Application of NFATc2 as biomarker to preparation of product for diagnosing, preventing or treating rheumatoid arthritis

Abstract

The invention belongs to the technical field of biology, and particularly relates to application of NFATc2 serving as a biomarker to preparation of a product for diagnosing, preventing or treating rheumatoid arthritis. It is found that compared with normal people, the level of NFATc2 in synovial tissues and synovial fibroblasts of rheumatoid arthritis patients is remarkably increased, and the NFATc2 can be used as a biomarker for diagnosis of rheumatoid arthritis. In addition, the invention finds that NFATc2 has the effect of promoting joint destruction. Therefore, the effect of relieving arthritis can be achieved by inhibiting the level of the NFATc2, and the NFATc2 can be used as a biomarker with the rheumatoid arthritis treatment effect. The invention provides an effective means for diagnosis and treatment of rheumatoid arthritis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to the application of NFATc2 as a biomarker in the preparation of products for diagnosing, preventing or treating rheumatoid arthritis. Background Art

[0002] Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by the production of autoantibodies, synovitis, and bone and cartilage destruction, and is known as the "incurable cancer". Early manifestations include migratory joint pain and dysfunction, and joint stiffness and severe deformity often occur in the late stage, with a high disability rate, seriously endangering the health of the general public. "Early detection, early diagnosis, and early treatment" are the main principles for controlling the progression of RA. Therefore, early and accurate diagnosis of RA is of great significance for patients to receive early intervention and delay the disease progression.

[0003] Currently, the clinical diagnosis of RA mainly relies on joint involvement, symptom duration, acute-phase reactants, and serum indicators such as serum rheumatoid factor (RF) and anti-citrullinated protein antibody (ACPA). However, due to the atypical and diverse early clinical symptoms of RA, joint involvement often occurs in the late stage, and combined with the non-specificity of acute-phase reactants, serological indicators are particularly important for the early and accurate diagnosis of RA. However, both RF and ACPA recommended by the current guidelines have certain limitations. RF and ACPA can be detected not only in RA patients but also widely in patients with autoimmune diseases such as osteoarthritis (OA) and systemic lupus erythematosus (SLE). Therefore, their sensitivity or specificity is relatively low, 69%, 85% and 67%, 95% respectively. The sensitivity and specificity of the combined diagnosis of RA are only 78% and 82%, respectively, and the misdiagnosis rate is as high as 19%. In addition, their performance in early RA is also unsatisfactory, with a positive rate of only 57%. Therefore, there is an urgent need in this field to find diagnostic indicators that can accurately diagnose RA patients early and are simple and convenient to operate. This research is of great significance for early detection, early diagnosis, and early treatment of RA patients, thereby slowing down the disease process, controlling clinical symptoms, and improving the quality of life. Summary of the Invention

[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and provide the application of NFATc2 as a biomarker in the preparation of products for diagnosing, preventing or treating rheumatoid arthritis.

[0005] In the first aspect provided by the present invention, there is provided the application of NFATc2 as a biomarker in the preparation of products for diagnosing, preventing or treating rheumatoid arthritis.

[0006] Among them, the products for diagnosing rheumatoid arthritis include detection reagents for detecting the expression of NFATc2 in biological samples.

[0007] The biological samples include synovial tissue and / or synovial fibroblasts.

[0008] The expression level of NFATc2 in the synovial tissue and / or synovial fibroblasts of patients with rheumatoid arthritis is significantly different from that of normal people. Specifically, the expression level of NFATc2 in synovial fibroblasts of synovial tissue in patients with rheumatoid arthritis is significantly higher than that of normal people.

[0009] Among them, the products for treating or preventing rheumatoid arthritis include drugs that inhibit the expression of NFATc2.

[0010] In a second aspect of the present invention, a diagnostic tool for rheumatoid arthritis is provided, including an NFATc2 detection reagent, which detects the expression level of NFATc2, and is an NFATc2 nucleic acid quantitative detection reagent or an NFATc2 protein quantitative detection reagent, and the NFATc2 protein is obtained by encoding from the NFATc2 gene.

[0011] The NFATc2 gene nucleic acid quantitative detection reagent includes at least one of a specific primer for the NFATc2 gene or transcript and a specific recognition probe for the NFATc2 gene or transcript; the NFATc2 protein quantitative detection reagent includes an antibody or antibody fragment that specifically binds to the NFATc2 protein.

