Application of peripheral T lymphocyte index in preparation of coronary heart disease risk assessment kit, kit and method

Abstract

The invention discloses application of a peripheral T lymphocyte index in preparation of a coronary heart disease risk assessment kit, the kit and a method. Prediction indexes of the kit comprise four T lymphocyte subgroups: CD3 + CD8 + CD28-, CD3 + CD8 + HLADR +, Tc1, CD3 + gamma delta + V delta 2 + NKG2D +. The invention finds that the four novel lymphocytes in the peripheral blood are risk factors of the coronary heart disease for the first time, and the four risk factors are combined as a combined prediction index. The kit has an optimal effect on evaluating the risk of the coronary heart disease, and has a wide application prospect in the aspect of predicting the risk of the coronary heart disease.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to the application of peripheral T lymphocyte indexes in the preparation of a coronary heart disease risk assessment kit, a kit and a method. Background Art

[0002] Coronary atherosclerotic heart disease, clinically known as coronary artery disease (CAD), refers to a heart disease caused by atherosclerosis of the body's coronary arteries, resulting in stenosis or occlusion of the lumen, and thus myocardial ischemia, hypoxia or necrosis. In recent years, with the rapid development of social modernization, the continuous increase in people's work pressure and the gradual severity of the aging population, the harm caused by cardiovascular diseases to human health has been increasing year by year, and there is a trend of younger age. It has become the most common disease causing death globally, especially in developed countries.

[0003] As a common cardiovascular disease in society, coronary heart disease and many other cardiovascular diseases such as atherosclerotic coronary heart disease have a common pathological basis. Currently, atherosclerosis is generally regarded clinically as a lipid-driven vascular wall immune inflammatory disease containing autoimmunity antigen components. According to the latest research, the immune system has been proven to be involved in almost every lesion stage of atherosclerotic diseases. It is known that the immune system can be divided into two subsystems: innate immunity and adaptive immune system. The main mediators of innate immunity include monocytes-macrophages, neutrophils, dendritic cells, NK cells and γδT cells, and the main mediators of adaptive immunity include T and B lymphocytes and their subsets. In the immune system, various immune cells directly express different immune phenotypes on their cell surfaces, or indirectly secrete various cytokines to influence and interact with each other, accelerating the secretion of anti-inflammatory cytokines or pro-inflammatory cytokines in plaques. The process mediated by them involves the occurrence, development and rupture of coronary artery plaques.

[0004] Previous studies on the immune function of coronary heart disease mainly focused on infiltrating lymphocytes in atherosclerotic plaques. At present, clinical studies on the relationship between the expression levels of various lymphocytes and their subsets in peripheral blood and coronary heart disease are a hot topic of concern. More and more studies hope to find that peripheral blood immune lymphocytes can be used as new biomarkers for evaluating the risk of coronary heart disease and targets for intervention and treatment. As mentioned above, inflammatory response and immune response play important roles throughout the process of atherosclerosis. Based on this, the present invention innovatively proposes to detect indexes such as the percentages, developmental stages, differentiation types and functional activities of T lymphocytes, NK cells, γδT cells and their subsets, in order to more comprehensively evaluate the risk of coronary heart disease, and thus provide a new solution for the current application of peripheral blood immune lymphocytes in the risk assessment of coronary heart disease. Summary of the Invention

[0005] In order to solve the deficiencies of the above-mentioned technologies, the present invention provides an application of peripheral T lymphocyte indexes in the preparation of a coronary heart disease risk assessment kit, a kit and a method.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is: the application of peripheral T lymphocyte indexes in the preparation of a coronary heart disease risk assessment kit, and the T lymphocyte subsets involved in the peripheral T lymphocyte indexes include:

[0007] CD3+CD8+CD28-, CD3+CD8+HLADR+, Tc1, CD3+γδ+Vδ2+NKG2D+.

[0008] A coronary heart disease risk assessment kit, which combines four indexes of CD3+CD8+CD28-, CD3+CD8+HLADR+, Tc1, and CD3+γδ+Vδ2+NKG2D+ as combined prediction indexes, and the AUC value for evaluating the risk of coronary heart disease is as high as 0.956.

[0009] Preferably, the kit includes the following monoclonal antibodies:

[0010] Anti-CD3 antibody, anti-CD8 antibody, anti-CD28 antibody, anti-HLADR antibody, anti-CXCR3 antibody, anti-CXCR5 antibody, anti-CCR4 antibody, anti-TCRγδ antibody, anti-Vδ2 antibody, anti-NKG2D antibody;

[0011] Each monoclonal antibody is labeled with a fluorescent label.

