Application of premature coronary heart disease marker MXRA5 in preparation of reagent and kit for diagnosing premature coronary heart disease

Abstract

The invention relates to the technical field of coronary heart disease detection reagents or kits, in particular to application of a premature coronary heart disease marker MXRA5 in preparation of a reagent and a kit for diagnosing premature coronary heart disease. It is found for the first time that the level of MXRA5 in a patient suffering from the premature coronary heart disease is remarkably increased, MXRA5 has a large area under a curve, MXRA5 can effectively distinguish healthy control from PCHD groups, it is prompted that application of MXRA5 to PCHD has high diagnostic value, and the application has great significance in diagnosis of the premature coronary heart disease and guidance of standardized treatment; the diagnostic index provided by the invention is serum metabolite, can be detected only by taking a small amount of blood, and is basically noninvasive.

Description

Technical Field

[0001] The present invention relates to the technical field of coronary heart disease detection reagents or kits, and is an application of an early-onset coronary heart disease marker MXRA5 in the preparation of reagents and kits for diagnosing early-onset coronary heart disease. Background Art

[0002] Coronary artery heart disease (CHD) is a type of myocardial hypoxia-ischemia disease caused by the narrowing and obstruction of the coronary arteries, which prevents the blood from carrying enough oxygen and nutrients to the heart. The 2016 European Epidemiology Report on Cardiovascular Diseases showed that cardiovascular disease accounted for 45% of all causes of death from diseases, and 25% of cardiovascular disease patients were under 75 years old, an increase from the previous period. The incidence of coronary heart disease in the young and middle-aged groups has been increasing year by year. According to the NECP-ATP III regulations, premature coronary artery heart disease (PCHD) is defined as patients with coronary heart disease whose onset age is ≤55 years for men and ≤65 years for women. The age group covered by PCHD is the main population in society, so early identification and early intervention are of great significance.

[0003] PCHD is a multifactorial disease that is not only closely related to genetic factors, but also affected by a variety of environmental factors. At present, obesity, hypertension, diabetes, abnormal lipid metabolism, etc. cannot fully explain the pathogenesis of PCHD. At present, the diagnosis of premature coronary heart disease mainly relies on coronary angiography and coronary CTA, but its accuracy and applicability are affected by many factors, such as the professional level of the operator and the patient's unwillingness to undergo invasive examinations. Understanding the risk factors of PCHD is the basis for identification and intervention. The particularity of PCHD risk factors, the diagnostic and therapeutic value of various inflammatory indicators and new inflammatory markers in PCHD patients are worthy of our in-depth discussion and research.

[0004] Extensive research has been dedicated to studying blood biomarkers for early-onset coronary heart disease to improve diagnosis and facilitate personalized treatment. In-depth research has been conducted from aspects such as gene polymorphisms, miRNAs, immune cells, etc., but their promotion and application in clinical practice are often limited due to complex detection methods and poor practicality. ELISA, the full name is Enzyme-Linked Immunosorbent Assay, is a commonly used biochemical analysis method, especially playing an important role in immunological detection. ELISA binds antigens or antibodies to the surface of a solid-phase carrier (such as a microplate), then adds the sample to be tested to allow it to undergo a specific immune reaction with the antigens or antibodies on the solid-phase carrier. Subsequently, enzyme-labeled antigens or antibodies are added to form an antigen-antibody-enzyme-labeled antigen / antibody complex. Finally, by adding a substrate, the enzyme catalyzes the substrate to develop color, and the content of the substance to be tested is quantitatively or qualitatively analyzed based on the depth of the color or the optical density value. This method is simple to operate, economical and applicable, can simultaneously and rapidly measure multiple samples, and has high specificity. Studying differential metabolites in serum and exploring metabolic markers are of great significance for achieving early diagnosis of early-onset coronary heart disease.

