CD39 detection kit for rheumatoid arthritis

Abstract

The invention belongs to the technical field of rheumatoid arthritis detection, and relates to a CD39 detection kit for rheumatoid arthritis, which comprises a component for detecting the expression quantity and / or concentration of CD39 in CD4T cells, CD8T cells and Treg cells in a sample. The CD39 protein is used as a marker for specific diagnosis, prognosis and / or treatment monitoring of rheumatoid arthritis. The meso-position fluorescence intensity and / or positive cell proportion expressed by CD39 in CD4T cells, CD8T cells and Treg cells are detected through a flow cytometry experiment, and disease progress and / or treatment monitoring of rheumatoid arthritis is carried out according to the expression level of CD39. The CD39 detection kit for rheumatoid arthritis provided by the invention has the advantages of high specificity and high sensitivity.

Description

Technical Field

[0001] The present invention belongs to the technical field of rheumatoid arthritis detection, and relates to a CD39 detection kit for rheumatoid arthritis. Background Art

[0002] Rheumatoid arthritis (RA) is an autoimmune disease mainly characterized by synovitis and vasculitis, often accompanied by multi-system damage. Its global incidence is about 1%. The main clinical manifestations of patients are chronic progressive synovitis. As the disease progresses, synovitis can lead to the destruction of articular cartilage, bone, and joint capsule, ultimately resulting in joint deformity and loss of function. RA is not only one of the main diseases leading to labor decline and disability, but also seriously affects the development of the national economy. At present, there is still no radical cure for the treatment of RA. Clinically, it mainly relies on anti-rheumatic drugs, glucocorticoids, and newly developed targeted biological agents. Although some patients can achieve remission, due to the heterogeneity of the disease, there are still a large number of patients who have not achieved immune homeostasis or obtained effective remission.

[0003] In recent years, the cellular and molecular mechanisms closely related to disease progression have been widely studied. Research has shown that metabolic disorders of T cells are one of the key factors in the pathological process of RA. Metabolic disorders of T cells can lead to their over-activation, massive secretion of pro-inflammatory factors, and excessive immune responses to autoantigens, thereby exacerbating the attack of the immune system on joint tissues. Therefore, finding an immune-related biomarker with high specificity and sensitivity is of great significance for the early diagnosis, disease assessment, and prognosis prediction of RA.

[0004] As a transmembrane ectonucleoside triphosphate diphosphohydrolase-1 (ENTPD-1), CD39 has two transmembrane domains and one extracellular domain, and plays an important role in the pathogenesis of RA. When cells are subjected to stress or injury, they release adenosine triphosphate (ATP) into the extracellular environment, thereby triggering an inflammatory response. In this process, CD39 regulates the immune response by hydrolyzing extracellular ATP into adenosine monophosphate (AMP), while CD73 further converts AMP into the immunosuppressive nucleoside adenosine (ADO). CD39 is considered to be the rate-limiting enzyme in this cascade reaction. It plays a key immunomodulatory role between the ATP-mediated pro-inflammatory state and the ADO-mediated immunosuppressive state, thereby enhancing immune balance and reducing organ damage caused by excessive pro-inflammatory responses. Existing research has shown that CD39 plays an important role in the pathophysiological process of RA. Summary of the Invention

[0005] In view of the deficiencies of the prior art, the present invention provides a CD39 detection kit for rheumatoid arthritis, providing a new and effective option for the specific diagnosis, prognosis and / or treatment monitoring of rheumatoid arthritis, with the advantages of high specificity and high sensitivity.

[0006] To solve the above technical problems, the object of the present invention is achieved by the following technical solutions:

[0007] A CD39 detection kit for rheumatoid arthritis, the CD39 detection kit includes components for detecting the expression level and / or concentration of CD39 in CD4+ T cells, CD8+ T cells, and Treg cells in a sample.

[0008] In the above-mentioned CD39 detection kit for rheumatoid arthritis, the CD39 detection kit is applied to: specific diagnosis, prognosis and / or treatment monitoring of rheumatoid arthritis.

[0009] In the above-mentioned CD39 detection kit for rheumatoid arthritis, the CD39 detection kit is applied to: detecting the median fluorescence intensity and / or the proportion of positive cells of CD39 expression in CD4+ T cells, CD8+ T cells, and Treg cells through flow cytometry experiments, and monitoring the disease progression and / or treatment of rheumatoid arthritis according to the expression level of CD39.