[0012] Preferably, the fluorescent label of the anti-CD3 antibody is PerCP-Cy5.5 or APC-H7;

[0013] The fluorescent label of the anti-CD8 antibody is BV421 or PerCP-Cy5.5;

[0014] The fluorescent label of the anti-CD28 antibody is PE-Cy7;

[0015] The fluorescent label of the anti-HLADR antibody is PE;

[0016] The fluorescent label of the anti-CXCR3 antibody is AF488;

[0017] The fluorescent label of the anti-CXCR5 antibody is AF647;

[0018] The fluorescent label of the anti-CCR4 antibody is BV421;

[0019] The fluorescent label of the anti-TCRγδ antibody is BV421;

[0020] The fluorescence label of the anti-Vδ2 antibody is PE;

[0021] The fluorescence label of the anti-NKG2D antibody is PE-Cy7.

[0022] Preferably, the kit further includes the isotype control antibodies corresponding to the respective monoclonal antibodies.

[0023] Preferably, the fluorescence label is PerCP-Cy TM 5.5 anti-CD3 antibody, and its isotype control antibody is PerCP-Cy TM 5.5 Mouse IgG1,k;

[0024] The anti-CD3 antibody with a fluorescence label of APC-H7, and its isotype control antibody is APC-H7 mouse IgG1;

[0025] The anti-CD8 antibody with a fluorescence label of BV510, and its isotype control antibody is BD Horizon TM BV510 MouseIgG1,k;

[0026] The anti-CD8 antibody with a fluorescence label of PerCP-Cy5.5, and its isotype control antibody is PerCP-Cy TM 5.5 MouseIgG1 k;

[0027] The anti-CD28 antibody with a fluorescence label of PE-Cy7, and its isotype control antibody is PE-Cy TM 7 Mouse IgG1,k;

[0028] The anti-HLA-DR antibody with a fluorescence label of PE, and its isotype control antibody is PE Mouse IgG2a,k;

[0029] The anti-CXCR3 antibody with a fluorescence label of AF488, and its isotype control antibody is Alexa 488 MouseIgG1 k;

[0030] The anti-CXCR5 antibody with a fluorescence label of AF647, and its isotype control antibody is Alexa 647 RatIgG2b,k;

[0031] The anti-CCR4 antibody with a fluorescence label of BV421, and its isotype control antibody is BV421 Mouse IgG1,k;

[0032] The anti-γδ antibody with a fluorescence label of BV421, BV421 Mouse IgG1,k;

[0033] An anti-TCR Vδ2 antibody labeled with PE, and its isotype control antibody is PE Mouse IgG2a,k;

[0034] An anti-NKG2D antibody labeled with PE-Cy7, and its isotype control antibody is PE-Cy TM 7Mouse IgG1,k.

[0035] A method for detecting peripheral T lymphocytes in a coronary heart disease risk assessment kit, comprising the following steps:

[0036] (1) Add erythrocyte lysate to a flow cytometry tube containing peripheral blood, mix well by pipetting and blowing, let it stand for lysis for 5 minutes, and centrifuge to remove the supernatant;

[0037] (2) Repeat the lysis step of the first step above, centrifuge to remove the supernatant;

[0038] (3) Wash the cells with 1×PBS, centrifuge to remove the supernatant;

[0039] (4) Repeat the washing step of the third step above, centrifuge to remove the supernatant;

[0040] (5) Resuspend the cells with PBS to make a lymphocyte suspension;

[0041] (6) Add monoclonal antibodies labeled with different fluorescences to the lymphocyte suspension:

[0042] CD3, CD4, CD8, CD28, CD127, CCR7, CD25, CD45RA, CD57, CD38, PD-1, HLA-DR, CD56, CD94, NKG2D, NKP30, NKP46, VD1, VD2, NKB1, γδ, CD183, CD196, CD194, CD279;

[0043] Gently mix and incubate in the dark;

[0044] (7) Terminate the staining, add 1×PBS to wash the cells, wash away the unbound antibodies, and centrifuge to remove the supernatant;

[0045] (8) Resuspend the cells, use a BD FACS Canto flow cytometer to detect the sample on the machine, and obtain the data of the lymphocyte subsets after fluorescence labeling.

[0046] Preferably, the final concentration of the lymphocyte suspension in step (5) is 1x10 6 / 100 μL.

[0047] Preferably, the ratio of the fluorescent antibody to the lymphocyte suspension added in step (6) is 1:200;

[0048] In step (6), the conditions for dark incubation are: dark incubation at 4°C for 15 - 20 minutes.

[0049] Preferably, the centrifugation conditions in steps (1), (2), (3), (4), and (7) are: centrifugation at 1500 rpm for 5 minutes.

