Biomarker for diagnosis of rheumatoid arthritis and application thereof

Abstract

The invention relates to the technical field of biological medicine, in particular to a biomarker for diagnosis of rheumatoid arthritis and application of the biomarker. The biomarker for diagnosis of rheumatoid arthritis, disclosed by the invention, is a neuronal glial antigen 2, namely NG2. The inventor finds that the soluble NG2 (sNG2) level is significantly up-regulated in serum of a rheumatoid arthritis patient (RA), and is significantly positively correlated with RA joint disease activity (DAS28) score, joint dysfunction grading, rheumatoid factors (RF) and inflammatory factors TNF-alpha; the sNG2 level in RA serum is obviously reduced after treatment of adalimumab, prednisone and leflunomide; in addition, the expression of the NG2 level in RA peripheral blood macrophages is up-regulated. Therefore, by detecting the NG2 gene expression level in the macrophage in the blood of the rheumatoid arthritis patient and / or the sNG2 level in the serum, the kit can be used for diagnosis and treatment prognosis indexes of the rheumatoid arthritis, has good sensitivity and specificity, and provides a new strategy for clinical diagnosis of the rheumatoid arthritis.

Description

Technical Field

[0001] The present invention relates to the application of NG2 in the field of biomedical technology, and in particular to a biomarker for diagnosing rheumatoid arthritis and an application thereof. Background Art

[0002] Rheumatoid arthritis (RA) is a common chronic inflammatory autoimmune disease with clinical symptoms including joint destruction, pain, and sports injuries, often accompanied by chronic joint inflammation, progressive bone destruction, and mononuclear cell infiltration. Previous literature has shown that the activation of myeloid cells, especially macrophages, in RA patients is one of the causes of repeated attacks of RA synovitis. There are many types of cells in the bone joints, including osteoblasts, osteoclasts, chondrocytes, synovial fibroblasts, etc. Among them, osteoclasts are multinuclear cells formed by the fusion of mononuclear macrophages differentiated from myeloid progenitor cells in the bone marrow. During the onset of the disease, the decrease in osteoblasts and the increase in osteoclasts will directly lead to reduced bone formation and increased bone erosion. This disruption of bone balance will eventually cause irreversible damage to the joints.

[0003] NG2 (neuronal glial antigen 2), also known as CSPG4 (chondroitin sulfate proteoglycan 4), is a multifunctional transmembrane proteoglycan involved in a variety of cellular processes such as migration, survival, apoptosis and differentiation. NG2 is frequently expressed in a variety of solid organ cancers. NG2 is involved in cancer cell adhesion, invasion, proliferation, angiogenesis, complement inhibition and signal transduction, and is closely related to malignant tumors such as liver cancer, pancreatic cancer, lung cancer and kidney cancer. In bone homeostasis, NG2 is involved in the initiation of bone metastasis and bone remodeling. NG2 expression is upregulated during intrachondral and intramembranous ossification and downregulated with the completion of ossification. Overexpression of lysyl oxidase-like protein 2 (LOXL2) in chondrocytes increases the level of chondroitin sulfate proteoglycan (NG2) mRNA. In addition, the molecule has multiple protein binding domains, including ERK1 / 2 and PKC-α, MUPP-1 and integrin, among which NG2 plays a key role in regulating the ERK1 / 2 signal cascade, and ERK1 / 2 also regulates chondrocyte differentiation, hypertrophy, and osteoblast differentiation. Existing studies have shown that metalloproteinases such as ADAM10, MMP-9, MMP-13 and MMP-14 have various relationships with the proliferation, migration, synovial inflammation, cartilage destruction and other pathological occurrences of synovial fibroblasts during the pathogenesis of rheumatoid arthritis. At the same time, it has been found that the above-mentioned proteases can hydrolyze NG2, forming soluble NG2 (sNG2) that is released outside the cell to exert its effect.

