Biomarker for diagnosing and distinguishing rheumatoid arthritis and knee osteoarthritis and application of biomarker

Abstract

The invention relates to the technical field of biological medicine, in particular to a biomarker for diagnosing and distinguishing rheumatoid arthritis and knee osteoarthritis and application of the biomarker. In order to solve the problems of insufficient sensitivity and specificity of rheumatoid arthritis (RA) and knee osteoarthritis (KOA) in clinical detection, the invention discloses a novel biomarker, namely a soluble interleukin 17 receptor D, namely sIL-17RD. The inventor finds that the sIL-17RD level is remarkably increased in serum of a patient with rheumatoid arthritis, the sIL-17RD level is not remarkably changed in serum of a patient with knee osteoarthritis, and the sIL-17RD level is remarkably increased in synovial fluid of the patient with knee osteoarthritis. Therefore, by measuring the sIL-17RD level in the serum sample of the rheumatoid arthritis patient and the sIL-17RD level in the synovial fluid and serum of the knee osteoarthritis patient, the rheumatoid arthritis and the knee osteoarthritis are diagnosed and distinguished, and a new strategy is provided for clinical diagnosis and distinguishing of the rheumatoid arthritis and the knee osteoarthritis.

Description

Technical Field

[0001] The present invention relates to the application of sIL-17RD in the field of biomedical technology, and in particular to a biomarker for diagnosing and distinguishing rheumatoid arthritis and knee osteoarthritis and an application thereof. Background Art

[0002] Rheumatoid arthritis (RA) is a common chronic inflammatory autoimmune disease with clinical symptoms including morning stiffness, joint swelling and pain, and limited mobility, often accompanied by chronic joint inflammation, progressive bone destruction, and mononuclear cell infiltration. Although the pathogenesis of RA is still unclear, previous literature has shown that TNF-α levels are significantly increased in the serum and synovial fluid of arthritis patients. In addition, TNF-α, which is mainly secreted by macrophages, plays an important role in the pathogenesis of RA. The TNF-α-induced mouse arthritis model exhibits all symptoms of inflammatory arthritis, including synovitis, pannus formation, and bone erosion. The activation of myeloid cells, especially macrophages, is the core of the pathogenesis of RA synovitis. Activated macrophages promote the production of proinflammatory factors (such as TNF-α, IL-6, and GM-CSF) in serum and synovial fluid, which are closely related to the induction and maintenance of synovitis and participate in the subsequent disease development process.

[0003] Osteoarthritis (KOA) is the most common joint disease in the world. Studies in North America and Europe have shown that approximately 30% of people aged ≥65 years suffer from knee OA, and the characteristic signs of OA include pain, transient morning stiffness, and cramps (grating sounds or sensations in the joints) during movement. Joint pain and loss of function can cause a huge economic burden, often accounting for 1-2.5% of GDP in developed countries. Osteoarthritis affects the entire joint, including articular cartilage, subchondral bone, and synovium. Although the pathological process can affect a single tissue, the entire joint is ultimately involved due to its intimate structural and functional relationship. Substantial changes occur in the articular cartilage matrix, which is caused and affected by chondrocytes. When chondrocytes fail to repair the damaged cartilage matrix, these cells begin to secrete matrix-degrading enzymes, reactive oxygen species, cytokines, and chemokines, among others. Then, synovial inflammation ensues, leading to cartilage degradation. Obesity and increased levels of associated adipokines also increase the inflammatory state. Remodeling of the subchondral plate reflects and causes changes in the mechanical loading of the cartilage and subchondral bone.