[0005] Human matrix-remodeling associated 5 (MXRA5) is a matrix-remodeling related protein. The essence of MXRA5 is a protein molecule with a molecular weight of 312 kDa and a vascular endothelial growth factor receptor, belonging to the MXRA family. The main role of the MXRA family in the human body is to participate in cell adhesion and matrix reconstruction, and its expression is up-regulated in the cartilage of patients with osteoarthritis. Therefore, MXRA5 is widely used in the diagnosis and evaluation of curative effects of orthopedic diseases (Lin Qiaofeng, et al. Expression and clinical significance of human matrix remodeling associated protein in feces of colon cancer patients [J]. Shenzhen Journal of Integrated Traditional Chinese and Western Medicine, Vol. 27, No. 8, April 2017, 3-4).

[0006] The Chinese patent document with the publication number CN114058693A discloses the application of MEG3 as a diagnostic marker for coronary heart disease. MEG3 is used as a marker for diagnosing different degrees of coronary artery stenosis. Compared with healthy people, the expression level of LncRNA MEG3 in the peripheral blood of CHD patients with coronary artery stenosis > 90% is significantly down-regulated statistically.

[0007] The Chinese patent document with the publication number CN115851910A discloses a marker, system and application for diagnosing or predicting coronary heart disease. The marker includes 12 intestinal bacterial species. Using the marker and system of the present invention, it is possible to accurately diagnose whether the test subject has coronary heart disease and predict whether the subject has the risk of suffering from coronary heart disease.

[0008] The Chinese patent document with the publication number CN113533737A discloses the application of NFAM1 in screening drugs for preventing and treating coronary heart disease or preparing a kit, and specifically discloses the application of a nuclear factor of activated T cells (NFAT) activating protein with immunoreceptor tyrosine-based activation motif 1 (NFAM1) in searching for or screening drugs or biological agents for preventing and treating coronary heart disease and coronary heart disease-related diseases, as well as in preparing a kit for early warning, early diagnosis or prognosis assessment. The present invention first confirms that the expression of NFAM1 in peripheral blood monocytes of patients with coronary heart disease is significantly increased. The present invention also discovers that inhibiting the level of NFAM1 can effectively reduce the expression of chemokine receptors and the activation of the p38 MAPK pathway, thereby inhibiting monocyte chemotaxis.

[0009] The Chinese patent document with the publication number CN110082535A discloses a molecular marker MYH11 for diagnosing chronic coronary heart disease and its application, and discovers that by detecting the concentration of MYH11 in a patient's blood sample, the occurrence of diseases caused by atherosclerosis such as chronic coronary heart disease can be predicted, and for diagnosing chronic coronary heart disease.

[0010] There is currently no report on MXRA5 as a marker for premature coronary heart disease. Summary of the Invention

[0011] The present invention provides the application of a premature coronary heart disease marker MXRA5 in preparing a reagent and a kit for diagnosing premature coronary heart disease. The present invention first discloses that the level of MXRA5 is significantly increased in patients with premature coronary heart disease. MXRA5 can effectively distinguish between healthy controls and the PCHD group, suggesting that MXRA5 has a high diagnostic value for PCHD and can be used as a marker for diagnosing premature coronary heart disease.

[0012] One of the technical solutions of the present invention is achieved by the following measures: the application of a premature coronary heart disease marker MXRA5 in preparing a reagent for diagnosing premature coronary heart disease.

[0013] Another technical solution of the present invention is achieved by the following measures: the application of a premature coronary heart disease marker MXRA5 in preparing a kit for diagnosing premature coronary heart disease.

[0014] The kit includes a reagent for detecting the content of metabolite MXRA5 in a test sample, and also includes instructions for using the kit to evaluate whether a subject has or is prone to premature coronary heart disease.

[0015] The following is a further optimization or / and improvement of the above-mentioned invention technical solution: The detection sample sources of the above-mentioned early-onset coronary heart disease marker MXRA5 include, but are not limited to, blood, serum, plasma, lymph fluid, sweat, saliva, vitreous humor, synovial fluid, lymph fluid, semen, cerebrospinal fluid, urine, cell culture medium, cells, or ex vivo tissue samples or organ samples.