[0004] The pathogenesis of RA and the prediction of the disease are still not very clear, and there is a lack of ideal biomarkers in clinical diagnosis. Erythrocyte sedimentation rate and C-reactive protein, CCP antibody, rheumatoid factor, etc. are commonly used serological indicators in the clinical diagnosis of rheumatoid arthritis, but the sensitivity of CCP antibody and the specificity of rheumatoid factor are not ideal. Therefore, the development of new biomarkers for early diagnosis and monitoring of the disease course is a key research direction for RA diagnosis and treatment. Summary of the invention

[0005] In view of the insensitivity and poor specificity of clinical detection methods for RA, in order to make up for the defects of the existing technology, the present invention proposes a NG2 protein from the blood that can be used as a biomarker for the onset of rheumatoid arthritis. By detecting the macrophage NG2 expression in the patient's blood and the soluble NG2 (sNG2) level in the serum, early diagnosis of rheumatoid arthritis patients and monitoring of the occurrence and development of the disease can be achieved.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a biomarker for clinical diagnosis and treatment prognosis of rheumatoid arthritis, wherein the marker is neuronal glial antigen 2 (NG2).

[0008] The present invention detects the NG2 expression level of macrophages in human blood and the sNG2 level secreted in serum, and identifies that it can be used as a biomarker for rheumatoid arthritis. The expression level of the marker NG2 is significantly upregulated in the peripheral blood macrophages of patients with rheumatoid arthritis, and sNG2 is significantly upregulated in the serum of patients with rheumatoid arthritis. It can be used for the diagnosis of rheumatoid arthritis and the monitoring of the course of the disease. It has strong specificity and high sensitivity, and provides a new strategy for the diagnosis of clinical rheumatoid arthritis.

[0009] Furthermore, as a preferred embodiment of the marker of the present invention, the marker exists in the blood of RA patients. Still further, the biomarker exists in the peripheral blood of rheumatoid arthritis patients.

[0010] In a second aspect, the present invention provides use of a reagent for measuring NG2 expression in preparing a tool for diagnosing rheumatoid arthritis.

[0011] As a preferred embodiment of the use described in the present invention, the detection of NG2 expression includes: one or more of ELISA method, Western blot method, flow cytometry method, gene chip detection method, and real-time fluorescence quantitative method.

[0012] As a preferred embodiment of the use described in the present invention, by detecting the NG2 level in the patient's peripheral blood monocytes and / or macrophages and the sNG2 level in the blood, it is determined whether the patient has rheumatoid arthritis, and the expression change level of sNG2 in the peripheral blood serum of rheumatoid arthritis patients before and after treatment is used to predict the therapeutic efficacy of rheumatoid arthritis.

[0013] Furthermore, the inventors found that NG2 expression was upregulated in RA blood macrophages, sNG2 expression was upregulated in RA serum, and sNG2 levels in RA patients were significantly positively correlated with clinical indicators of DAS28 and RF as well as inflammatory factor TNF-α. sNG2 in RA serum was significantly downregulated after treatment with adalimumab, prednisone and leflunomide.

[0014] As a preferred embodiment of the use of the present invention, the diagnosis of rheumatoid arthritis comprises the following steps:

[0015] (1) Collecting samples from test subjects and collecting control samples;

[0016] (2) detecting and comparing the expression levels of NG2 in the test subject samples and control samples;

[0017] If the expression level of NG2 in the sample of the test subject is increased compared to the expression level of NG2 in the control sample, the test subject is diagnosed as having rheumatoid arthritis or being at risk of having rheumatoid arthritis.

[0018] As a preferred embodiment of the use described in the present invention, the control sample is derived from a healthy population or a healthy tissue of a subject to be tested, and the sample of the subject to be tested is one or both of serum and whole blood.

[0019] As a preferred embodiment of the use described in the present invention, in step (1), a sample of a test subject and a control sample are collected, and the specific operations include: (a) obtaining peripheral blood by centrifugation; or (b) obtaining peripheral blood by density gradient centrifugation, magnetic bead separation, and immunoseparation technology.

