Application of beta2-microglobulin in preparation of product for diagnosis and / or prognosis evaluation of acute exacerbation of chronic obstructive pulmonary disease

Abstract

The invention provides application of beta2-microglobulin in preparation of a product for diagnosing and / or prognostically evaluating acute exacerbation of chronic obstructive pulmonary disease, and relates to the technical field of biology. By detecting the level of the beta2-microglobulin in the plasma sample of the subject, the result shows that the level of the beta2-microglobulin in the plasma sample of the patient at the acute exacerbation stage of chronic obstructive pulmonary disease is obviously increased compared with that of a healthy control group. According to the method, after patients in the acute exacerbation period of chronic obstructive pulmonary disease are discharged from hospital for one year, the re-exacerbation condition and the survival condition of the patients are counted, the patients are divided into a high-value group and a low-value group according to the beta2 microglobulin level during group entering, and a multi-factor analysis result shows that the content of the beta2 microglobulin in the group is higher than that in the prior art. The low-concentration beta2 microglobulin is an independent risk factor of the acute exacerbation risk of the patient suffering from the end-stage chronic obstructive pulmonary disease again. A test result shows that the beta2-microglobulin has diagnosis and prognosis evaluation values in acute exacerbation of chronic obstructive pulmonary disease.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to an application of β2-microglobulin in the preparation of a product for diagnosing and / or prognosticating acute exacerbation of chronic obstructive pulmonary disease. Background Art

[0002] Chronic obstructive pulmonary disease (COPD) is a chronic airway disease characterized by persistent airway symptoms and airflow limitation. It is a common and frequently occurring respiratory disease. Acute exacerbation of COPD refers to a continuous deterioration beyond the patient's daily condition in a short period of time, such as worsening cough, shortness of breath or wheezing, increased sputum volume, purulent or mucopurulent sputum, and fever, which may be accompanied by a significant increase in symptoms. It is necessary to change the routine medication for COPD. Acute exacerbation of COPD is the main reason for the deterioration of the patient's disease and increased mortality. At present, the diagnosis of acute exacerbation of COPD is based solely on the experience of clinicians and lacks quantitative indicators. Therefore, there is an urgent need to discover objective indicators for diagnosing and predicting acute exacerbation of COPD.

[0003] At present, there are many studies on acute exacerbation of COPD at home and abroad, but the achievements in predicting acute exacerbation biomarkers are very limited. Some studies have shown that certain biomarkers (such as C-reactive protein, fibrinogen, blood eosinophils, surfactant protein D, fetoprotein A, lipofuscin and interleukin 6, etc.) have a certain role in predicting the risk of future acute exacerbations in COPD patients, but the above indicators have not been confirmed in large-scale clinical trials, so they have not been widely used in clinical practice. In 2017, Jason D et al. analyzed 90 clinically rare plasma biomarkers in patients in the two major cohorts of SPIROMICS and COPDGene and found that even if some markers in the two cohorts were linearly correlated with acute exacerbation of symptoms, the reproducibility between the two cohorts was very poor, and these markers only played a very small role in suggesting prediction on the basis of the above commonly used biomarkers, and their clinical application value was very limited.

[0004] β2-Microglobulin (β2-MG) belongs to the light chain of MHC I. MHC I is composed of heavy chain (hypervariable region) and light chain (constant region), and is involved in CD8 T cell-mediated anti-infection immunity. β2-MG is highly expressed in all immune cells. The complex of MHC I and β2-MG is secreted into the blood after being hydrolyzed. Under normal circumstances, β2-MG is maintained at a relatively constant level. In addition, β2-MG is reabsorbed and decomposed by the proximal tubules of the kidneys. Under normal circumstances, most of it is cleared by the kidneys, and the plasma β2-MG level is low. Therefore, β2-MG is associated with the onset of infection, lymphoproliferative blood system diseases, and kidney diseases, but its diagnostic and prognostic value in patients with acute exacerbation of COPD remains unclear. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide an application of β2-microglobulin in the preparation of a product for diagnosis and / or prognosis evaluation of acute exacerbation of COPD, which can assist in the early identification, disease assessment, and prognosis judgment of acute exacerbation of COPD.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides an application of β2-microglobulin in the preparation of a product for diagnosing and / or prognosticating acute exacerbation of chronic obstructive pulmonary disease.