[0016] The sample source of the above-mentioned early-onset coronary heart disease marker MXRA5 is serum.

[0017] In the above-mentioned detection samples, the level of the early-onset coronary heart disease marker MXRA5 is significantly increased.

[0018] In the serum of the above-mentioned early-onset coronary heart disease patients, the level of the early-onset coronary heart disease marker MXRA5 is significantly increased.

[0019] The present invention first discovered that the level of MXRA5 is significantly increased in patients with early-onset coronary heart disease, and MXRA5 has a very high area under the curve. MXRA5 can effectively distinguish healthy controls and the PCHD group, indicating that the application of MXRA5 to PCHD has high diagnostic value and is of great significance for the diagnosis and guiding standardized treatment of PCHD; moreover, the diagnostic index provided by the present invention is a serum metabolite, and only a small amount of blood needs to be taken for detection, which is basically non-invasive. Description of the Drawings

[0020] Appendix Figure 1 It is a bar chart of the MXRA5 concentration in the early-onset coronary heart disease group and the healthy control group.

[0021] Appendix Figure 2 It is an ROC curve analysis chart of MXRA5 for detecting early-onset coronary heart disease. Detailed Embodiments

[0022] The present invention is not limited by the following embodiments, and the specific implementation manners can be determined according to the technical solutions of the present invention and the actual situation.

[0023] The present invention will be further described below in conjunction with the embodiments: Embodiment 1: Application of an early-onset coronary heart disease marker MXRA5 in the preparation of a reagent for diagnosing early-onset coronary heart disease.

[0024] Embodiment 2: Application of an early-onset coronary heart disease marker MXRA5 in the preparation of a kit for diagnosing early-onset coronary heart disease.

[0025] Embodiment 3: As an optimization of the above embodiments, the detection sample sources of the early-onset coronary heart disease marker MXRA5 include, but are not limited to, blood, serum, plasma, lymph fluid, sweat, saliva, vitreous humor, synovial fluid, lymph fluid, semen, cerebrospinal fluid, urine, cell culture medium, cells, or ex vivo tissue samples or organ samples.

[0026] Example 4: As an optimization of the above Example 3, the sample source of the early-onset coronary heart disease marker MXRA5 is serum.

[0027] Example 5: As an optimization of the above example, in the test sample, the level of the early-onset coronary heart disease marker MXRA5 is significantly increased.

[0028] Example 6: As an optimization of the above Example 5, in the serum of the early-onset coronary heart disease patients, the level of the early-onset coronary heart disease marker MXRA5 is significantly increased.

[0029] The relevant research content on the application of the early-onset coronary heart disease marker MXRA5 of the present invention in the preparation of reagents and kits for diagnosing early-onset coronary heart disease is as follows: 1. Materials and Reagents Reagents Distilled water or deionized water (H 2 O) Universal Diluent Standard Concentrated biotinylated detection antibody 100X (Biotin-antibody(100X)) Concentrated enzyme conjugate 100X (Streptavidin-HRP(100X)) 20X Wash Buffer (Wash Buffer(20X)) TMB Substrate Stop Solution Plate Sealer Instruments Microplate reader Centrifuge in a 37°C incubator 2. Experimental Methods 2.1 Sample Collection Collect serum samples from 80 patients with early-onset coronary heart disease and 80 healthy controls.

[0030] Centrifuge the whole blood specimens collected in serum separator tubes at 3500 rpm for 15 min at 2°C to 8°C within 30 minutes after collection, and take the supernatant, or store the supernatant (i.e., serum) at -80°C to avoid repeated freezing and thawing.

[0031] Inclusion criteria for the early-onset coronary heart disease group: 1) The subject has signed an informed consent form.

[0032] 2) According to the NCEP-ATPⅢ criteria, premature coronary artery heart disease (PCHD) is defined as coronary heart disease patients with onset age ≤ 55 years for men and ≤ 65 years for women.