[0050] The present invention discloses an application of peripheral T lymphocyte indexes in the preparation of a coronary heart disease risk assessment kit, a kit, and a method. It is first discovered that in adult peripheral blood, CD3+CD8+CD28-, CD3+CD8+HLADR+, Tc1, and CD3+γδ+Vδ2+NKG2D+ cells have a certain correlation with the occurrence of coronary heart disease in terms of the level of their expression ratios; their combined indexes can be used as prediction indexes for coronary heart disease and have important guiding significance for the risk assessment of coronary heart disease. Moreover, the present invention provides a detection method for peripheral blood immune cells. Through this detection method, the percentage of novel peripheral blood lymphocyte subsets can be accurately detected, and it has broad prospects in the risk assessment of coronary heart disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 It is the ROC curve of the peripheral T lymphocyte indexes of the present invention.

[0052] Figure 2 It is the flow cytometry data analysis chart of the expression ratio of CD8+ T cells in peripheral blood of coronary heart disease patients and healthy people of the present invention.

[0053] Figure 3 It is the flow cytometry data analysis chart of the expression ratio of Tc cells in peripheral blood of coronary heart disease patients and healthy people of the present invention.

[0054] Figure 4 It is the flow cytometry data analysis chart of the expression ratio of γδT cells in peripheral blood of coronary heart disease patients and healthy people of the present invention.

[0055] Figure 5 It is the flow cytometry data analysis chart of the expression ratio of NT cells in peripheral blood of coronary heart disease patients and healthy people of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0056] The present invention will be further described in detail below with reference to the drawings and specific embodiments.

[0057] As an important part of the immune system, T lymphocytes play a key role in the occurrence and development of coronary heart disease. They can recognize and bind to antigens on the coronary artery wall, such as oxidized low-density lipoproteins, etc., thereby triggering an immune response. This immune response may lead to damage to the coronary artery wall and exacerbation of the inflammatory response, thus promoting the development of coronary heart disease. Therefore, detecting the number, ratio, and functional status of T lymphocytes can be used to evaluate the risk of coronary heart disease.

[0058] In summary, the association between T lymphocytes and coronary heart disease risk assessment is specifically reflected in the following three aspects:

[0059] 1. Inflammatory response: Coronary heart disease is a disease caused by coronary atherosclerosis, and its pathogenesis involves complex inflammatory responses. T lymphocytes play an important role in the inflammatory response, participating in the inflammatory process and affecting the stability and progression of plaques.

[0060] 2. Immune response and damage: T lymphocytes trigger an immune response by recognizing and responding to antigens on the coronary artery wall, such as oxidized low-density lipoproteins. This response may lead to damage to the coronary artery wall and exacerbation of the inflammatory response, thereby promoting the development of coronary heart disease.

[0061] 3. Memory and secondary response: Some T lymphocytes (such as memory T cells) can survive in the body for a long time. When they encounter the same antigen again, they can rapidly proliferate and differentiate into effector T cells, thus accelerating the speed and intensity of the immune response. This memory function may make an individual more likely to have an immune reaction when exposed to coronary heart disease-related antigens, increasing the risk of disease.

[0062] Based on this, the present invention uses advanced immunodetection techniques, such as flow cytometry or multicolor immunofluorescence, to accurately detect T lymphocyte subsets in peripheral blood. These subsets include CD4+ T cells, CD8+ T cells, γδ T cells, etc., each of which has different immune functions and characteristics.

[0063] By monitoring the quantity and proportion of peripheral blood T lymphocyte subsets, the overall immune status of an individual is evaluated, including the activation degree of the immune system, the strength of the inflammatory response, and the balance state of immune regulation. These indicators can reflect an individual's immune response ability and inflammatory response state to coronary heart disease-related antigens.

[0064] Subsequently, a coronary heart disease risk assessment model can be constructed in combination with the detection results of peripheral blood T lymphocyte subsets. This model uses statistical and machine learning algorithms to quantitatively evaluate an individual's coronary heart disease risk. By comparing the risk scores of different individuals, the risk of an individual developing coronary heart disease in a future period can be predicted.

[0065] Specifically for the present invention, for the first time, the expression differences of novel T lymphocyte indicators in the peripheral blood of coronary heart disease patients and healthy controls were discovered, and their expression conditions are related to the assessment of the risk of coronary heart disease. Flow cytometry was used to detect the percentage levels of various novel lymphocytes and their subsets in human peripheral blood, and the expression differences between the healthy control group and the coronary heart disease group were analyzed; each independent risk factor and the combination of independent risk factors were used as combined prediction indicators, and ROC curves were drawn to evaluate the risk of coronary heart disease

[0066] The T lymphocyte subsets involved in the determined peripheral blood lymphocyte indexes include: CD3+CD8+CD28-, CD3+CD8+HLADR+, Tc1, CD3+γδ+Vδ2+NKG2D+.

[0067] Using the above T lymphocyte subsets as the peripheral blood lymphocyte detection indexes of the coronary heart disease risk assessment kit, its application in the preparation of the coronary heart disease risk assessment kit is realized.