[0004] The interleukin 17 receptor D (IL-17RD) gene is also known as Sef. Our research group cloned the IL-17RD gene of humans and mice. Previous studies have found that IL-17RD functionally interacts with TNFR2 through its extracellular domain to activate the NF-B signaling pathway. It is important to find that TNFR2 and IL-17RD form a stable complex. In addition, TNF-α was observed to directly bind to IL-17RD and promote the interaction between IL-17RD and TNFR2. In order to further explore how IL-17RD regulates TNF-α-mediated inflammatory signaling pathways through its extracellular domain, and whether it plays an important role in the inflammatory disease RA, we conducted further in-depth research and made the following series of important discoveries (Cellular & Molecular Immunology, 2021): 1) A soluble IL-17RD (which we named sIL-17RD) was first discovered in the serum of arthritis rat PIA model and arthritis mouse CIA model, and was significantly upregulated compared with the control; 2) sIL-17RD produced a glycosylated dimer by cleavage of the IL-17RD extracellular domain (between P289 and V290) by TACE enzyme; 3) Under inflammatory conditions, TNF-α, rather than IL-17A and IL-1β, promoted the shedding of the IL-17RD extracellular domain in macrophages; 4) sIL-17RD promoted the activation of TNF-α inflammatory signaling pathway NF-κB, thereby promoting TNF-α-induced macrophage inflammatory response; 5) sIL-1 7) RD promotes TNF-α-mediated dimerization of TNF-α homologous (or heterologous) receptors TNFR1 / 1, TNFR2 / 2, and TNFR1 / 2, thereby promoting TNF-α pathway activation; 6) sIL-17RD directly binds to TNF-α and its receptors TNFR1 and TNFR2, and forms sIL-17RD-TNF-α-TNFR1 and sIL-17RD-TNF-α-TNFR2 heteromeric complexes; 7) Intraperitoneal injection of sIL-17RD promotes the development of arthritis in CIA mice; sIL-17RD promotes the production of proinflammatory factors IL-6, TNF-α, and IL-17, and reduces the inflammatory response of the anti-inflammatory factor IL-10, thereby promoting the inflammatory response of arthritis mice. Combined with the above findings, we propose a new concept of sIL-17RD immune regulation in RA: TNF-α, an important cytokine in the inflammatory immune response, can activate the TNF-α-TNFRs signaling pathway by promoting the shedding of IL-17RD mediated by the TACE enzyme, and exert the immune regulatory function of IL-17RD. The positive feedback regulation of pro-inflammatory mechanism by TNF-α is an important supplement to the mechanism of RA pathogenesis regulated by TNF-α.

[0005] 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.

[0006] Similarly, the pathogenesis of KOA is still unclear, and there is no unified gold standard for its diagnosis, staging and treatment. The current clinical diagnosis of KOA is based on a comprehensive judgment based on the patient's clinical symptoms and imaging examinations. The clinical symptoms are mainly pain, limited knee joint movement and narrow joint space. Radiographic X-rays and MRI examinations are the main diagnostic methods for KOA at this stage, and there is currently a lack of more specific laboratory test indicators for KOA. The sIL-17RD protein we found was significantly increased in the synovial fluid of KOA and was significantly correlated with the severity of KOA, suggesting that sIL-17RD protein can be used as an auxiliary indicator for the clinical diagnosis of KOA and the judgment of its severity. Summary of the invention

[0007] In view of the characteristics of insensitivity and poor specificity of clinical detection methods for RA and KOA, in order to make up for the defects of the existing technology, the present invention proposes a sIL-17RD protein from blood / knee joint fluid (synovial fluid) that can be used as a biomarker for the onset of rheumatoid arthritis. By detecting the macrophage IL-17RD expression in the patient's blood and the sIL-17RD level in the serum, early diagnosis of rheumatoid arthritis patients and monitoring of the occurrence and development of the disease can be achieved. By detecting the sIL-17RD protein content in the serum and synovial fluid of KOA patients, early diagnosis of knee osteoarthritis patients and monitoring of the occurrence and development of the disease can be achieved.

[0008] In order to achieve the above object, the present invention adopts the following technical solution:

[0009] In a first aspect, the present invention provides a biomarker for clinical diagnosis, differentiation and treatment prognosis of rheumatoid arthritis and osteoarthritis, wherein the biomarker is interleukin 17 receptor D (IL-17RD).

[0010] Furthermore, the biomarker is interleukin 17 receptor D, namely IL-17RD; including IL-17RD in cells and tissues and soluble IL-17RD in serum and joint fluid, namely sIL-17RD.

[0011] The present invention detects the content of IL-17RD expressed by macrophages in human blood and the soluble IL-17RD (sIL-17RD) secreted in serum and synovial fluid, and identifies that it can be used as a biomarker for rheumatoid arthritis and knee osteoarthritis.