[0020] In a third aspect, the present invention provides a kit for diagnosing rheumatoid arthritis.

[0021] Furthermore, the kit comprises a reagent for detecting NG2 expression.

[0022] As a preferred embodiment of the kit of the present invention, the sample to be detected is serum and / or peripheral blood mononuclear cells.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The present invention proposes for the first time that the expression level of sNG2 in serum significantly changes (high expression) in the peripheral blood of RA patients, and that the expression of NG2 in RA blood macrophages is significantly upregulated. This molecular marker can be used for the diagnosis of RA, has strong specificity and high sensitivity, can conveniently and quickly obtain test samples, and can obtain test results economically and effectively, which can not only reduce the test cost, but also shorten the test time, improve the test efficiency and accuracy, reduce the patient's pain and economic costs, and shorten the cycle of predicting the therapeutic effect. Therefore, it can be used as a biomarker for the diagnosis of rheumatoid arthritis; in addition, the rheumatoid arthritis biomarker of the present invention can be repeatedly tested, which is convenient for re-examination and can improve the accuracy of the test results; the present invention predicts the therapeutic effect of rheumatoid arthritis based on the downregulation of sNG2 levels after rheumatoid arthritis treatment, can provide data support for the diagnosis of rheumatoid arthritis treatment results, and provide a new strategy for the diagnosis of rheumatoid arthritis in clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 sNG2 was significantly upregulated in RA serum and positively correlated with the grade of RA joint dysfunction.

[0026] Figure 2 The sNG2 in RA serum was significantly positively correlated with the clinical indicators DAS28 and RF.

[0027] Figure 3 TNF-α promotes NG2 expression and sNG2 production in RA macrophages.

[0028] Figure 4 TNF-α promotes EGR1-mediated upregulation of NG2 expression in macrophages.

[0029] Figure 5 To inhibit the activity of metalloproteinases and reduce TNF-α-induced sNG2 production in macrophages. DETAILED DESCRIPTION

[0030] The present invention will be further described below in conjunction with specific examples. The following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The following examples are convenient for a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples, unless otherwise specified, are conventional methods, and the materials, reagents, etc. used can all be obtained from commercial sources.

[0031] Example 1

[0032] This example introduces the application of a biomarker for rheumatoid arthritis.

[0033] The rheumatoid arthritis biomarker of this embodiment is neuronal glial antigen 2 (sNG2), including NG2 expressed by RA blood macrophages and soluble NG2 (sNG2) in serum.

[0034] 1. Sample collection

[0035] Thirty-one patients with rheumatoid arthritis admitted to the First Affiliated Hospital of Anhui Medical University (North District) were collected. All enrolled cases were diagnosed with RA by clinical and related rheumatoid tests, with an age range of (23-79) years. At the same time, 43 healthy volunteers were recruited as the control group, with an age range of (19-55) years. The diagnosis of all patients in the case group met the 1987 RA classification diagnostic criteria of the American College of Rheumatology. The control group was healthy subjects who underwent physical examinations during the same period. All the research subjects were unrelated Han people. This study has been approved by the Ethics Committee of the First Affiliated Hospital of Anhui Medical University (North District), with approval number PJ-YX2024-012. All patients and healthy volunteers have obtained informed consent. The basic information and clinical data of the 31 RA patients and 43 normal controls are summarized in Table 1.

[0036] 2. Serum extraction

[0037] 4 mL of peripheral blood was collected from 31 RA patients and 43 normal subjects by venous blood sampling. The blood collection tube used in this embodiment is a blood collection tube with a blood anticoagulant. Within 30 minutes after specimen collection, centrifuge at 4°C and 1000g for 20 minutes. The upper pale yellow liquid after centrifugation is serum. If the color is reddish, hemolysis may occur. Hemolyzed specimens will affect the final test results. Therefore, hemolyzed specimens are not suitable for this test. Carefully draw serum into EP tubes. If no subsequent operation is performed immediately, store the specimen at -20°C or -80°C. Repeated freezing and thawing should be avoided.