[0068] As Figure 1 shown, in order to analyze the diagnostic value of the four independent risk factors of CD3+CD8+CD28-, CD3+CD8+HLADR+, Tc1, CD3+γδ+Vδ2+NKG2D+ for predicting coronary heart disease, the ROC curves were plotted with the sensitivity of predicting coronary heart disease of these four independent risk factors as the ordinate and 1-specificity as the abscissa.

[0069] The results of ROC curve analysis are as follows: The AUC value of CD3+CD8+CD28- is 0.622, that is: when the CD3+CD8+CD28- index of the patient's peripheral T lymphocytes is less compared with that of healthy people, the possibility of suffering from coronary heart disease increases, and the predicted AUC value is 0.622.

[0070] The AUC value of CD3+CD8+HLADR+ is 0.623, that is: when the CD3+CD8+HLADR+ index of the patient's peripheral T lymphocytes is more compared with that of healthy people, the possibility of suffering from coronary heart disease increases, and the predicted AUC value is 0.623.

[0071] The AUC value of Tc1 is 0.586, that is: when the Tc1 index of the patient's peripheral T lymphocytes is more compared with that of healthy people, the possibility of suffering from coronary heart disease increases, and the predicted AUC value is 0.586.

[0072] The AUC value of CD3+γδ+Vδ2+NKG2D+ is 0.803, that is: when the CD3+γδ+Vδ2+NKG2D+ index of the patient's peripheral T lymphocytes is more compared with that of healthy people, the possibility of suffering from coronary heart disease increases, and the predicted AUC value is 0.803.

[0073] Among them, the AUC value of CD3+γδ+Vδ2+NKG2D+ is the highest among the four independent risk factors, and the AUC value is 0.803, indicating that CD3+γδ+Vδ2+NKG2D+ has a moderate diagnostic value for predicting coronary heart disease.

[0074] Combine the above four independent risk factors as a combined prediction index, and then by plotting the ROC curve, analyze the diagnostic significance of this combined prediction index for predicting coronary heart disease. The results show that the AUC value of the combined prediction index is 0.956, 95% CI = 0.926 - 0.985, p < 0.001. Combining the above results indicates that the combined prediction index has a better prediction effect and high diagnostic value, indicating that the coronary heart disease prediction system is effective.

[0075] In addition, it should be noted that, as Figures 2 - 5 shown in the flow cytometry data analysis chart of, it shows the differences between healthy people and coronary heart disease patients in a certain index. In the figure, HC represents healthy physical examination subjects; CAD represents coronary heart disease patients. Figure 2 It is a flow cytometry data analysis chart of the expression ratio of peripheral blood CD8+ T cells in healthy people and coronary heart disease patients, representing the index in helper T cells and cytotoxic T cells; Figure 3 It is a flow cytometry data analysis chart of the expression ratio of peripheral blood Tc cells in healthy people and coronary heart disease patients, representing the T lymphocyte factor index; Figure 4 It is a flow cytometry data analysis chart of the expression ratio of peripheral blood γδT cells in healthy people and coronary heart disease patients, representing the index in γδT lymphocytes; Figure 5 It is a flow cytometry data analysis chart of the expression ratio of peripheral blood NK cells in healthy people and coronary heart disease patients, representing the index in NK cells.

[0076] In addition, Figures 2 - 5 in, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, **** indicates P < 0.0001, and ns indicates P > 0.05.

[0077] Furthermore, in the detection of coronary heart disease, monoclonal antibodies are closely related to T lymphocytes, which are mainly reflected in the following aspects:

[0078] 1. Detection of T lymphocyte subsets: Using monoclonal antibodies to label and isolate T lymphocytes, different subsets of T lymphocytes can be detected, such as CD4+ T cells, CD8+ T cells, γδT cells, etc. These subsets play different roles in the occurrence and development of coronary heart disease, so their numbers and proportions can be used as important indicators for evaluating the risk of coronary heart disease.

[0079] 2. Evaluation of T lymphocyte function: Monoclonal antibodies can also be used to evaluate the functional status of T lymphocytes. For example, by detecting indicators such as T lymphocyte proliferation, activation, and cytokine secretion, the immune response ability and inflammatory reaction status of T lymphocytes in coronary heart disease can be understood. This information is of great significance for formulating personalized treatment plans and predicting disease progression.

[0080] 3. Development of a coronary heart disease risk assessment kit: Based on the association between monoclonal antibodies and T lymphocytes, the development of a coronary heart disease risk assessment kit can be achieved. The coronary heart disease risk assessment kit uses monoclonal antibodies to label and detect T lymphocytes, and constructs a coronary heart disease risk assessment model. Through this model, the coronary heart disease risk of an individual can be quantitatively evaluated, providing strong support for clinical diagnosis and treatment.