[0008] Preferably, the β2-microglobulin level is significantly elevated, indicating that the subject is at risk of acute exacerbation of COPD.

[0009] Preferably, when the β2-microglobulin level concentration is <2.1 mg / L, it indicates that patients with end-stage COPD are at risk of another acute exacerbation.

[0010] Preferably, the product comprises a reagent for detecting the level of β2-microglobulin in a sample or a kit for detecting the level of β2-microglobulin in a sample.

[0011] More preferably, the sample is selected from a plasma sample or a serum sample.

[0012] More preferably, the detection method comprises radioimmunoassay or enzyme-linked immunosorbent assay.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] The present invention detects the level of β2-microglobulin in the plasma samples of the subjects, and the results show that the level of β2 microglobulin in the plasma samples of patients with acute exacerbation of COPD is significantly increased compared with the healthy control group. The present invention follows up the patients with acute exacerbation of COPD for one year after discharge, statistics the patients' re-exacerbation and survival status, and divides them into high-value group and low-value group according to the β2 microglobulin level at the time of enrollment. The results of multivariate analysis show that low concentration of β2 microglobulin is an independent risk factor for the risk of re-acute exacerbation in patients with end-stage COPD. The present invention uses β2 microglobulin as an objective indicator to assist in the early identification of acute exacerbation of COPD, disease assessment, and prognosis judgment, and provide direction for clinical treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a volcano plot of the differences in plasma protein between patients with acute exacerbation of COPD and patients with stable COPD;

[0016] Figure 2 This is a schematic diagram of the quantitative detection results of plasma β2-microglobulin levels in patients with acute exacerbation of COPD, patients with stable COPD and healthy controls; in the figure, the left figure shows the plasma β2-MG levels obtained by proteomics method, with 30 people in each group; the right figure shows the plasma β2-MG levels obtained by ELISA method, with 50 people in the acute exacerbation group of COPD, 20 people in the stable group and 20 people in the healthy group;

[0017] Figure 3 The ROC curve for diagnosing acute exacerbation of COPD by plasma β2-MG level;

[0018] Figure 4 The Kaplan-Meier survival curves of acute exacerbation and re-hospitalization of COPD patients stratified by different β2-microglobulin levels; in the figure, A is a patient with early COPD (GOLD1-2); B is a patient with late COPD (GOLD3-4); C is a patient with late COPD (after correction for previous acute exacerbations);

[0019] Figure 5 This is the COX survival curve of acute exacerbation and re-hospitalization of COPD patients stratified by different β2-microglobulin levels; in the figure, A is a patient with early COPD (GOLD1-2); B is a patient with late COPD (GOLD3-4);

[0020] Figure 6 Figure 5 Kaplan-Meier time-to-exacerbation curves for severe exacerbations stratified by β2-MG levels in SPIROMICS patients with end-stage COPD. DETAILED DESCRIPTION

[0021] The present invention provides an application of β2-microglobulin in preparing a product for treating acute exacerbation of chronic obstructive pulmonary disease.

[0022] The present invention provides an application of β2-microglobulin in preparing a product for prognosis assessment of acute exacerbation of chronic obstructive pulmonary disease.

[0023] In the present invention, the β2-microglobulin level in the subject's sample is increased compared to the β2-microglobulin level in the healthy control sample, indicating that the subject is at risk of acute exacerbation of COPD. In the present invention, the β2-microglobulin level refers to the total β2-microglobulin level, that is, the amount of total β2-microglobulin contained in the sample. In the present invention, the term "healthy control" refers to a healthy subject who is determined in advance not to suffer from a disease including COPD.