[0012] Furthermore, the expression level of the marker IL-17RD is significantly upregulated in the peripheral blood macrophages of patients with rheumatoid arthritis, and sIL-17RD is significantly upregulated in the serum of patients with rheumatoid arthritis. It can be used for the diagnosis of rheumatoid arthritis and 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.

[0013] Furthermore, the expression level of the marker IL-17RD is upregulated in the articular cartilage and synovial fluid of patients with knee osteoarthritis, and sIL-17RD has no obvious changes in the serum of patients with knee osteoarthritis. It can be used for the diagnosis of knee osteoarthritis inflammation, and also has the characteristics of monitoring the course of the disease, strong specificity and high sensitivity, and can provide a new strategy for the clinical diagnosis of knee osteoarthritis.

[0014] Furthermore, as a preferred embodiment of the marker of the present invention, the marker is present in RA serum, KOA joint fluid and serum, as well as RA peripheral blood monocytes, macrophages and KOA chondrocytes.

[0015] In a second aspect, the present invention provides a use of a reagent for detecting IL-17RD expression in preparing a tool for diagnosing and differentiating rheumatoid arthritis and knee osteoarthritis.

[0016] As a preferred embodiment of the use described in the present invention, the detection of IL-17RD expression includes: one or more of ELISA method, Western blot method, immunofluorescence method, flow cytometry method, gene chip detection method, and real-time fluorescence quantitative method.

[0017] As a preferred embodiment of the use of the present invention, by detecting the expression level of IL-17RD in the serum and blood macrophages of RA patients, it is determined whether it is rheumatoid arthritis, and the expression level of IL-17RD in the serum of RA patients before and after treatment is used to predict the therapeutic efficacy of rheumatoid arthritis. Wherein, the treatment includes but is not limited to the use of adalimumab, prednisone, and leflunomide for treatment.

[0018] Furthermore, the expression of IL-17RD was upregulated in the serum of RA patients and in RA blood macrophages, and the expression level of sIL-17RD was significantly positively correlated with the clinical indicators of DAS28 and RF and the inflammatory factor TNF-α; the expression of sIL-17RD in serum was significantly downregulated after treatment with adalimumab, prednisone and leflunomide.

[0019] As a preferred embodiment of the use of the present invention, by detecting the expression level of IL-17RD in the serum and cartilage of KOA patients, it is determined whether it is knee osteoarthritis, and the expression level of IL-17RD in the serum of KOA patients before and after treatment is used to predict the therapeutic effect of knee osteoarthritis. Wherein, the treatment includes but is not limited to the use of adalimumab, prednisone, and leflunomide for treatment.

[0020] Furthermore, the expression of IL-17RD did not change significantly in the serum of KOA patients, but was upregulated in cartilage. The expression level of sIL-17RD was significantly correlated with the minimum joint space (mJSW) and the inflammatory factor TNF-α, and the sIL-17RD content in serum was significantly positively correlated with the patient's clinical KL grade.

[0021] In a third aspect, the present invention provides a kit for detecting rheumatoid arthritis and knee osteoarthritis, comprising a reagent for detecting the expression level of IL-17RD.

[0022] As a preferred embodiment of the detection kit of the present invention, the sample to be detected is blood or synovial fluid. The sample to be detected is further selected from at least one of serum, peripheral blood mononuclear cells, macrophages and knee joint fluid.

[0023] As a preferred embodiment of the detection kit of the present invention, the detection of IL-17RD expression water comprises the following steps:

[0024] (1) Collecting samples from test subjects and collecting control samples;

[0025] (2) detecting and comparing the expression levels of IL-17RD in the test subject samples and control samples;

[0026] If the expression level of IL-17RD in the sample of the subject to be tested is compared with the expression level of IL-17RD in the control sample, IL-17RD in peripheral blood macrophages is significantly upregulated, sIL-17RD in serum is significantly increased, and sIL-17RD in serum is significantly downregulated after drug treatment, then the subject to be tested is diagnosed as having rheumatoid arthritis or being at risk of having rheumatoid arthritis; if the expression level of IL-17RD in the sample of the subject to be tested is compared with the expression level of IL-17RD in the control sample, IL-17RD in cartilage tissue is significantly upregulated, sIL-17RD in serum is unchanged, and sIL-17RD in synovial fluid is significantly increased with increasing KL stage, then the subject to be tested is diagnosed as having knee osteoarthritis or being at risk of having knee osteoarthritis.