[0081] Therefore, based on the existing technical basis of peripheral blood immune lymphocytes in the assessment of coronary heart disease risk, the monoclonal antibodies used in the coronary heart disease risk assessment kit determined by the present invention include the following: anti-CD3 antibody, anti-CD8 antibody, anti-CD28 antibody, anti-HLA-DR antibody, anti-CXCR3 antibody, anti-CXCR5 antibody, anti-CCR4 antibody, anti-TCRγδ antibody, anti-Vδ2 antibody, anti-NKG2D antibody.

[0082] By consulting a large number of literatures and materials, the antigens to be detected are identified. Subsequently, appropriate fluorescent dyes are paired with the antigens to be detected. And through a large number of preliminary experiments, concentration titration experiments of monoclonal antibodies are carried out to screen out the optimal concentration, and fluorescent labels are determined for the monoclonal antibodies used in the monoclonal antibody coronary heart disease risk assessment kit.

[0083] Among them, the fluorescent label of the anti-CD3 antibody is PerCP-Cy5.5 or APC-H7;

[0084] The fluorescent label of the anti-CD8 antibody is BV421 or PerCP-Cy5.5;

[0085] The fluorescent label of the anti-CD28 antibody is PE-Cy7;

[0086] The fluorescent label of the anti-HLA-DR antibody is PE;

[0087] The fluorescent label of the anti-CXCR3 antibody is AF488;

[0088] The fluorescent label of the anti-CXCR5 antibody is AF647;

[0089] The fluorescent label of the anti-CCR4 antibody is BV421;

[0090] The fluorescent label of the anti-TCRγδ antibody is BV421;

[0091] The fluorescent label of the anti-Vδ2 antibody is PE;

[0092] The fluorescent label of the anti-NKG2D antibody is PE-Cy7.

[0093] In the coronary heart disease risk assessment kit, for each monoclonal antibody with different fluorescent labels, corresponding isotype control antibodies need to be equipped:

[0094] The fluorescent label is PerCP-Cy TM Anti-CD3 antibody with a concentration of 5.5, and its isotype control antibody is PerCP-Cy TM 5.5 Mouse IgG1,k, and the antibody catalog number is 550795;

[0095] Anti-CD3 antibody fluorescently labeled with APC-H7, and its isotype control antibody is APC-H7 mouse IgG1, and the antibody catalog number is 560167;

[0096] Anti-CD8 antibody fluorescently labeled with BV510, and its isotype control antibody is BD Horizon TM BV510 Mouse IgG1,k, and the antibody catalog number is 562946;

[0097] Anti-CD8 antibody fluorescently labeled with PerCP-Cy5.5, and its isotype control antibody is PerCP-Cy TM 5.5 Mouse IgG1 k, and the antibody catalog number is 550795;

[0098] Anti-CD28 antibody fluorescently labeled with PE-Cy7, and its isotype control antibody is PE-Cy TM 7 Mouse IgG1,k, and the antibody catalog number is 557872;

[0099] Anti-HLA-DR antibody fluorescently labeled with PE, and its isotype control antibody is PE Mouse IgG2a,k, and the antibody catalog number is 555574;

[0100] Anti-CXCR3 antibody fluorescently labeled with AF488, and its isotype control antibody is Alexa 488 Mouse IgG1 k, and the antibody catalog number is 557702;

[0101] Anti-CXCR5 antibody fluorescently labeled with AF647, and its isotype control antibody is Alexa 647 Rat IgG2b,k, and the antibody catalog number is 557691;

[0102] Anti-CCR4 antibody fluorescently labeled with BV421, and its isotype control antibody is BV421 Mouse IgG1,k, and the antibody catalog number is 562438;

[0103] Anti-γδ antibody fluorescently labeled with BV421, BV421 Mouse IgG1,k, and the antibody catalog number is 562438;

[0104] Anti-TCR Vδ2 antibody labeled with PE, and its isotype control antibody is PE Mouse IgG2a,k, with the antibody catalog number 555574;

[0105] Anti-NKG2D antibody labeled with PE-Cy7, and its isotype control antibody is PE-Cy TM 7Mouse IgG1,k, with the antibody catalog number 557872.

[0106] In summary, the present invention first discovers that four novel lymphocytes in peripheral blood are risk factors for coronary heart disease, namely CD3+CD8+CD28-, CD3+CD8+HLADR+, Tc1, and CD3+γδ+Vδ2+NKG2D+. Combining these four risk factors as a combined prediction index for a coronary heart disease risk assessment kit can achieve the best risk assessment effect, and draw an ROC curve for evaluating the risk of coronary heart disease, which has important guiding significance for the risk assessment of coronary heart disease.