[0010] Preferably, the CD39 detection kit is applied to: differentiating rheumatoid arthritis patients from healthy controls and differentiating between effective and ineffective treatment groups among patients through the detected median fluorescence intensity of CD39 and / or the proportion of positive cells.

[0011] Preferably, the CD39 detection kit is applied to: identifying rheumatoid arthritis patients with CD39 expression in T cells higher than the critical value, which is determined by receiver operating characteristic (ROC) curve analysis, to provide a highly sensitive and specific diagnosis of rheumatoid arthritis.

[0012] Preferably, the CD39 detection kit is applied to: predicting the disease progression of rheumatoid arthritis through the detected median fluorescence intensity of CD39; its effective prediction index is: the median fluorescence intensity value of CD39 in CD4+ T cells / CD8+ T cells / Treg cells is higher than 1149.4 / 644.35 / 1298.2.

[0013] In the above-mentioned CD39 detection kit for rheumatoid arthritis, the detection method of the CD39 detection kit includes the following steps:

[0014] (1)Collect samples and add fluorescently labeled antibodies against CD45, CD3, CD4, CD8, CD25, and CD127 to obtain CD4 T cells, CD8 T cells, and Treg cells;

[0015] (2)Stain T cells with a fluorescently labeled antibody against CD39;

[0016] (3)Perform flow cytometry analysis on the CD39-stained T cells using a flow cytometer to detect the fluorescent signal of the CD39 protein and collect data.

[0017] In step (1) above, the sample includes plasma, serum, or blood extracts, brushings, biopsies, or surgically resected tissues or fluid samples from a subject. Preferably, the sample is a peripheral blood sample.

[0018] The present invention also provides the use of CD39 protein as a biomarker in the preparation or screening of reagents or kits for the specific diagnosis, prognosis, and / or treatment monitoring of rheumatoid arthritis.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention provides a CD39 detection kit for rheumatoid arthritis, using CD39 protein as a highly sensitive and specific diagnostic indicator for rheumatoid arthritis, and performing specific diagnosis, prognosis, and / or treatment monitoring of rheumatoid arthritis based on the expression level of CD39 protein. Its advantages are that by detecting biomarkers in the blood for the clinical diagnosis of the occurrence and development of rheumatoid arthritis, it not only reduces the diagnostic cost, but also is easy to operate, has a high patient acceptance rate, and is relatively easy to perform large-scale screening of diseases, thus improving the detection efficiency, increasing the accuracy of the results, and having practical application value. The present invention can be used to guide clinical screening, research on the pathogenic mechanism of rheumatoid arthritis, screening of rheumatoid arthritis treatment drugs, and screening of new biomarkers and potential new drug targets for rheumatoid arthritis, and has broad application prospects. Description of the Drawings

[0021] Figure 1 Is a heatmap of the Mendelian randomization analysis of immune cells and rheumatoid arthritis;

[0022] Figure 2 Is a forest plot of the Mendelian randomization analysis of immune cells and rheumatoid arthritis;

[0023] Figure 3 Is the gating strategy for T cells and the expression of CD39 by fluorescence flow cytometry in a cohort of rheumatoid arthritis patients;

[0024] Figure 4Gating strategy for T cells and CD39 expression by fluorescence flow cytometry in the healthy control cohort;

[0025] Figure 5 Clinical characteristics and laboratory results of the study subjects;

[0026] Figure 6 Wilcoxon rank sum test was used to analyze the expression of CD39 in healthy controls and rheumatoid arthritis;

[0027] Figure 7 Receiver operating characteristic (ROC) curve analysis;

[0028] Figure 8 Correlation between CD39 expression and clinical indicators in RA patients. Detailed implementation

[0029] The present invention will be further described with specific examples in conjunction with the accompanying drawings.

[0030] The present invention will explore the expression of CD39 in T cells of RA patients through Mendelian randomization and flow cytometry, and further analyze its correlation with RA clinical indicators and disease activity, providing potential immunobiomarkers for the early identification, disease condition and prognosis prediction of RA.

[0031] First, the present invention uses Mendelian randomization to analyze the causal relationship between immune cells and rheumatoid arthritis.

