200. Mu.L of sample diluent was added to each vial.

(3) 100. Mu.L of Std 1 was added to Std2 and gently mixed, and then 100. Mu.L of Std2 was added to Std3, and the mixture was diluted to Std7 in a gradient, whereby a standard solution was obtained.

(3) 100. Mu.L of the sample dilution was drawn into another new centrifuge tube, labeled CT, as negative control.

(4) The completely dried slide chip was removed, 100. Mu.L of sample dilution was added to each well, and incubated on a shaker at room temperature for 1 hour, and the quantitative antibody chip was blocked. Followed by washing with buffer.

(5) The buffer in each well was aspirated, 60 μl of standard solution and sample (sample 2-fold dilution loading) were added to the wells and incubated overnight at 4 ℃.

(6) The slide was washed using a chip washer. In this application, a Thermo Scientific Well Wash Versa chip plate washer is used to wash slides, which is essentially two steps, first with 1 Xwash I, 10 times with 250. Mu.L 1 Xwash I per well, 10 seconds per shake, with a high selection of shaking intensity, and dilution of 20 Xwash I with deionized water. Then, the 1 Xwashing reagent II channel was changed to wash, and 250. Mu.L of 1 Xwashing reagent II per well was washed 6 times with 10s shaking for each time, the shaking intensity was selected to be high, and 20 Xwashing reagent II was diluted with deionized water.

(7) Incubating the antibody mixture

The mixture containing NSE, erbB3, MIF and HGF R antibodies was centrifuged in eppendorf tubes, then 1.4ml of sample dilution was added, mixed well, centrifuged again rapidly, and 80. Mu.L of adiponectin antibody was added to each well, and incubated on a shaker at room temperature for 2 hours.

(8) And (6) cleaning again, wherein the step is the same as the step (6).

(9) Incubation of Cy 3-streptavidin

Cy 3-streptavidin eppendorf tube was centrifuged, followed by 1.4ml of sample dilution, mixed well and centrifuged again rapidly. mu.L of Cy 3-streptavidin was added to each well and the quantitative protein chip was incubated in a dark place with aluminum foil paper and 1 hour on a shaking table at room temperature.

(10) And (6) cleaning again, wherein the step is the same as the step (6).

(11) The chips were placed in a desiccator, centrifuged at 1000rpm and thoroughly dried.

(12) And (5) performing fluorescence detection on the dried glass chip. In this embodiment, the InnoScan 300Microarray Scanner fluorescence scanner is used to scan the signals, and the scanning parameters are: wavelength: 532nm; resolution:10 μm. Signal values were captured using Mapix software using Cy3 or green channel (excitation frequency=532 nm).

S3 data analysis was performed using QAH-CUST data analysis software to obtain ErbB3, MIF, NSE and HGF R protein expression levels in plasma samples. The method comprises the following steps:

firstly, carrying out normalization processing on original detection data of a protein chip experiment, namely removing chip background from original data obtained by chip scanning, averaging data of a standard substance, and carrying out normalization processing. Because the original measurement data is distributed in a biased state, the original measurement data is subjected to double log conversion (log 2) to be distributed in a normal state and then used for subsequent statistical analysis. In addition, the degree of dispersion of the data was analyzed, and data statistics were not taken into account for data other than the mean ± 2 standard errors (SD). In this example, the primary measurement of differential expression of NSE, erbB3, MIF and HGF R proteins and the discrete values are shown in Table 1.

Table 14 raw measurements of plasma proteins and values after double log conversion

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Note that: the italic numbers in the above table are discrete values, i.e. outside the mean ± 2 Standard Deviations (SDs), considered as outliers.

S4 the statistical results of the present invention are shown in fig. 2, where NSE, erbB3, MIF and HGF R plasma proteins are significantly differentially expressed in healthy control population and in the depressive group, where NSE, erbB3 and MIF protein expression levels in the depressive group are significantly increased, and HGF R protein expression levels are significantly decreased, and the differences are statistically significant (in this example, "x" P <0.001 in fig. 1).

In addition, as shown in fig. 2, the results of ROC curve analysis show that the AUC and 95% confidence intervals of NSE, erbB3, MIF and HGF R proteins for identifying depression and healthy control are 0.795 (0.711-0.879), 0.805 (0.724-0.886), 0.749 (0.655-0.842) and 0.760 (0.669-0.853), respectively, which indicates that NSE, erbB3, MIF and HGF R proteins have extremely excellent differential diagnosis efficacy as objective biomarkers for depression.

The detection of ErbB3, MIF, NSE and HGF R protein levels in the invention can accurately diagnose anxiety depression patients, avoid affecting the accuracy and pertinence of a treatment scheme due to misdiagnosis and missed diagnosis, and improve the working efficiency.

In this specification, the invention has been described with reference to specific embodiments thereof. It will be apparent, however, that various modifications and changes may be made without departing from the spirit and scope of the invention. The description is thus to be regarded as illustrative instead of limiting.

Claims (4)

1. Use of a biomarker for the manufacture of a product for the assisted diagnosis of depression, wherein the biomarker is one or more of ErbB3, MIF, NSE and HGF R proteins.

2. Use of a reagent for detecting biomarker content in the preparation of a kit for assisting in diagnosis of depression, wherein the biomarker is one or more of ErbB3, MIF, NSE and HGF R proteins.

3. The use of a kit for the assisted diagnosis of depression, wherein the kit is for detecting the level of a biomarker selected from one or more of ErbB3, MIF, NSE and HGF R proteins.

4. The use according to claim 3, wherein the test sample is plasma.

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