[0038] Table 1

[0039]

[0040] 3. Index detection

[0041] In this example, the Dot-bolt method and the conventional double antibody sandwich method (sandwich ELISA) were used to detect the NG2 content in the sample.

[0042] Dot-blot:

[0043] Serum samples from RA patients (n=6) and controls were applied to nitrocellulose membranes, blocked with 5% BSA in 0.1% PBST, and then incubated with NG2 antibody and β-actin antibody (Proteintech, Cat. No.: 66009-1-Ig) at room temperature for 1 h. The bound antibodies were probed with enzyme-labeled secondary antibodies, and finally probed with ECL chemiluminescence kit (Pierce, Rockford, IL, USA). Dot-blot was used to detect the content of sNG2 in the serum of healthy controls and RA patients (6 vs. 6). The results are shown in Table 1. Figure 1 A. The intensity of each point was quantified using Image J software, and the results are shown in Figure 1 As shown in B, the quantitative statistical chart of the Dot-blot results shows that the sNG2 content in RA serum increased significantly.

[0044] Sandwich ELISA:

[0045] (a) On the first day, the capture antibody (NG2 monoclonal antibody) was diluted to 2.0 μg / mL in PBS. 100 μL of the diluted capture antibody was immediately added to each well of the ELISA plate, the plate was sealed with a sealing film, and incubated at 4°C overnight.

[0046] (b) Perform the test on the next day. Discard the liquid, pat dry on absorbent paper, add 300 μL of washing solution to each well and let stand for 1 min, discard the liquid, pat dry on absorbent paper, and repeat the above operation three times.

[0047] (c) Add 300 μL of blocking solution to each well and incubate at room temperature for 1 hour.

[0048] (d) Repeat the wash in step (b) and prepare to add the sample when completed.

[0049] (e) 100 μL of serum sample diluted 1:5 with PBS and serially diluted standards were added to each well, the plate was sealed with a sealing film, and incubated at room temperature for 2 hours.

[0050] (f) Repeat the wash in (b).

[0051] (g) 100 μL of 0.5 μg / mL horseradish peroxidase (HRP)-conjugated NG2 polyclonal antibody was added to each well, the plate was sealed with a sealing film, and incubated at room temperature for 1 hour.

[0052] (h) Repeat the wash in (b).

[0053] (j) Add 150 μL of TMB substrate solution to each well and incubate at room temperature in the dark for 20 minutes. Avoid placing the plate in direct sunlight.

[0054] (k) Add 50 μL of stop solution (2M H2SO4) to each well and tap gently to ensure thorough mixing.

[0055] (l) Immediately use an ELISA reader to measure the optical density of the well plate at 450 nm. The obtained value needs to be further analyzed and processed.

[0056] 4. Concentration calculation

[0057] Calculate the average absorbance of each group of standards, controls, and samples. Construct a standard curve by plotting the absorbance of each standard on the y-axis and the concentration on the x-axis, and draw a best-fit curve through these points on the graph. To determine the concentration of the sample, find the absorbance value of the sample on the y-axis and draw a horizontal line to the standard curve. Draw a vertical line to the x-axis at the intersection and read the concentration. The read concentration multiplied by 5 (dilution factor) is the actual concentration of the sample. In addition to the above method, computer-based curve fitting statistical software can also be used to calculate the concentration of the sample. ELISA was used to detect the content of sNG2 in the serum of RA patients (n=31) and healthy controls (n=43). The results are shown in Figure 2. Figure 1 As shown in C, Figure 1 C shows that the sNG2 content in the serum of rheumatoid arthritis patients increased significantly. In this example, we also collected serum from patients treated with existing clinical drugs. The sNG2 content in the serum of rheumatoid arthritis patients after receiving adalimumab (2 cases), prednisone (3 cases), and leflunomide (2 cases) was as follows: Figure 1 As shown in D, Figure 1 D shows that the sNG2 content in the patient's serum decreased after treatment. In addition, we found that compared with the control group, the relationship between the serum sNG2 level in RA patients and joint damage (X-ray stage = 0) or damage (X-ray stage ≥ 1) was Figure 1 As shown in E, Figure 1 E shows that serum sNG2 levels in RA patients with joint damage are significantly increased. At the same time, the correlation results between serum sNG2 levels and the classification of joint dysfunction in RA patients are as follows Figure 1 As shown in F, Figure 1 From F, we can see that there is a significant correlation.