[0032] Specifically, it includes the following steps:

[0033] S1: Read the GWAS data of exposure factors and outcome variables; specifically, reading the GWAS data of exposure factors is to obtain genome-wide association data related to immune cells; reading the GWAS data of outcome variables is to obtain genome-wide association data related to seropositive rheumatoid arthritis; the data is sourced from the UK Biobank genome-wide association study database (https: / / gwas.mrcieu.ac.uk / ) and the FinnGen database (https: / / www.finngen.fi / en / access_results), and the IDs of GWAS data related to immune cells (GCST90001391 to GCST90002121) and the ID of GWAS data related to seropositive rheumatoid arthritis (finn-b-RHEUMA_SEROPOS_OTH) are selected.

[0034] S2: Select appropriate SNPs as instrumental variables in the GWAS data; specifically, first select SNPs with P-values lower than 5×10^-8. And remove the instrumental variables with linkage disequilibrium, which is mainly measured by two parameters r2 and Kb. r2 = 1 indicates a complete linkage disequilibrium relationship between two SNPs, and r2 = 0 indicates a complete linkage equilibrium between two SNPs, that is, the distribution of these two SNPs is completely random; kb: refers to the regional length considering linkage disequilibrium. In the present invention, SNPs with an r2 value less than 0.001 within the range of 10000 kb are mainly concerned. Finally, identify weak instrumental variables. If the F value is less than 10 (this is a common threshold), then this instrumental variable can be regarded as a weak instrumental variable.

[0035] S3: Conduct Mendelian randomization analysis, and perform FDR correction (False Discovery Rate), sensitivity analysis, pleiotropy check, and heterogeneity analysis; specifically, use Mendelian randomization for causal relationship analysis, use the "TwoSampleMR" package in R software for analysis, and combine "MREgger regression" for pleiotropy test and heterogeneity analysis. To control the false positive risk brought by multiple hypothesis testing, perform FDR correction on the P-values. For the heterogeneity analysis, if the P-value of the heterogeneity test < 0.05, it indicates that there is heterogeneity in the results. For the pleiotropy check, adopt the MRPRESSO test, and identify possible pleiotropic instrumental variables by evaluating the contribution of each instrumental variable to the results, so as to perform correction. For the sensitivity analysis, by removing each instrumental variable one by one and observing whether the results change significantly, evaluate the stability and influence of each instrumental variable on the causal relationship.

[0036] Result analysis: Refer to Appendix Figure 1 , which shows the heatmap of the Mendelian randomization analysis of immune cells and rheumatoid arthritis. The outer circle is the immune cell phenotype, and the inner circle uses different colors to represent the P-values of the five Mendelian randomization analysis methods. The attached figure shows that there is a causal relationship between immune cells related to CD39 protein, such as CD39 on CD39+activated Treg cells, CD39+secreting Treg%CD4 Treg cells, CD39+secreting Treg%secreting Treg cells, CD39 onCD39+CD8br cells and rheumatoid arthritis.

[0037] Refer to Appendix Figure 2 , the forest plot of the Mendelian randomization analysis of immune cells and rheumatoid arthritis shows the results after correction by the main Mendelian randomization analysis method Inverse variance weighted, including FDR correction, heterogeneity test, and pleiotropy test. Figure 2More rigorous evidence shows a significant causal relationship between rheumatoid arthritis and immune cells related to CD39 protein, namely CD39 on CD39+CD8br cells (OR: 0.923 (0.860 - 0.990), P: 0.025), CD39+ secreting Treg% CD4 Treg cells (OR: 0.954 (0.918 - 0.992), P: 0.019), and CD39 on CD39+ activated Treg cells (OR: 0.919 (0.871 - 0.970), P: 0.02).

[0038] According to the above-mentioned Mendelian randomization result analysis, the expression of CD39 in T cells is an important predictive indicator for patients with rheumatoid arthritis.

[0039] Secondly, the present invention detects the expression of CD39 by flow cytometry, and specifically illustrates it through the following examples and experimental steps.