[0033] 3) Exclusion criteria: those with severe liver and kidney function impairment, patients with malignant tumors, hematological system, autoimmune diseases, etc.; pregnant and lactating women; patients under 18 years old.

[0034] Inclusion criteria for the healthy control group: 1) The subjects have signed the informed consent form.

[0035] 2) Those with good health.

[0036] 3) Exclusion criteria: those with severe infections, at risk of bleeding; those who have undergone coronary intervention; those who are receiving immunosuppressive therapy; those with severe liver and kidney insufficiency, severe heart failure, severe valvular heart disease; patients in the active stage of malignant tumors; pregnant and lactating patients; Collect 4 mL to 5 mL of fasting venous blood from all subjects, centrifuge at 3500 rpm / 15 min at 4°C. After centrifugation, carefully take out the blood collection tube, avoid inversion, and immediately separate the upper serum for standby.

[0037] 2.2 Detection of metabolites (i.e., MXRA5) 1) Take out reagents such as concentrated biotinylated detection antibody, concentrated enzyme conjugate, and standard from the refrigerator and equilibrate to room temperature; 2) Preparation of standard gradient working solution: Add 1 mL of universal diluent to the lyophilized standard, let it stand for 15 minutes until completely dissolved, and then gently mix (concentration is 4000 pg / mL). Dilute according to the following concentrations: 4000 pg / mL; 2000 pg / mL; 1000 pg / mL; 500 pg / mL; 250 pg / mL; 125 pg / mL; 62.5 pg / mL; 0 pg / mL.

[0038] Serial dilution method: Take 7 EP tubes, add 500 μL of universal diluent to each tube. Pipette 500 μL from the 4000 pg / mL standard working solution into the first EP tube and mix to prepare a 2000 pg / mL standard working solution. Follow this step to pipette and mix successively. The last tube is directly used as the blank well and no liquid needs to be pipetted from the penultimate tube.

[0039] 3) Pipette 100 μL of the sample into an EP tube, add 100 μL of universal diluent (sample:diluent = 1:1 (V / V)), and vortex for 30 s.

[0040] 2.3 Metabolite detection 1) After equilibration at room temperature for 10 minutes, take out the required strips from the aluminum foil bag, seal the remaining strips in a ziplock bag and place them back at 4°C.

[0041] 2) Sample addition: Add 100 μL of the serum sample to be tested or the standard of different concentrations into the corresponding wells, add 100 μL of universal diluent into the blank wells, cover with the sealing plate and incubate at 37°C for 1 hour.

[0042] 3) Add biotinylated antibody: Take out the microplate reader, discard the liquid, and do not wash. Add 100 μL of biotinylated antibody working solution directly to each well, cover with the sealing mold and incubate at 37℃ for 1 hour.

[0043] 4) Wash the plate: discard the liquid, add 300 μL 1x washing solution to each well, let it stand for 1 minute, shake off the washing solution, pat dry on absorbent paper, and repeat this washing process 3 times.

[0044] 5) Add enzyme conjugate working solution: Add 100 μL of enzyme conjugate working solution to each well, cover with sealing plate mold and incubate at 37°C for 30 minutes.

[0045] 6) Wash the plate: discard the liquid and wash the plate 5 times according to the washing method in step 4.

[0046] 7) Add substrate: Add 90 μL of substrate (TMB) to each well, cover with sealing mold, and incubate at 37°C in the dark for 15 minutes.

[0047] 8) Add stop solution: Take out the ELISA plate, add 50 μL of stop solution directly to each well, and immediately measure the OD value of each well at a wavelength of 450 nm.

[0048] 2.4 Data Analysis The original data was copied from the microplate reader and converted into Excel format. The standard curve was drawn with concentration as the ordinate and OD value as the abscissa. When R 2 When >0.99, this formula can be used to calculate the concentration.