[0058] In summary, the sNG2 content in the serum of rheumatoid patients is closely related to the disease.

[0059] Example 2

[0060] This example introduces the correlation analysis between the above-mentioned biomarker for rheumatoid arthritis (sNG2) and clinical indicators. In order to find more evidence to prove that sNG2 is a potential marker for the diagnosis of rheumatoid arthritis, we conducted a correlation analysis between the sNG2 content in the patient's serum and the common clinical indicators of the patient in clinical diagnosis. This example is based on computer software (GraphPad Prism 8) to calculate the correlation between the content of sNG2 and clinical indicators, and the steps are as follows:

[0061] (a) Import data: Create a new XY table in GraphPad Prism 8 and input the clinical data of each patient into the table one by one.

[0062] (b) Normality test: Click the Analysis button on the toolbar to enter the Analyze Data interface. Select the Normality and Lognormality Tests option in Column analyses, keep the default parameter settings, and click OK.

[0063] (c) Correlation analysis: Click the Analysis button on the toolbar to enter the Analyze Data interface. Select the Correlation option in XYanalyses. According to the normality test results in step (b), if both variables obey the normal distribution, the Pearson correlation analysis in the parameters is used. If one or both of the two variables are rank variables or do not satisfy the normal distribution, the Spearman correlation analysis is required. Keep the default options for other parameter settings and click OK.

[0064] (d) The results of the correlation analysis can be viewed in Results: If the P value is less than 0.05, there is a significant difference in the correlation.

[0065] The analysis results are as follows Figure 2 As shown by Figure 2 It can be seen that the sNG2 content in the serum of patients with rheumatoid arthritis has a significant positive correlation with DAS28 and RF indicators. Figure 2 A is the correlation analysis between sNG2 and DAS28; Figure 2 B is the correlation analysis between sNG2 and RF; Figure 2 C is the correlation analysis between sNG2 and CCP; Figure 2 D is the correlation analysis between sNG2 and ESR; Figure 2 E is the correlation analysis between sNG2 and CRP; Figure 2F is the correlation analysis between sNG2 and Sharp score.

[0066] In clinical diagnosis, DAS28 is an important indicator for judging the degree of disease activity. Its calculation is based on the examination of 28 joints, including the joints of both hands and wrist joints. This indicator takes into account the degree of joint swelling and joint sensitivity, as well as the concentration of CRP in the blood. These factors are combined to evaluate the disease activity level of RA patients. In addition, RF (rheumatoid factor) is another important indicator for diagnosing rheumatoid arthritis. It is associated with the progression and severity of the disease. Although RF is not a specific marker, its positive result can support the diagnosis of RA. High levels of RF may indicate more serious disease progression and joint damage.

[0067] In summary: The level of sNG2 in patient serum is significantly correlated with two important indicators in the clinical diagnosis of rheumatoid arthritis (DAS28 and RF), which further demonstrates that NG2 is a potential marker for the diagnosis of rheumatoid arthritis.

[0068] Example 3

[0069] This example describes the expression of the above-mentioned rheumatoid arthritis biomarker (sNG2) in monocyte-derived macrophages.