[0027] Furthermore, in step (1), collecting samples from the test subjects and collecting control samples, the specific operations include:

[0028] (a) Centrifuge peripheral blood / synovial fluid to obtain; or

[0029] (b) peripheral blood / synovial fluid is collected and subjected to density gradient centrifugation, magnetic bead-based separation, and immunoseparation technology to prepare the product.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] The present invention proposes for the first time that the expression level of sIL-17RD changes significantly (high expression) in the serum of peripheral blood of RA patients, and the expression level of sIL-17RD does not change significantly in the serum of peripheral blood of KOA patients; the expression of IL-17RD in RA blood macrophages is significantly upregulated, and the sIL-17RD in KOA synovial fluid is significantly upregulated. The molecular marker can be used for the diagnosis of RA and KOA, has strong specificity and high sensitivity, can conveniently and quickly obtain test samples, and 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; in addition, the rheumatoid arthritis and knee osteoarthritis biomarkers 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 sIL-17RD 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

[0032] Figure 1 Quantitative analysis of IL-17RD gene expression in serum, peripheral blood, and monocyte-induced macrophages from healthy controls and RA patients.

[0033] Figure 2 This is a correlation analysis chart between the clinical diagnostic indicators of RA patients and the sIL-17RD content in serum.

[0034] Figure 3 This is an analysis chart of the expression of IL-17RD in the cartilage of patients with knee osteoarthritis.

[0035] Figure 4 This is an analysis chart of sIL-17RD levels in serum and synovial fluid of healthy people and patients with knee osteoarthritis.

[0036] Figure 5 This is a correlation analysis chart between the clinical diagnostic indicators of KOA patients and the levels of sIL-17RD and TNF-α in the synovial fluid. DETAILED DESCRIPTION

[0037] 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.

[0038] Example 1

[0039] This example introduces the application of a biomarker for rheumatoid arthritis.

[0040] The first biomarker introduced in this example is the expression of soluble interleukin 17 receptor D (sIL-17RD) in serum.

[0041] 1. Sample collection

[0042] Thirty patients with rheumatoid arthritis admitted to the First Affiliated Hospital of Anhui Medical University (North District) were collected. All enrolled cases were diagnosed as RA patients by clinical and related rheumatoid tests, aged 23 to 79 years old. At the same time, 43 healthy volunteers were recruited as the control group, aged 19 to 55 years old. The diagnosis of all patients in the case group met the 1987 RA classification diagnostic criteria of the American College of Rheumatology, and the control group was healthy subjects who underwent physical examinations during the same period. The research subjects were all unrelated Han people. This study has been approved by the Ethics Committee of Anhui Medical University, with approval number 20200486. All patients and healthy volunteers have obtained informed consent.

[0043] 2. Serum extraction

[0044] 4 mL of peripheral blood was collected from 30 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 light 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.

[0045] 3. Index detection

[0046] In this example, the conventional double antibody sandwich method (sandwich ELISA) was used to detect the content of sIL-17RD in the sample.

[0047] (a) On the first day, the capture antibody (IL-17RD 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.

[0048] (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.

[0049] (c) Add 300 μL of blocking solution to each well and incubate at room temperature for 1 hour.

[0050] (d) Repeat the wash in step (b) and prepare to add the sample when completed.

[0051] (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.

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

[0053] (g) 100 μL of 0.5 μg / mL horseradish peroxidase (HRP)-conjugated IL-17RD polyclonal antibody was added to each well, the plate was sealed with a sealing film, and incubated at room temperature for 1 hour.

[0054] (h) Repeat the wash in (b).

[0055] (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.

[0056] (k) Add 50 μL of stop solution (2M H2SO4) to each well and tap gently to ensure thorough mixing.

[0057] (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.