[0040] 1. Inclusion and exclusion criteria for samples

[0041] Patient group: The present invention analyzes 37 patients diagnosed with rheumatoid arthritis collected from Enze Hospital of Taizhou Enze Medical Center (Group) from July to August 2024. The inclusion criteria for the patient group are based on the rheumatoid arthritis diagnostic criteria issued by the American College of Rheumatology (ACR) or the European League Against Rheumatism (EULAR). The exclusion criteria are as follows: 1) Patients participating in other drug trials and other research projects and receiving drug treatment; 2) Pregnant or lactating women; 3) Cancer patients; 4) Patients with blood diseases; 5) Immunodeficient patients such as those with human immunodeficiency virus (HIV).

[0042] Healthy control group: 56 healthy individuals recruited from the physical examination center of Enze Hospital of Taizhou Enze Medical Center (Group).

[0043] All human blood samples in this study were approved by the Medical Ethics Committee of Enze Hospital of Taizhou Enze Medical Center (Group) (K20240709).

[0044] 2. Flow cytometry detection method

[0045] Collect 2 ml of peripheral blood samples from each participant in a test tube containing EDTA anticoagulant, and invert to mix. Take 50 μl of whole blood from each test tube, add CD25-PE / Cy7, CD127-PE, CD8-AF700, CD39-APC, CD4-FITC, CD3-PerCP, and CD45-KO antibodies for staining, shake well, and incubate in the dark at room temperature for 15 min. After incubation, add erythrocyte lysing solution, shake well, and incubate in the dark at room temperature for 10 min, then centrifuge (1500 rpm, 5 min) to remove impurities outside the cell membrane. Add enough buffer, shake well, and then centrifuge (1500 rpm, 5 min). Discard the supernatant, repeat the washing step until clean and a high-quality cell pellet is obtained, and perform flow cytometry analysis. The panel of flow cytometry is shown in Table 1 below. Flow cytometry analysis is performed on a DxFLEX flow cytometer for collection and analysis, and at least 105 cells are collected for analysis. After screening out the lymphocyte population through forward scatter / side scatter characteristics, refer to Figure 3 and Figure 4 sorting gating strategies to sort CD4 T cells, CD8 T cells, and Treg cells from the lymphocyte population, and further gate CD39+CD4 T cells, CD39+CD8 T cells, or CD39+Treg cells to analyze the data using the software CytExpert. Specifically, Figure 3 shows the gating strategy for T cells by fluorescence flow cytometry and the expression of CD39 in the rheumatoid arthritis patient cohort, where A-D are the gating strategies for CD4 T cells, CD8 T cells, and Treg cells, E is the proportion of CD39+Treg cells in Treg cells, F is the proportion of CD39+CD4 T cells in CD4 T cells, and G is the proportion of CD39+CD8 T cells in CD8 T cells. Figure 4 shows the gating strategy for T cells by fluorescence flow cytometry and the expression of CD39 in the healthy control cohort, where A-D are the gating strategies for CD4 T cells, CD8 T cells, and Treg cells, E is the proportion of CD39+Treg cells in Treg cells, F is the proportion of CD39+CD4 T cells in CD4 T cells, and G is the proportion of CD39+CD8 T cells in CD8 T cells.

[0046] Table 1: Immunofluorescence flow cytometry panel

[0047]

[0048] 3. Detection, collection, and analysis of clinical data

[0049] The biochemical parameters of the samples to be tested, such as C-reactive protein (CRP), C3, C4, anti-cyclic citrullinated peptide antibody (anti-CCP antibody), rheumatoid factor (RF), immunoglobulins (IgG, IgA, IgM), etc., were detected using a biochemical analyzer (Beckman AU5800, Germany). The white blood cell count and other hematological parameters were detected using an automated hematology analyzer (Sysmex Corporation, Japan). The erythrocyte sedimentation rate was measured using an erythrocyte sedimentation rate analyzer (ALIFAX TEST1, Italy). All data were obtained from the hospital laboratory information system and medical record system.

[0050] The laboratory and clinical data of the patient group (Rheumatoid Arthritis, RA) were integrated and statistically analyzed with those of the healthy control group (Health control, HC). Clinical data were collected from electronic medical records in a standardized form, including demographic data, laboratory data, and clinical symptoms. The clinical characteristics of the study population are shown in Table 1. The DAS28 score (disease activity score for rheumatoid arthritis) was used to evaluate the clinical manifestations, laboratory tests, and joint swelling and pain of RA patients. At the same time, based on the clinical data of RA patients, the patients were divided into a treatment-effective group (RA effective) and a treatment-ineffective group (RA ineffective).