[0107] Example

[0108] This example discloses a method for detecting the percentage levels of various novel lymphocytes and their subsets in human peripheral blood by flow cytometry according to the present invention, specifically as follows:

[0109] (1) Blood is collected on the premise of signing an informed consent form with the patient. All specimens are taken from heparinized venous blood on an empty stomach in the early morning. In this example, 79 cases of coronary heart disease and 100 cases of healthy adult samples are collected. This part of the research protocol has been approved by the ethics committee of this unit. At the same time, clinical data such as clinical indicators of coronary heart disease patients are collected.

[0110] (2) Flow immunodetection of lymphocytes. Add red blood cell lysate to the flow cytometry tube containing peripheral blood, mix well by pipetting and let it stand for lysis, then centrifuge at 1500 rpm for 5 minutes. After centrifugation, pour out the supernatant, repeat the lysis step of the first step above, and continue to centrifuge at 1500 rpm for 5 minutes. After centrifugation, pour out the supernatant, and use a pipette tip to suck out the liquid at the tube mouth, add 1 mL of 1×PBS for washing, and centrifuge at 1500 rpm for 5 minutes. After centrifugation, pour out the supernatant, repeat the above washing step, and continue to centrifuge at 1500 rpm for 5 minutes. After centrifugation, pour out the supernatant, and resuspend the cells with 600 μL of 1×PBS.

[0111] According to the flow antibody staining scheme designed for the experimental research direction, the flow antibody was diluted at a ratio of 1:200 and mixed to prepare 5 tubes of premixed solution, which were placed on ice for later use. 100 μL of the above-mentioned resuspended cells were added to each flow tube, and 10 μL of antibody premixed solution was added to the corresponding five flow tubes according to the designed 5 sets of staining schemes. The flow tube without any antibody was used as a blank control group. The above six flow tubes were placed on an oscillator to oscillate to ensure that the antibodies and cells were fully mixed, and then the flow tubes were incubated at 4°C in the dark for 15-20 minutes. After staining, 1×PBS was added to each flow tube to wash away the surface antibodies that were not bound to the cells, and centrifuged at 1500rpm for 5 minutes. After centrifugation, the supernatant was discarded, and 200 μL of 1×PBS was added to each flow tube to resuspend the cells. The samples of each flow tube were tested on the BD FACS Canto flow cytometer.

[0112] (3) Statistical analysis of flow cytometry data. Flow Jo 10.8.1 was used for analysis, and the data were entered into Microsoft Excel 2019 for integration into a database. Graph pad Prism 9.4.0 was used to perform univariate t-test analysis on immune lymphocyte indicators between the healthy control group and the CHD group; One-way ANOVA test was used to compare the differences between the CHD groups; the correlation between the clinical-related indicators of CHD patients and the two continuous variables of immune lymphocytes in their peripheral blood was analyzed by Pearson correlation coefficient. SPSS26.0 software was used to analyze the count data in the general clinical data of CHD patients using chi-square test; multivariate binary logistic regression analysis was used to screen out independent risk factors for CHD; the accuracy of independent risk factors for CHD risk assessment was calculated by drawing ROC curves to complete this analysis. If the analysis results showed P < 0.05, it was unanimously considered that the difference was statistically significant.

[0113] Furthermore, Table 1 shows the results of the analysis of various immune cell subsets (marked in the "risk factor" column), including statistical indicators: p value (P value), ROC value (ROC value) and 95% confidence interval (95% CI).

[0114] Risk factors: Specific immune cell subsets are listed, differentiated by combinations of cell surface markers, such as CD3, CD4, CD8, γδ (γδ T cells), Vδ2, PD-1, HLA-DR, etc., which indicate cell type, activation state, and differentiation state.

[0115] P value: It represents the statistical significance of the association between each cell subset and the research results (such as disease severity, risk, etc.). Generally, a p value less than 0.05 is considered statistically significant, meaning there is a meaningful association between the cell subset and the research results.

[0116] ROC value: It represents the area under the receiver operating characteristic curve and is used to measure the discrimination ability of each cell subset.

[0117] Cell subsets with ROC values close to 1 have better predictive performance, while ROC values close to 0.5 indicate poor discrimination ability.

[0118] 95% confidence interval: It reflects the credibility of the ROC value, that is, the actual ROC value fluctuates within a 95% probability range. The narrower the interval, the higher the accuracy of the ROC value.

[0119] Table 1

[0120]

[0121] It can be seen from this that the present invention also discloses a method for detecting peripheral T lymphocytes for coronary heart disease risk assessment, comprising the following steps:

[0122] (1) Add red blood cell lysate to a flow cytometry tube containing peripheral blood, mix well by pipetting and blowing, let it stand for lysis for 5 minutes, centrifuge at 1500 rpm for 5 minutes, and discard the supernatant.

[0123] Among them, the peripheral blood should be collected from patients diagnosed with coronary heart disease uniformly before undergoing coronary artery stent surgery.