[0058] 4. Concentration calculation

[0059] 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 methods, computer-based curve fitting statistical software can also be used to calculate the concentration of the sample. We used ELISA to analyze the content of sIL-17RD in the serum of healthy controls (Control, 43 cases) and RA patients (30 cases), as well as quantitative analysis of the gene expression of IL-17RD during in vitro induction of macrophage differentiation of peripheral blood monocytes from healthy controls and patients. The results are shown in Figure 2. Figure 1 As shown. Figure 1 A shows that the sIL-17RD content in the serum of the RA group was significantly higher than that in the healthy control group; in this example, we also collected serum from patients treated with existing clinical drugs, such as Figure 1 As shown in B, Figure 1 B shows that the serum sIL-17RD level of patients with rheumatoid arthritis decreased significantly after receiving adalimumab, prednisone, and leflunomide treatment. Figure 1 C shows that the gene expression level of IL-17RD is upregulated during the differentiation of monocyte-derived macrophages. Figure 1 D shows that the gene expression level of IL-17RD is upregulated in monocyte-derived macrophages of patients with rheumatoid arthritis.

[0060] In summary, the level of sIL-17RD in the serum of rheumatoid patients increased significantly and decreased with treatment.

[0061] This example then introduces the IL-17RD gene expression of the above-mentioned rheumatoid arthritis biomarker sIL-17RD in monocyte-derived macrophages.

[0062] Previous literature has shown that the activation of myeloid cells, especially macrophages, in arthritis patients is the core of the persistence 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 disease process will lead to increased bone erosion, thereby causing irreversible damage to the joints. In order to further illustrate the relationship between sIL-17RD and macrophages, we induced healthy human PBMC (peripheral blood mononuclear cells) to differentiate into macrophages and then detected the gene expression of IL-17RD. The steps are as follows.

[0063] 1. PBMC extraction: Ficoll density gradient centrifugation was used to separate and purify peripheral blood mononuclear cells.

[0064] (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.

[0065] (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.

[0066] (c) Centrifuge at 900 g in a swinging rotor for 30 min at room temperature.

[0067] (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.

[0068] (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.

[0069] (f) Discard the supernatant, add 10 mL of PBS to resuspend the cells, and centrifuge at 250 g for 10 min to obtain PBMCs.

[0070] 2. PBMC induction: Use 40ng / mL M-CSF to induce monocytes to differentiate into macrophages. Resuspend the cells obtained in step 1 with α-MEM 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.

[0071] 3. Gene expression detection: Trizol was used to extract RNA from uninduced PBMC cells and induced macrophages, and QPCR was used to detect IL-17RD gene expression. This experiment used 2-ΔΔct value for relative quantification to analyze the relative fold change of target gene expression between the experimental group and the normal control group. Figure 1 As shown in C, the expression of IL-17RD increased after induction into macrophages. In addition, up-regulation of IL-17RD expression was also detected in macrophages derived from peripheral blood monocytes of healthy controls and patients with rheumatoid arthritis.

[0072] In conclusion, IL-17RD gene expression is upregulated in monocyte-derived macrophages from patients with rheumatoid arthritis.

[0073] Example 2

[0074] This example introduces the correlation analysis between the above biomarker sIL-17RD and clinical indicators of rheumatoid arthritis.

[0075] In order to find more evidence to prove that sIL-17RD is a potential marker for the diagnosis of rheumatoid arthritis, we conducted a correlation analysis between the sIL-17RD 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 sIL-17RD content and the clinical indicators, and the steps are as follows:

[0076] (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.

[0077] (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.

[0078] (c) Correlation analysis: Click the Analysis button on the toolbar to enter the Analyze Data interface. Select the Correlation option in XY analyses. According to the normality test results in step (b), if both variables follow a 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 meet the normal distribution, the Spearman correlation analysis is required. Keep the default options for other parameter settings and click OK.

[0079] (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.

[0080] The results of correlation analysis between clinical diagnostic indicators of RA patients and serum sIL-17RD levels are as follows Figure 2 As shown. Among them, Figure 2 A is the result of correlation analysis between sIL-17RD and TNF-α; Figure 2 B is the result of correlation analysis between sIL-17RD and RF; Figure 2 C is the result of correlation analysis between sIL-17RD and DAS-28; Figure 2 D is the result of correlation analysis between sIL-17RD and CRP; Figure 2E is the result of correlation analysis between sIL-17RD and ESR; Figure 2 F is the result of correlation analysis between sIL-17RD and CCP; it can be seen that the sIL-17RD content in the serum of patients with rheumatoid arthritis is significantly correlated with DAS28 and RF indicators.