[0024] In the present invention, the product includes a reagent for detecting the level of β2-microglobulin in a sample or a kit for detecting the level of β2-microglobulin in a sample. In the present invention, as an practicable method, the reagent for detecting the level of β2-microglobulin in a sample is well known to those skilled in the art, and includes any reagent that can specifically bind to total β2-microglobulin and detect its content, including but not limited to anti-β2-microglobulin antibodies.

[0025] In the present invention, the method for detecting the level of β2-microglobulin in the sample of the subject can be detected by any method known to those skilled in the art. In the present invention, as an operative method, the detection method includes but is not limited to radioimmunoassay and enzyme-linked immunosorbent assay. For example, radioimmunosorbent test (IRST), double antibody sandwich ELISA, indirect hemagglutination test, etc.

[0026] In the present invention, the sample can be any biological sample derived from a subject. In the present invention, the sample comprises a liquid (including blood, urine, semen), a tissue, a cell sample, an organ, a biopsy sample, etc. In the present invention, the sample is preferably selected from a serum sample and a plasma sample.

[0027] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0028] Example 1

[0029] (1) Sampling: Collect venous blood from patients, use EDTA for anticoagulation, centrifuge at 3000 rpm and 4°C for 10 min, take 0.2 mL / tube of the supernatant and dispense it into 1.5 mL cryovials, store at -80°C to avoid repeated freezing and thawing until proteomics or ELISA analysis is performed.

[0030] (2) DIA quantitative proteomics detection

[0031] Take out the sample from the -80℃ refrigerator, transfer it to a 1.5ml centrifuge tube, add an appropriate amount of protein dissolving solution (8M urea, 100mM TEAB, pH=8.5), and shake to mix. Centrifuge at 4℃, 12000g for 15min, take the supernatant, add 10mM DTT, react at 56℃ for 1h, then add sufficient IAM, react at room temperature in the dark for 1h. Take the protein sample, add protein dissolving solution (8M urea, 100mMTEAB, pH=8.5) to make up the volume to 100μL, add trypsin and 100mM TEAB buffer, mix well, digest at 37℃ for 4h, then add trypsin and CaCl2 to digest overnight. Formic acid was added to adjust the pH to less than 3, and the mixture was centrifuged at room temperature and 12000g for 5 minutes. The supernatant was slowly passed through a C18 desalting column, and then the column was continuously washed three times with a cleaning solution (0.1% formic acid, 3% acetonitrile). An appropriate amount of eluent (0.1% formic acid, 70% acetonitrile) was added, and the filtrate was collected and freeze-dried. Mass spectrometry and data analysis of the collected freeze-dried samples were performed by Beijing Novogene Technology Co., Ltd.

[0032] (3) ELISA analysis

[0033] The plasma samples were diluted according to the instructions, and the concentration of β2 microglobulin in the plasma was detected using an ELISA kit (purchased from Abcam, product number ab181423).

[0034] Example 2

[0035] The subjects were a multicenter clinical cohort consisting of 30 patients with acute exacerbation of COPD, 30 patients with stable COPD, and 30 age- and sex-matched healthy controls enrolled in Peking Union Medical College Hospital and its affiliated medical alliance hospitals. Inclusion criteria for COPD patients: (1) met the diagnostic criteria for COPD (stable phase, acute exacerbation phase); (2) aged ≥ 40 years; Exclusion criteria: (1) severe heart, liver, and kidney dysfunction; (2) combined with malignant tumors; (3) combined with other respiratory diseases; (4) lost to follow-up or dropped out.

[0036] Venous blood samples were collected from COPD patients and healthy controls according to the method of Example 1 for proteomics and ELISA analysis. This study was approved by Peking Union Medical College Hospital (Approval No.: B306). All subjects signed informed consent before inclusion in the cohort.

[0037] Proteomics results identified 21 differentially expressed proteins, including 15 up-regulated proteins and 6 down-regulated proteins ( Figure 1 ). The results of proteomics and ELISA tests showed that the levels of β2-microglobulin in serum samples of patients with acute exacerbation of COPD were significantly increased compared with those in the control group ( Figure 2 ).