[0124] (2) Repeat the lysis step of the first step above, centrifuge and discard the supernatant.

[0125] (3) Wash the cells with 1×PBS, centrifuge at 1500 rpm for 5 minutes, and discard the supernatant.

[0126] (4) Repeat the washing step of the third step above, centrifuge and discard the supernatant.

[0127] (5) Resuspend the cells with PBS to make a lymphocyte suspension; the final concentration of the lymphocyte suspension is 1x10 6 / 100 μL;

[0128] (6) Add monoclonal antibodies labeled with different fluorescences to the lymphocyte suspension:

[0129] CD3, CD4, CD8, CD28, CD127, CCR7, CD25, CD45RA, CD57, CD38, PD-1, HLA-DR, CD56, CD94, NKG2D, NKP30, NKP46, VD1, VD2, NKB1, γδ, CD183, CD196, CD194, CD279;

[0130] The proportion of the added fluorescently labeled monoclonal antibody was 1:200. After gently mixing with the lymphocyte suspension, it was incubated in the dark at 4°C for 15 - 20 minutes;

[0131] (7) Terminate the staining, add 1×PBS to wash the cells, wash away the unbound antibody, centrifuge at 1500 rpm for 5 minutes, and discard the supernatant;

[0132] (8) Resuspend the cells, and use a BD FACS Canto flow cytometer to detect the samples on the machine to obtain the data of the lymphocyte subsets after fluorescence labeling.

[0133] (9) Result analysis: Perform statistical analysis on the lymphocyte subset data, analyze the correlation between lymphocytes and their clinical indicators, screen out the independent risk factors for coronary heart disease, and form a coronary heart disease risk assessment model by combining each independent risk factor.

[0134] The four independent risk factors screened out, CD3+CD8+CD28-, CD3+CD8+HLADR+, Tc1, CD3+γδ+Vδ2+NKG2D+, were combined as a combined prediction index, and the AUC value for assessing the risk of coronary heart disease was as high as 0.956.

[0135] It can be seen from this that although the immune system has been proven to be involved in almost every pathological stage of atherosclerotic diseases, the research on the immune function of coronary heart disease mainly focuses on the infiltrating lymphocytes in atherosclerotic plaques. However, the changes and roles of lymphocytes in peripheral blood in the occurrence and development of coronary heart disease have been less studied, and their relationship as risk factors with coronary heart disease is not yet fully understood. At present, more and more research hopes to discover new biomarkers for coronary heart disease risk assessment and target points for intervention and treatment in peripheral blood immune lymphocytes. Therefore, this study explores the role of novel lymphocytes and their subsets in peripheral blood in coronary heart disease risk assessment and constructs a coronary heart disease risk assessment model from the perspective of immunology.

[0136] The present invention has the following advantages and effects compared with the prior art:

[0137] 1) The present inventors first discovered that there is a certain correlation between the high or low expression ratios of CD3+CD8+CD28-, CD3+CD8+HLADR+, Tc1, and CD3+γδ+Vδ2+NKG2D+ cells in adult peripheral blood and the occurrence of coronary heart disease; the combined index can be used as a predictive index for coronary heart disease and has important guiding significance for the risk assessment of coronary heart disease.

[0138] 2) The present invention provides a detection method for peripheral blood immune cells. Through this detection method, the percentage of novel peripheral blood lymphocyte subsets can be accurately detected, and it has broad prospects in the risk assessment of coronary heart disease.

[0139] The above embodiments are not limitations on the present invention, and the present invention is not limited to the above examples. Changes, modifications, additions, or substitutions made by those skilled in the art within the scope of the technical solution of the present invention also fall within the protection scope of the present invention.

Claims

1. Use of peripheral T lymphocyte indexes in the preparation of a coronary heart disease risk assessment kit, characterized in that: The T lymphocyte subsets involved in the peripheral T lymphocyte index include: CD3+CD8+CD28-, CD3+CD8+HLADR+, Tc1, CD3+γδ+Vδ2+NKG2D+.

2. A coronary heart disease risk assessment kit, characterized in that: The kit combines four indicators, CD3+CD8+CD28-, CD3+CD8+HLADR+, Tc1, and CD3+γδ+Vδ2+NKG2D+, as combined prediction indicators, and the AUC value for evaluating the risk of coronary heart disease is as high as 0.

956.

3. The coronary heart disease risk assessment kit according to claim 2, characterized in that: The kit includes the following monoclonal antibodies: Anti-CD3 antibody, anti-CD8 antibody, anti-CD28 antibody, anti-HLADR antibody, anti-CXCR3 antibody, anti-CXCR5 antibody, anti-CCR4 antibody, anti-TCRγδ antibody, anti-Vδ2 antibody, anti-NKG2D antibody; Each monoclonal antibody is labeled with a fluorescent label.