[0051] It can be seen from Figure 5 the results that the lymphocytes in the RA patient group were significantly lower than those in the healthy control group, while the neutrophils were significantly higher than those in the healthy control group. The CRP, ESR, DAS28 score, and IgG in the treatment-ineffective group were all significantly higher than those in the treatment-effective group.

[0052] 4. Analysis of the expression of CD39 on different cells

[0053] Statistical analysis: The Wilcoxon rank-sum test was used to analyze the differences between the healthy controls and rheumatoid arthritis patients and between the patient groups. The statistical significance was determined as a p-value less than 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. All clinical data were expressed as the median (interquartile range).

[0054] In this example, flow cytometry was used to detect the expression of CD39 on the surface of CD4 T cells, CD8 T cells, and Treg cells in the peripheral blood of RA patients and healthy controls. The sorting strategy and the expression of CD39 in the RA patient group are shown in Figure 3 and those in the healthy control group are shown in Figure 4 .

[0055] Figure 6The Wilcoxon rank sum test was used to analyze the expression of CD39 in healthy controls and rheumatoid arthritis. Among them, A and G show the differential analysis of the proportion of CD39+CD4 T cells in RA patients and healthy controls. B and H show the differential analysis of the median fluorescence intensity of CD39 on CD4 T cells in RA patients and healthy controls. C and I show the differential analysis of the proportion of CD39+CD8 T cells in RA patients and healthy controls. D and J show the differential analysis of the median fluorescence intensity of CD39 on CD8 T cells in RA patients and healthy controls. E and K show the differential analysis of the proportion of CD39+Treg cells in RA patients and healthy controls. F and L show the differential analysis of the median fluorescence intensity of CD39 on Treg cells in RA patients and healthy controls.

[0056] Specifically:

[0057] 1) Differential analysis of the expression of CD39+CD4 T cells in the RA patient group and the healthy control group.

[0058] As Figure 6 shown, the statistical analysis results show that the proportion of CD39+CD4 T cells in the RA patient group is significantly higher than that in the healthy control group (P<0.001, Figure 6 A), and the median fluorescence intensity of CD39 on CD4 T cells is also significantly higher than that in the healthy control group (P<0.001, Figure 6 B). The proportion of CD39+CD4 T cells in the effective and ineffective RA treatment groups is significantly higher than that in the healthy control group (P<0.001, Figure 6 G), while there is no significant difference between the RA patient groups. The median fluorescence intensity of CD39 on CD4 T cells is also significantly higher than that in the healthy control group (RA effective P<0.001, RA ineffective P<0.001, Figure 6 H), and the effective RA group is higher than the ineffective RA group between groups (P<0.05, Figure 6 H).

[0059] 2) Differential analysis of the expression of CD39+CD8 T cells in the RA patient group and the healthy control group.

[0060] As Figure 6 shown, the statistical analysis results show that the proportion of CD39+CD8 T cells in the RA patient group is significantly higher than that in the healthy control group (P<0.001, Figure 6 C), and the median fluorescence intensity of CD39 on CD8 T cells is also significantly higher than that in the healthy control group (P<0.001, Figure 6 D). The proportion of CD39+CD8 T cells in the effective and ineffective RA treatment groups is significantly higher than that in the healthy control group (P<0.001, Figure 6I), and there were no significant differences among the RA patient groups. The median fluorescence intensity of CD39 on CD8 T cells was also significantly higher than that in the healthy control group (RA effective P < 0.001, RA ineffective P < 0.01, Figure 6 J), and there were no significant differences among the RA patient groups.

[0061] 3) Analysis of the differences in the expression of CD39+ Treg cells between the RA patient group and the healthy control group.

[0062] As Figure 6 shown, the statistical analysis results showed that the proportion of CD39+ Treg cells in the RA patient group was significantly higher than that in the healthy control group (P < 0.001, Figure 6 E), and the median fluorescence intensity of CD39 on Treg cells was also significantly higher than that in the healthy control group (P < 0.001, Figure 6 F). The proportion of CD39+ Treg cells in the effective and ineffective RA treatment groups was significantly higher than that in the healthy control group (P < 0.01, Figure 6 K), and there were no significant differences among the RA patient groups. The median fluorescence intensity of CD39 on Treg cells was also significantly higher than that in the healthy control group (P < 0.001, Figure 6 L), and the effective RA group was higher than the ineffective RA group between groups (P < 0.05, Figure 6 L).