[0038] Example 3 Application of β2-microglobulin in diagnosing acute exacerbation of chronic obstructive pulmonary disease

[0039] The receiver operating characteristic (ROC) curve of Example 2 was drawn, and the area under the curve (AUC) was used to evaluate the diagnostic efficacy of plasma β2 microglobulin for acute exacerbation of COPD. P<0.05 was considered statistically significant.

[0040] according to Figure 3 The results of ROC analysis showed that β2-microglobulin had diagnostic value in acute exacerbation of COPD (AUC=0.988).

[0041] Example 4 Application of β2-microglobulin in prognostic assessment of acute exacerbation of chronic obstructive pulmonary disease

[0042] Follow-up of the patients in Example 2 for relapse within 1 year

[0043] The patients with acute exacerbation of COPD were followed up for 1 year after discharge, and the re-exacerbation and survival of the patients were recorded. According to the β2 microglobulin level at the time of enrollment, they were divided into a high value group (>2.1mg / L) and a low value group (<2.1mg / L). Kaplan-Meier and Cox regression were used to draw survival curves, and the related factors affecting the prognosis of patients with acute exacerbation of COPD were analyzed.

[0044] according to Figure 4 The Kaplan-Meier survival curve showed that in patients with advanced COPD (GOLD3-4), patients with low β2-microglobulin levels had a poor prognosis ( Figure 4 B. Figure 4 C), while the opposite results were obtained in patients with early COPD (GOLD1-2) ( Figure 4 A) The results of multivariate analysis are as follows Figure 5 As shown in the data, low concentration of β2-microglobulin was an independent risk factor for the risk of recurrent acute exacerbation in patients with end-stage COPD (HR=9.478, P<0.05).

[0045] Example 5 Validation of the use of β2-microglobulin in the diagnosis and / or prognostic assessment of acute exacerbation of COPD by external large database

[0046] (1) SPIROMICS cohort verifies differences in β2-microglobulin between different groups

[0047] The Subpopulations and Intermediate Outcome Measures in COPD Study (SPIROMICS) study data were applied for validation from the National Heart, Lung, and Blood Institute Biospecimen and Data Information Coordinating Center. The study is a multicenter prospective cohort study that includes patients with COPD, smokers with normal lung function, and non-smokers, and the participants are regularly followed up clinically.

[0048] (2) Prognosis of acute exacerbation of COPD based on grouping based on plasma β2-microglobulin levels

[0049] A total of 1399 patients were included, and peripheral blood β2 microglobulin levels were detected and recorded. According to the median β2 microglobulin level (2.1 mg / L), they were divided into high-value group and low-value group, and the effects of demographic characteristics, clinical data, and previous exacerbation history on the occurrence of severe COPD acute exacerbation during the follow-up period were comprehensively considered.

[0050] (3) Results analysis

[0051] After adjusting for potential confounding factors such as age, gender, and race, it was found that low concentrations of β2-microglobulin were still a risk factor for future severe acute exacerbations in patients with end-stage COPD (GOLD3-4). Figure 6 ).

[0052] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of β2-microglobulin in the preparation of products for diagnosis and / or prognosis assessment of acute exacerbation of COPD.

2. The use according to claim 1, characterized in that: The significantly elevated β2-microglobulin level indicates that the subject is at risk of acute exacerbation of COPD.

3. The use according to claim 1, characterized in that: When the β2-microglobulin level concentration is <2.1 mg / L, it indicates that patients with end-stage COPD are at risk of another acute exacerbation.

4. The use according to claim 1, characterized in that: The product includes a reagent for detecting the level of β2-microglobulin in a sample or a kit for detecting the level of β2-microglobulin in a sample.

5. The use according to claim 4, characterized in that: The sample is selected from a plasma sample or a serum sample.

6. The use according to claim 4, characterized in that: The detection method includes radioimmunoassay or enzyme-linked immunosorbent assay.

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