[0081] 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.

[0082] In summary: The content of sIL-17RD in patient serum is significantly correlated with three important indicators in the clinical diagnosis of rheumatoid arthritis (DAS28, RF and CRP), which further demonstrates that sIL-17RD is a potential marker for the diagnosis of rheumatoid arthritis.

[0083] Example 3

[0084] This example introduces the expression of the above biomarker sIL-17RD in articular cartilage of patients with osteoarthritis.

[0085] 1. Sample collection

[0086] Patients with normal knee joints (amputation patients) and 3 cases of knee osteoarthritis of different grades admitted to the First Affiliated Hospital of Anhui Medical University (North District) were collected. All included cases were diagnosed with OA by clinical and related knee bone examinations, and their ages ranged from (23 to 79) years old. The diagnosis of all case groups was based on the diagnostic criteria of the "Guidelines for the Diagnosis and Treatment of Osteoarthritis" of the Chinese Medical Association, and the research subjects were all unrelated Han people.

[0087] 2. Cartilage protein extraction

[0088] Weigh about 0.5g of cartilage into a grinding tube, add 1mL RIPA lysis buffer, grind thoroughly and pour into a 1.5mL EP tube. After 40% power ultrasound (10s working, 20s pause, cycle 3 times), let stand on ice for 20 minutes, centrifuge at 4℃, 12000rpm for 20 minutes, and collect the supernatant into a new EP tube.

[0089] 3. Index detection

[0090] In this example, the Western blot method was used to detect the expression of IL-17RD in mouse cartilage (3F12 antibody was used to detect the extracellular domain, and ICD antibody was used to detect the intracellular domain).

[0091] (a) Prepare the separation gel and stacking gel according to the purchased Western blot kit

[0092] (b) Assemble the electrophoresis mold, pour in the electrophoresis solution and load the sample, first 80V for 30 minutes, wait for the sample to run out of the concentrated gel, then 120V for 1 hour to end the electrophoresis

[0093] (c) Assemble the mold in the order of black gel and white film, pour in the transfer solution, 300mA, transfer in an ice bath for 2 hours

[0094] (d) After the transfer, the NC membrane was removed and placed in 5% skim milk powder (1 g skim milk powder + 20 mL 1×TBST) and blocked at room temperature for 1 h.

[0095] (e) Dilute the antibody according to the recommended ratio and incubate at 4°C overnight (antibody dilution ratio: NG2, 1:1000; MMP13, 1:1000; Actin, 1:5000)

[0096] (f) On the next day, the NC membrane was removed and washed three times with 1× TBST, 5 min each time.

[0097] (g) Incubate with HRP-conjugated secondary antibodies of different species at room temperature for 1 hour

[0098] (h) Wash the membrane 3 times with 1×TBST, 5 min / time, and then develop and count

[0099] Western blot results and IL-17RD immunostaining results showed that IL-17RD was expressed in cartilage of patients with knee osteoarthritis. Figure 3 As shown. Among them, Figure 3 A and Figure 3 C is the expression of IL-17RD in cartilage of healthy subjects and patients with different grades of knee osteoarthritis (3F12 antibody detects the extracellular domain of IL-17RD, and ICD antibody detects the intracellular domain of IL-17RD); Figure 3 B and Figure 3 D is the quantitative analysis of the first two respectively; Figure 3 E is the ICD fluorescence staining of articular cartilage in healthy people and patients with knee osteoarthritis; Figure 3 F is Figure 3 Quantitative statistical results in E. It can be seen that the expression of IL-17RD is upregulated in the articular cartilage of patients with knee osteoarthritis.

[0100] Example 4

[0101] This example describes the expression of sIL-17RD in the serum and synovial fluid of patients with knee osteoarthritis.