[0049] If the OD value of the sample is higher than the upper limit of the standard curve, it should be appropriately diluted and re-measured and multiplied by the corresponding dilution factor when calculating the sample concentration.

[0050] 3. Results After normality and variance homogeneity tests were performed on the concentrations of MXRA5 in the premature coronary heart disease group and the healthy control group, the t-test was used to compare whether there were any differences between the two groups.

[0051] The results showed that compared with the healthy control group, MXRA5 was significantly increased in the premature coronary heart disease group (Table 1, Figure 1as shown), and ROC curve analysis found that MXRA5 had good predictive value, with an AUC value of 0.921 (as Figure 2 shown).

[0052] In summary, the present invention first discovered that the level of MXRA5 was significantly increased in patients with premature coronary heart disease, and MXRA5 had a high area under the curve. MXRA5 could effectively distinguish healthy controls from the PCHD group, suggesting that the application of MXRA5 to PCHD had high diagnostic value, which was of great significance for the diagnosis and guiding standardized treatment of PCHD; moreover, the diagnostic index provided by the present invention was a plasma metabolite, and only a small amount of blood needed to be taken for detection, which was basically non-invasive.

[0053] The above technical features respectively constitute the embodiments of the present invention, which have strong adaptability and implementation effects. Non-essential technical features can be added or subtracted according to actual needs to meet the requirements of different situations.

Claims

1. Application of MXRA5, a marker for premature coronary heart disease, in the preparation of reagents for diagnosing premature coronary heart disease.

2. The use of the premature coronary heart disease marker MXRA5 according to claim 1 in the preparation of a reagent for diagnosing premature coronary heart disease, characterized in that: The detection sample sources of the premature coronary heart disease marker MXRA5 include but are not limited to blood, serum, plasma, lymph, sweat, saliva, vitreous fluid, synovial fluid, lymph, semen, cerebrospinal fluid, urine, cell culture fluid, cells or in vitro tissue samples or organ samples.

3. Use of the premature coronary heart disease marker MXRA5 according to claim 2 in the preparation of a reagent for diagnosing premature coronary heart disease, characterized in that: The sample source of the premature coronary heart disease marker MXRA5 is serum.

4. Use of the premature coronary heart disease marker MXRA5 according to claim 2 or 3 in the preparation of a reagent for diagnosing premature coronary heart disease, characterized in that: In the test sample, the level of MXRA5, a marker for premature coronary heart disease, was significantly increased.

5. Use of the premature coronary heart disease marker MXRA5 according to claim 4 in the preparation of a reagent for diagnosing premature coronary heart disease, characterized in that: In the serum of the patients with premature coronary heart disease, the level of the premature coronary heart disease marker MXRA5 is significantly increased.

6. Use of MXRA5, a marker for premature coronary heart disease, in the preparation of a kit for diagnosing premature coronary heart disease.

7. Use of the premature coronary heart disease marker MXRA5 according to claim 6 in preparing a kit for diagnosing premature coronary heart disease, characterized in that: The detection sample sources of the premature coronary heart disease marker MXRA5 include but are not limited to blood, serum, plasma, lymph, sweat, saliva, vitreous fluid, synovial fluid, lymph, semen, cerebrospinal fluid, urine, cell culture fluid, cells or in vitro tissue samples or organ samples.

8. Use of the premature coronary heart disease marker MXRA5 according to claim 7 in preparing a kit for diagnosing premature coronary heart disease, characterized in that: The sample source of the premature coronary heart disease marker MXRA5 is serum.

9. Use of the premature coronary heart disease marker MXRA5 according to claim 7 or 8 in preparing a kit for diagnosing premature coronary heart disease, characterized in that: In the test sample, the level of MXRA5, a marker for premature coronary heart disease, was significantly increased.

10. Use of the premature coronary heart disease marker MXRA5 according to claim 9 in preparing a kit for diagnosing premature coronary heart disease, characterized in that: In the serum of the patients with premature coronary heart disease, the level of the premature coronary heart disease marker MXRA5 is significantly increased.

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