[0070] Literature in the prior art shows that the activation of myeloid cells, especially macrophages, in arthritis patients is the core of the pathogenesis of RA synovitis. Osteoclasts in bone joints are mainly multinuclear cells formed by the fusion of mononuclear macrophages. The increase in osteoclasts during the onset of the disease will lead to increased bone erosion, thereby causing irreversible damage to the joints. In order to further illustrate the relationship between sNG2 and macrophages, we induced healthy human PBMC (peripheral blood mononuclear cells) to differentiate into macrophages, and then detected the gene expression and extracellular domain shedding of NG2. The steps are as follows:

[0071] 1. PBMC extraction: Ficoll density gradient centrifugation was used to separate and purify peripheral blood mononuclear cells.

[0072] (a) Take fresh anticoagulated whole blood (EDTA, sodium citrate or heparin anticoagulation is acceptable) and dilute the whole blood with an equal volume of whole blood and tissue diluent or PBS.

[0073] (b) Add an equal volume of separation solution to the diluted whole blood into the centrifuge tube, use a Pasteur pipette to draw up the blood, and spread the diluted blood evenly on the surface of the separation solution, taking care to keep a clear interface between the two liquid surfaces.

[0074] (c) Centrifuge at 900 g in a swinging rotor for 30 min at room temperature.

[0075] (d) After centrifugation, obvious stratification will appear: the top layer is the diluted plasma layer, the middle layer is the transparent separation fluid layer, the white film layer between the plasma and the separation fluid is the lymphocyte layer, and the bottom of the centrifuge tube is the red blood cells and granulocytes.

[0076] (e) Carefully pipette the buffy coat cells into a 15 mL clean centrifuge tube, add 10 mL PBS to wash the buffy coat cells, and centrifuge at 250 g for 10 min.

[0077] (f) Discard the supernatant, add 10 mL of PBS to resuspend the cells, and centrifuge at 250 g for 10 min to obtain PBMCs.

[0078] 2. PBMC induction: Use 40ng / mL M-CSF to induce monocytes to differentiate into macrophages. Resuspend the cells obtained in step 1 with αMEM medium containing 40ng / mL M-CSF, and seed them into the well plate. Change the medium every three days. The medium contains 40ng / mL M-CSF. After 7 days, monocyte-derived macrophages can be obtained.

[0079] 3. Gene expression detection: Trizol was used to extract RNA from uninduced PBMC cells and induced macrophages, and Q-PCR was used to detect NG2 gene expression. -ΔΔct The relative quantification of the values ​​was performed to analyze the relative fold changes in the expression of the target gene between the experimental group and the normal control group. The results of RT-Q-PCR detection of monocyte-derived macrophages (MDMs) in healthy human controls (5 cases) are as follows Figure 3 As shown in A, Figure 3 As shown in A, the expression level of NG2 increased after induction into macrophages.

[0080] 4. Detection of sNG2 content in cell supernatant: The ELISA method was used to detect the cell supernatant in the same manner as the index detection method in Example 1. The results of ELISA analysis of sNG2 levels in the supernatant of healthy control PBMCs and MDMs (5 cases) are as follows Figure 3 As shown in B, Figure 3 B shows that there is no significant difference in the sNG2 content in the cell supernatant before and after induction.

[0081] 5. In addition, the results of RT-Q-PCR detection of NG2 mRNA content in MDMs of controls (4 cases) and RA patients (4 cases) are as follows Figure 3 As shown in C, Figure 3 C shows that the level of human NG2 mRNA was increased in MDMs of RA patients compared with that in controls.

[0082] 6. We analyzed the serum TNF-α level in patients and its correlation with sNG2. Figure 3As shown in D, Figure 3 D shows that TNF-α and sNG2 are significantly positively correlated.

[0083] 7. RT-Q-PCR detection of NG2 gene expression in macrophages stimulated with TNF-α for 24 hours Figure 3 As shown in E, Figure 3 E shows that compared with the control group, TNF-α in MDMs significantly induced NG2 mRNA levels; the results of sNG2 content in the cell supernatant after macrophages were stimulated with TNF-α for 24 hours are as follows Figure 3 As shown in F, Figure 3 As shown in F, the production of sNG2 in the supernatant of MDMs treated with TNF-α increased compared with that in the control group.