[0102] 1. Sample collection

[0103] Ten healthy subjects and 40 patients with knee osteoarthritis (KOA) admitted to the First Affiliated Hospital of Anhui Medical University (North District) were collected. Among them, there were 8 males and 2 females in the 10 normal subjects, with an average age of 42.20 years old, and none of them had bad habits; among the 40 KOA patients, there were 12 males and 28 females. All enrolled cases were diagnosed with KOA by clinical manifestations and related bone and joint imaging examinations, and their ages were (50-83) years old. The diagnosis of all case groups was based on the diagnostic criteria of the "Guidelines for the Diagnosis and Treatment of Osteoarthritis" of the Chinese Medical Association, and the research subjects were all unrelated Han people. This study has been approved by the Ethics Committee of the First Affiliated Hospital of Anhui Medical University (North District), with the approval number PJ-YX2024-022. All patients and healthy volunteers have obtained informed consent.

[0104] 2. Extraction of synovial fluid and blood The synovial fluid of 10 healthy people and 40 OA patients was collected by syringe extraction. This operation was performed before the OA patients underwent joint replacement surgery. After the patients were anesthetized, 2-3 mL of blood / synovial fluid was first extracted and then injected into a clean blood collection tube. Within 30 minutes after the specimen was collected, it was centrifuged at 4°C and 3000g for 20 minutes. The upper light yellow clear liquid after centrifugation is the serum / synovial fluid. 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 the synovial fluid and dispense it into sterile EP tubes and store it at -80°C. Repeated freezing and thawing should be avoided.

[0105] 3. Index detection

[0106] In this example, ELISA was used to detect the content of sIL-17RD in serum or synovial fluid, and the specific scheme was the same as that in Example 1.

[0107] Analysis of sIL-17RD levels in serum and synovial fluid of healthy subjects and patients with knee osteoarthritis Figure 4 As shown. Among them, Figure 4 A is the sIL-17RD content in the serum of healthy controls and KOA patients; Figure 4 B is the content of sIL-17RD in the synovial fluid of patients with different degrees of knee osteoarthritis; Figure 4 C CT images of knee joints corresponding to different KL grades in patients with knee osteoarthritis; Figure 4 D is the content of sIL-17RD in the synovial fluid of osteoarthritis patients with different KL grades. Figure 4 A- Figure 4 D shows that in the serum of patients with knee osteoarthritis, the sIL-17RD content has no significant change compared with normal people, while the sIL-17RD content in their synovial fluid is increased and increases with the increase of KL grade of knee osteoarthritis.

[0108] Example 5

[0109] This example introduces the correlation between the sIL-17RD content in the synovial fluid of patients with osteoarthritis and the TNF-α content and the minimum joint gap. This example is based on computer software (GraphPadPrism8) to calculate the correlation analysis. The specific implementation plan is the same as that in Example 1.

[0110] Correlation analysis between clinical diagnostic indicators of KOA patients and the levels of sIL-17RD and TNF-α in synovial fluid Figure 5 As shown. Among them, Figure 5 A is the correlation analysis between sIL-17RD and the minimum joint space, which shows that: sIL-17RD in the synovial fluid of patients with knee joints is negatively correlated with the minimum joint space; Figure 5 B is the quantitative graph of TNF-α in the synovial fluid of patients with different KL grades of knee osteoarthritis. It can be seen that the TNF-α content in the synovial fluid of patients with knee osteoarthritis has nothing to do with the severity of knee osteoarthritis; Figure 5 C is the correlation analysis between sIL-17RD and TNF-α content, which shows that the sIL-17RD content in the synovial fluid of patients with knee osteoarthritis is positively correlated with the TNF-α content; Figure 5 D is the correlation analysis between TNF-α and minimum joint space, which shows that there is no correlation between TNF-α content and minimum joint space.

[0111] In summary, sIL-17RD is expressed in the synovial fluid of patients with knee osteoarthritis and is positively correlated with the severity of osteoarthritis. Compared with TNF-α, it can be used as a better diagnosis of osteoarthritis.