4. The kit for evaluating the risk of coronary heart disease according to claim 3, wherein: The fluorescent label of the anti-CD3 antibody is PerCP-Cy5.5 or APC-H7; The fluorescent label of the anti-CD8 antibody is BV421 or PerCP-Cy5.5; The fluorescent label of the anti-CD28 antibody is PE-Cy7; The fluorescent label of the anti-HLADR antibody is PE; The fluorescent label of the anti-CXCR3 antibody is AF488; The fluorescent label of the anti-CXCR5 antibody is AF647; The fluorescent label of the anti-CCR4 antibody is BV421; The fluorescent label of the anti-TCRγδ antibody is BV421; The fluorescent label of the anti-Vδ2 antibody is PE; The fluorescent label of the anti-NKG2D antibody is PE-Cy7.

5. The coronary heart disease risk assessment kit according to claim 4, characterized in that: The kit also includes isotype control antibodies corresponding to the monoclonal antibodies.

6. The kit for evaluating the risk of coronary heart disease according to claim 5, wherein: The fluorescent label is PerCP-Cy TM Anti-CD3 antibody with a fluorescence intensity of 5.5, and its isotype control antibody is PerCP-Cy TM 5.5 Mouse IgG1,k; For the anti-CD3 antibody with a fluorescent label of APC-H7, its isotype control antibody is APC-H7 mouse IgG1; Anti-CD8 antibody labeled with BV510, and its isotype control antibody is BD Horizon TM BV510Mouse IgG1,k; Anti-CD8 antibody labeled with PerCP-Cy5.5, and its isotype control antibody is PerCP-Cy TM 5.5 Mouse IgG1k; Anti-CD28 antibody labeled with PE-Cy7, and its isotype control antibody is PE-Cy TM 7 Mouse IgG1,k; For the anti-HLADR antibody with a fluorescent label of PE, its isotype control antibody is PE Mouse IgG2a,k; Anti-CXCR3 antibody labeled with AF488, and its isotype control antibody is Alexa 488 Mouse IgG1 k; Anti-CXCR5 antibody labeled with AF647, and its isotype control antibody is Alexa 647 Rat IgG2b,k; For the anti-CCR4 antibody with a fluorescent label of BV421, its isotype control antibody is BV421 Mouse IgG1,k; For the anti-γδ antibody with a fluorescent label of BV421, BV421 Mouse IgG1,k; For the anti-TCR Vδ2 antibody with a fluorescent label of PE, its isotype control antibody is PE Mouse IgG2a,k; Anti-NKG2D antibody fluorescently labeled with PE-Cy7, and its isotype control antibody is PE-Cy TM 7Mouse IgG1,k.

7. A method for detecting peripheral T lymphocytes in a coronary heart disease risk assessment kit, characterized in that: It includes the following steps: (1) Add red blood cell lysate to the flow tube containing peripheral blood, mix well with a pipette and let it stand for lysis for 5 minutes, then centrifuge to remove the supernatant; (2) Repeat the lysis step of the first step above, and centrifuge to remove the supernatant; (3) Wash the cells with 1×PBS and centrifuge to remove the supernatant; (4) Repeat the washing step of the third step above, and centrifuge to remove the supernatant; (5) Resuspend the cells with PBS to make a lymphocyte suspension; (6) Add monoclonal antibodies labeled with different fluorescences to the lymphocyte suspension: CD3, CD4, CD8, CD28, CD127, CCR7, CD25, CD45RA, CD57, CD38, PD-1, HLA-DR, CD56, CD94, NKG2D, NKP30, NKP46, VD1, VD2, NKB1, γδ, CD183, CD196, CD194, CD279; Gently mix and incubate in the dark; (7) Terminate the staining, add 1×PBS to wash the cells, wash away the unbound antibodies, and centrifuge to remove the supernatant; (8) Resuspend the cells, and use a BD FACS Canto flow cytometer to detect the samples on the machine to obtain the data of the lymphocyte subsets after fluorescence labeling.

8. The method for detecting peripheral T lymphocytes of the coronary heart disease risk assessment kit according to claim 7, characterized in that: The final concentration of the lymphocyte suspension described in step (5) is 1x10 6 / 100 μL.

9. The method for detecting peripheral T lymphocytes of the coronary heart disease risk assessment kit according to claim 8, characterized in that: The ratio of the fluorescent antibody to the lymphocyte suspension added in step (6) is 1:200; The dark incubation condition in step (6) is: incubate in the dark at 4°C for 15 - 20 minutes.

10. The method for detecting peripheral T lymphocytes of the coronary heart disease risk assessment kit according to claim 8, characterized in that: The centrifugation conditions in steps (1), (2), (3), (4), and (7) are: centrifuge at 1500 rpm for 5 minutes.

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