[0084] Example 4

[0085] To further analyze the differentially expressed genes (DEGs) of transcription factors that regulate NG2 expression, we used a single-cell sequencing dataset of synovial tissue from three RA patients and three controls. The single-cell transcriptome profiles of the six samples were analyzed using the Seurat V4R package and merged into a Seurat object. We performed quality control on the Seurat object, with the screening criteria of nFeature_RNA>200 and nFeature_RNA<4000 and mt<20%. Next, we used the Harmony R package to eliminate batch effects between samples, ensure that cells from different sample sources could be clustered correctly, and set the resolution to 1 for cell clustering, completing the basic steps of quality control and dimensionality reduction clustering of single-cell data. Subsequently, we used the FindMarkers function in Seurat to analyze the expression differences of each transcription factor between the control group and the RA group based on the Wilcoxon rank sum test.

[0086] The volcano plot was used to analyze the differentially expressed genes of NG2-related transcription factors in synovial macrophages between the control group (3 cases) and RA patients (3 cases). Figure 4 As shown in A, Figure 4 A shows that among the 317 transcription factors reported in the Genecards database, there was no significant difference in the expression of 196 transcription factors in RA synovial macrophages compared with the control group, 7 transcription factors were significantly downregulated, and 114 transcription factors were significantly upregulated. The violin plot shows the top 4 transcription factors upregulated in the synovial macrophages of the control group and RA patients. The results are as follows Figure 4 As shown in B, Figure 4B shows that compared with the control group, the expression levels of four transcription factors, EGR1, FOS, STAT1 and KLF10, in RA synovial macrophages were significantly upregulated. The results of RT-Q-PCR analysis of the expression of the first four upregulated transcription factors related to NG2 in MDMs treated with or without TNF-α are shown in Figure 2. Figure 4 As shown in C, Figure 4 C, we confirmed that EGR1 was significantly increased in TNF-α stimulated MDMs compared with control MDMs, while FOS, STAT1 and KLF10 were not significantly increased. Most importantly, RT-Q-PCR analysis of NG2 mRNA expression in TNF-α induced RAMDMs (4D) or U937 derived macrophages (4E) was performed as shown in Figure 4 Figure 4 D- Figure 4 As shown in F, Western blot and quantitative analysis results of NG2 and β-actin in TNF-α-induced U937-derived macrophages treated with or without the EGR1-specific inhibitor ML264 are shown in Figure 4 As shown in F, Figure 4 D- Figure 4 As can be seen from F, we found that inhibition of EGR1 using the specific inhibitor ML264 could significantly reduce the mRNA and protein expression of NG2 in RAMDMs and U937-derived macrophages.

[0087] These results indicate that TNF-α-induced upregulation of NG2 in MDMs is mainly through transcriptional regulation associated with EGR1.

[0088] Example 5

[0089] NG2 ectodomain shedding is released by sequential processing by metalloproteinases MMP-9, MMP-13, MMP-14, and ADAM10 to release multiple fragments. To further analyze which metalloproteinase is required for NG2 ectodomain shedding in TNF-α-stimulated macrophages, we performed RT-Q-PCR detection of these metalloproteinases in TNF-α-treated or untreated human MDMs. The results of RT-Q-PCR detection of MMP9, MMP13, MMP14, ADAM8, ADAM9, and ADAM10 mRNA expression in MDMs stimulated with or without TNF-α are shown in Figure 2. Figure 5 As shown in A, we found that MMP9 and MMP14 were significantly increased in TNF-α treated MDMs compared with controls, while MMP13, ADAM8, ADAM9, and ADAM10 were not significantly increased. Most interestingly, RT-Q-PCR analysis of MMP14 and MMP9 mRNAs in MDMs from healthy controls (n=4) and RA patients (n=4) showed Figure 5 B- Figure 5 (C), we found that MMP14, but not MMP9, was significantly enhanced in MDMs from RA patients compared with controls.