[0063] 5. Receiver operating characteristic (ROC) curve analysis

[0064] In this example, the receiver operating characteristic (ROC) curve analysis was used to evaluate the effectiveness of CD39+ T cells as a diagnostic indicator for RA.

[0065] The process of drawing this receiver operating characteristic (ROC) curve was as follows: The proportions and median fluorescence intensities (MFI) of CD39+ CD4 T cells, CD39+ CD8 T cells, and CD39+ Treg cells were used as diagnostic indicators to distinguish between RA patients and healthy controls, effective RA groups and healthy controls, and ineffective RA groups and healthy controls. The true positive rate (sensitivity) was used as the vertical axis, and the false positive rate (1 - specificity) was used as the horizontal axis to draw the ROC curve. Each point corresponded to a cut-off point of the diagnostic test, and connecting these points would draw the ROC curve.

[0066] The ROC curve was as Figure 7 shown, including the ROC curves of the proportions of CD39+ CD4 T, CD39+ CD8 T, and CD39+ Treg cells and the ROC curves of the median fluorescence intensities of CD39 expression on CD4 T cells, CD8 T cells, and Treg cells. The results showed that in the RA patient group and the healthy control group ( Figure 6A-C) CD39+ CD4 T cells (cell proportion AUC (area under the ROC curve) = 0.8734, median fluorescence intensity AUC = 0.8133), CD39+ CD8 T cells (cell proportion AUC = 0.8621, median fluorescence intensity AUC = 0.7673), CD39+ Treg cells (cell proportion AUC = 0.7441, median fluorescence intensity AUC = 0.8322), in the effective RA group and the healthy control group ( Figure 6 D-F) CD39+ CD4 T cells (cell proportion AUC (area under the ROC curve) = 0.9317, median fluorescence intensity AUC = 0.8785), CD39+ CD8 T cells (cell proportion AUC = 0.9201, median fluorescence intensity AUC = 0.8414), CD39+ Treg cells (cell proportion AUC = 0.7731, median fluorescence intensity AUC = 0.8975), in the ineffective RA group and the healthy control group ( Figure 6 G-I) CD39+ CD4 T cells (cell proportion AUC (area under the ROC curve) = 0.8289, median fluorescence intensity AUC = 0.7637), CD39+ CD8 T cells (cell proportion AUC = 0.8179, median fluorescence intensity AUC = 0.7108), CD39+ Treg cells (cell proportion AUC = 0.7219, median fluorescence intensity AUC = 0.7824), demonstrating that CD39 has high efficacy as a diagnostic marker.

[0067] 6. The correlation between the expression of CD39 in RA patients and clinical indicators.

[0068] Figure 8 Shows the correlation between the proportions and median fluorescence intensities (MFI) of CD39+ CD4 T cells, CD39+ CD8 T cells, and CD39+ Treg cells in RA patients and clinical laboratory parameters. Among them, A is the correlation between the median fluorescence intensity of CD39 expression in CD4 T cells and IgG. B is the correlation between the median fluorescence intensity of CD39 expression in Treg cells and IgA.

[0069] To further explore the clinical significance of the increased proportions of CD39+ CD4 T cells, CD39+ CD8 T cells, and CD39+ Treg cells in RA patients, in this example, the correlations between the proportions of CD39+ CD4 T cells, CD39+ CD8 T cells, and CD39+ Treg cells and inflammation and immune-related indicators were analyzed.

[0070] Such as Figure 8As shown, the results showed that the median fluorescence intensity of CD39 expression in CD4 T cells was significantly negatively correlated with the levels of serum IgG (R=-0.43, P=0.04) and serum IgA (R=-0.49, P=0.035). Figure 7 A, B).