[0112] In summary, the present invention proposes for the first time that the expression level of sIL-17RD in the peripheral blood serum of patients with rheumatoid arthritis changes significantly (high expression), and this molecular marker can be used for the diagnosis of rheumatoid arthritis, can provide data support for the diagnosis of the treatment results of rheumatoid arthritis, and provide a new strategy for the diagnosis of rheumatoid arthritis in the clinic; the present invention proposes for the first time that the expression level of sIL-17RD in the synovial fluid of patients with knee osteoarthritis changes significantly (high expression), but in the peripheral blood serum, the expression level of sIL-17RD in patients with knee osteoarthritis has no obvious change compared with that of healthy people. Therefore, this molecule can be used to distinguish between rheumatoid arthritis and knee osteoarthritis, can provide data support for the diagnosis of the treatment results of knee osteoarthritis, and provide a new strategy for the diagnosis of knee osteoarthritis in the clinic.

[0113] 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 diagnosing and differentiating rheumatoid arthritis and knee osteoarthritis, characterized in that: The biomarker is interleukin 17 receptor D, namely IL-17RD; including IL-17RD in cells and tissues and soluble IL-17RD in serum and joint fluid, namely sIL-17RD.

2. The biomarker according to claim 1, characterized in that The biomarkers are present in RA serum, RA peripheral blood mononuclear cells, RA macrophages, KOA joint fluid, KOA serum and KOA chondrocytes.

3. A kit for diagnosing and differentiating rheumatoid arthritis and knee osteoarthritis, characterized in that: The kit comprises reagents for detecting IL-17RD expression.

4. The kit according to claim 3, characterized in that The sample to be tested is selected from at least one of serum, peripheral blood monocytes or macrophages, and knee joint fluid.

5. Use of a reagent for detecting the expression of IL-17RD according to claim 1 in the preparation of a tool for diagnosing and differentiating rheumatoid arthritis and knee osteoarthritis.

6. The use according to claim 5, characterized in that The detection of IL-17RD expression includes: determining whether it is rheumatoid arthritis by detecting the serum sIL-17RD level of RA patients and the expression level of IL-17RD in blood macrophages, and predicting the therapeutic efficacy of rheumatoid arthritis by the change in the level of sIL-17RD in the serum of RA patients before and after treatment; and / or determining whether it is knee osteoarthritis by detecting the sIL-17RD level in the serum and synovial fluid of KOA patients and the expression level of IL-17RD in cartilage; wherein the treatment includes treatment with adalimumab, prednisone, and leflunomide.

7. The use according to claim 6, characterized in that The sIL-17RD expression level is significantly positively correlated with clinical indicators, which include: at least one of rheumatoid factor (RF), 28-joint disease activity score (DAS28), C-reactive protein (CRP) and inflammatory factor TNF-α.

8. The use according to claim 7, characterized in that The detection of the biomarker sIL-17RD and / or IL-17RD expression in claim 1 includes: one or more of ELISA method, Western blot method, immunofluorescence method, flow cytometry method, gene chip detection method, and real-time fluorescence quantitative method.

9. The use according to any one of claims 5 to 8, characterized in that: The diagnosis and differentiation of rheumatoid arthritis and knee osteoarthritis includes the following steps: (1) Collecting samples from test subjects and collecting control samples; (2) detecting and comparing the expression levels of IL-17RD in the test subject samples and control samples; If the expression level of IL-17RD in the sample of the subject to be tested is compared with the expression level of IL-17RD in the control sample, IL-17RD in peripheral blood macrophages is significantly upregulated, sIL-17RD in serum is significantly increased, and sIL-17RD in serum is significantly downregulated after drug treatment, then the subject to be tested is diagnosed as having rheumatoid arthritis or being at risk of having rheumatoid arthritis; if the expression level of IL-17RD in the sample of the subject to be tested is compared with the expression level of IL-17RD in the control sample, IL-17RD in cartilage tissue is significantly upregulated, sIL-17RD in serum is unchanged, and sIL-17RD in synovial fluid is significantly increased with increasing KL stage, then the subject to be tested is diagnosed as having knee osteoarthritis or being at risk of having knee osteoarthritis.

10. The use according to claim 9, 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 / knee joint fluid by centrifugation; or (b) obtaining peripheral blood / knee joint fluid by density gradient centrifugation, magnetic bead separation, and immunoseparation technology to obtain.

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