[0090] The above results indicated that the expression of MMP14 was upregulated in macrophages of RA patients treated with TNF-α compared with that in the control group.

[0091] GM6001 is a broad-spectrum matrix metalloproteinase (MMP) inhibitor that targets MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-12, MMP-14, and MMP-26.

[0092] Western-blot detection of NG2 protein expression in U937-derived macrophages stimulated by TNF-α in the presence or absence of GM6001. Figure 5 As shown in D; Figure 5 The quantitative results of NG2 / β-actin relative density in D are as follows Figure 5 E; ELISA analysis of sNG2 levels in the supernatant of U937-derived TNF-α-stimulated macrophages in the presence or absence of GM6001. Figure 5 F Figure 5 D- Figure 5 F As can be seen, we found that GM6001 treatment significantly reduced TNF-α-induced NG2 ectodomain shedding and soluble NG2 release from macrophages derived from the human monocytic cell line U937, indicating that GM6001 treatment inhibits TNF-α-induced NG2 ectodomain shedding. Therefore, these results suggest that MMP14 may be required for NG2 ectodomain shedding in TNF-α-stimulated RA macrophages.

[0093] In summary, the present invention proposes for the first time that the expression level of sNG2 in the peripheral blood serum of patients with rheumatoid arthritis changes significantly (high expression), and is related to DAS28 and RF. TNF-α mediates the expression of NG2 in MDMs and U937 by inducing EGR1, and produces high-level sNG2 secretion. This molecular marker can be used for the diagnosis of rheumatoid arthritis, can provide data support for the diagnosis of rheumatoid arthritis treatment results, and provide a new strategy for the diagnosis of rheumatoid arthritis in clinical practice.

[0094] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A biomarker for the diagnosis of rheumatoid arthritis, It is characterized in that The biomarker is neuronal glial antigen 2, namely NG2; including NG2 expressed by RA blood macrophages and soluble NG2 in serum, namely sNG2.

2. The biomarker according to claim 1, It is characterized in that The biomarkers are present in the peripheral blood of patients with rheumatoid arthritis.

3. A kit for diagnosing rheumatoid arthritis, It is characterized in that The kit comprises reagents for detecting NG2 expression.

4. The kit according to claim 3, It is characterized in that The samples tested are serum and / or peripheral blood mononuclear cells.

5. Use of a reagent for detecting NG2 expression in the preparation of a tool for diagnosing rheumatoid arthritis.

6. The use according to claim 5, It is characterized in that By detecting the NG2 level in the patient's peripheral blood monocytes and / or macrophages and the sNG2 level in the blood, it is determined whether the patient has rheumatoid arthritis. The expression change level of sNG2 in the peripheral blood serum of patients with rheumatoid arthritis before and after treatment can be used to predict the therapeutic efficacy of rheumatoid arthritis.

7. The use according to claim 5, It is characterized in that The detection of NG2 expression includes: one or more of ELISA method, Western blot method, flow cytometry method, gene chip detection method, and real-time fluorescence quantitative method.

8. The use according to claim 5, It is characterized in that The diagnosis of rheumatoid arthritis includes the following steps: (1) Collecting samples from test subjects and collecting control samples; (2) detecting and comparing the expression levels of NG2 in the test subject samples and control samples; If the expression level of NG2 in the sample of the test subject is increased compared to the expression level of NG2 in the control sample, the test subject is diagnosed as having rheumatoid arthritis or being at risk of having rheumatoid arthritis.

9. The use according to claim 8, It is characterized in that In step (1), samples of the test subjects and control samples are collected, and the specific operations include: (a) obtaining peripheral blood by centrifugation; or (b) obtaining peripheral blood by density gradient centrifugation, magnetic bead separation, and immunoseparation technology.

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