[0071] In summary, although there are reports on the expression of CD39 in various diseases, there is currently no study on the role of T cell CD39 in rheumatoid arthritis. The present invention first uses the Mendelian randomization method to analyze and illustrate the causal relationship between T cells related to CD39 and rheumatoid arthritis. Subsequently, through the analysis of the application of flow cytometry in clinical practice, it is verified that the expression of CD39 in the T cells of rheumatoid arthritis patients is significantly higher than that in the healthy control group. By comparing the median fluorescence intensity, it is also found that the expression of CD39 in the effective group of RA in CD4 T cells and Treg cells is significantly higher than that in the ineffective group of RA. Further study the clinical application of CD39, use the expression of CD39 in patients as a clinical diagnostic index, and evaluate its predictive value in rheumatoid arthritis. Perform ROC curve analysis, and the results show that CD39 has good sensitivity and specificity in distinguishing RA patients from healthy individuals. The median fluorescence intensity values of CD39 in CD4 T cells / CD8 T cells / Treg cells in flow cytometry higher than 1149.4 / 644.35 / 1298.2 are effective predictive indicators for the disease progression of rheumatoid arthritis. At the same time, the expression of CD39 is associated with clinical routine immune indexes (such as clinical indexes such as IgG and IgA), indicating that it may be closely related to the disease activity of rheumatoid arthritis. The above research shows that CD39 is expected to become a new marker for RA diagnosis, activity evaluation and a potential target for treating RA. Generally speaking, the present invention provides insights into the dynamic characteristics of immune cell subsets and their potential relevance in the pathogenesis and treatment of rheumatoid arthritis. In particular, the changes in the expression of CD39 on T cells are emphasized, indicating that these changes may vary depending on the disease progression, and suggesting its immunomodulatory role in the pathophysiological process of rheumatoid arthritis.

[0072] The above embodiments are only preferred embodiments of the present invention, and do not limit the protection scope of the present invention accordingly. Therefore, all equivalent changes made according to the structure, shape, and principle of the present invention should be covered within the protection scope of the present invention.

Claims

1. A CD39 detection kit for rheumatoid arthritis, characterized in that: The CD39 detection kit includes components for detecting the expression amount and / or concentration of CD39 in CD4 T cells, CD8 T cells, and Treg cells in a sample.

2. A CD39 detection kit for rheumatoid arthritis according to claim 1, characterized in that: The CD39 detection kit is used for specific diagnosis, prognosis and / or treatment monitoring of rheumatoid arthritis.

3. A CD39 detection kit for rheumatoid arthritis according to claim 2, characterized in that: The CD39 detection kit is used to detect the median fluorescence intensity and / or positive cell ratio of CD39 expression in CD4T cells, CD8T cells, and Treg cells through flow cytometry experiments, and to monitor the disease progression and / or treatment of rheumatoid arthritis according to the expression level of CD39.

4. A CD39 detection kit for rheumatoid arthritis according to claim 3, characterized in that: The CD39 detection kit is used to distinguish rheumatoid arthritis patients from healthy controls, and to distinguish effective treatment groups from ineffective treatment groups among patient groups through the CD39 median fluorescence intensity and / or positive cell ratio obtained by detection.

5. A CD39 detection kit for rheumatoid arthritis according to claim 3, characterized in that: The CD39 detection kit is used to identify rheumatoid arthritis patients whose T cell CD39 expression is higher than a critical value, which is determined by receiver operating characteristic curve analysis to provide a highly sensitive and specific diagnosis of rheumatoid arthritis.

6. A CD39 detection kit for rheumatoid arthritis according to claim 3, characterized in that: The CD39 detection kit is used to predict the disease progression of rheumatoid arthritis by detecting the CD39 median fluorescence intensity; its effective prediction index is: the CD39 median fluorescence intensity value of CD4T cells / CD8T cells / Treg cells is higher than 1149.4 / 644.35 / 1298.

2.

7. A CD39 detection kit for rheumatoid arthritis according to claim 3, characterized in that: The detection method of the CD39 detection kit comprises the following steps: (1) Collect samples and add fluorescently labeled antibodies for CD45, CD3, CD4, CD8, CD25, and CD127 to obtain CD4 T cells, CD8 T cells, and Treg cells; (2) staining T cells using fluorescently labeled antibodies against CD39; (3) Perform flow cytometric analysis on the CD39-stained T cells using a flow cytometer to detect the fluorescence signal of the CD39 protein and collect data.

8. A CD39 detection kit for rheumatoid arthritis according to claim 7, characterized in that: The sample is a peripheral blood sample.

9. Use of CD39 protein as a marker in the preparation or screening of reagents or kits for specific diagnosis, prognosis and / or treatment monitoring of rheumatoid arthritis.

Images