[0012] That is, the diagnostic tool for rheumatoid arthritis of the present invention can specifically directly detect the NFATc2 nucleic acid level, such as Northern blot, real-time fluorescence quantitative PCR (qPCR), gene chip, etc., and can also quantitatively detect the protein encoded by the NFATc2 gene, such as Western blot, Immunohistochemistry, Immunofluorescence, immunochromatography, enzyme-linked immunosorbent assay (ELISA), electrochemiluminescence detection (ECL), etc. Therefore, the diagnostic tool for rheumatoid arthritis of the present invention includes at least detection products based on the above methods, and its type can be a kit, a chip, a test strip, a high-throughput sequencing platform. Among them, the kit also includes an instruction manual for diagnosing or predicting early rheumatoid arthritis.

[0013] In a third aspect of the present invention, there is provided a product for preventing or treating rheumatoid arthritis, comprising an NFATc2 inhibitor. The NFATc2 inhibitor can reduce the expression of the NFATc2 gene. The NFATc2 inhibitor can be a chemical reagent or a biological agent, such as a monoclonal antibody or a polyclonal antibody that specifically recognizes NFATc2, a small interfering RNA (siRNA) or a short hairpin RNA (shRNA) against the NFATc2 gene.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0015] The present invention discloses a biomarker for the diagnosis, prevention and treatment of rheumatoid arthritis, and the biomarker is NFATc2. The present invention finds that the levels of NFATc2 in synovial tissues and synovial fibroblasts of patients with rheumatoid arthritis are significantly different from those of normal people, and can be used as a biomarker for the diagnosis of rheumatoid arthritis. The present invention also discloses the application of the anti-NFATc2 autoantibody, a diagnostic biomarker for rheumatoid arthritis, including its application in the preparation of detection reagents for diagnosing rheumatoid arthritis and in the preparation of drugs for targeted treatment of rheumatoid arthritis. In addition, the present invention finds that NFATc2 has a promoting effect on joint destruction. Therefore, by inhibiting the level of NFATc2, the effect of relieving arthritis can be achieved, and it can be used as a biomarker with a therapeutic effect on rheumatoid arthritis. In summary, the present invention provides an effective means for the diagnosis and treatment of rheumatoid arthritis. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, obtaining other drawings without creative efforts still belongs to the scope of the present invention.

[0017] Figure 1 For RT-qPCR detection of the anti-NFATc2 level in synovial fibroblasts of patients with rheumatoid arthritis and healthy individuals;

[0018] Figure 2 For Western blot detection of the anti-NFATc2 level in synovial fibroblasts of patients with rheumatoid arthritis and healthy individuals;

[0019] Figure 3 For immunofluorescence detection of the NFATc2 level in synovial fibroblasts of patients with rheumatoid arthritis and healthy individuals;

[0020] Figure 4To detect the level of NFATc2 in synovial tissues of patients with rheumatoid arthritis and healthy individuals by immunohistochemistry;

[0021] Figure 5 To detect the level of NFATc2 after knockdown by shRNA using Western blot;

[0022] Figure 6 To detect the migratory and invasive abilities of synovial fibroblasts after reducing the level of NFATc2 by Transwell assay;

[0023] Figure 7 To detect the migratory ability of synovial fibroblasts after reducing the level of NFATc2 by scratch assay;

[0024] Figure 8 To measure the degree of joint swelling in arthritis mice after reducing the level of NFATc2;

[0025] Figure 9 To measure the joint score in arthritis mice after reducing the level of NFATc2;

[0026] Figure 10 To detect joint destruction in arthritis mice after reducing the level of NFATc2 by H&E staining and safranin-O-fast green staining. Detailed implementation manners

[0027] To make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings.

[0028] Example 1 Detection of the level of NFATc2 in synovial fibroblasts of patients with rheumatoid arthritis:

[0029] The synovium is the main pathological site in patients with rheumatoid arthritis, which is composed of synovial fibroblasts (Fibroblast-like synoviocytes, FLS). Their excessive proliferation causes the formation of invasive pannus in the synovium, leading to cartilage destruction, which is an important cause of joint destruction in patients. Synovial tissues were collected from 10 patients with rheumatoid arthritis and 10 healthy volunteers. The inclusion criteria for patients with rheumatoid arthritis met the 2010 ACR / EULAR classification criteria for rheumatoid arthritis. This study was approved by the Ethics Committee of Wenzhou Medical University, and each patient was informed of the use of the samples and signed an informed consent form.

[0030] I. To detect the level of NFATc2 in synovial fibroblasts by RT-qPCR, the specific process is as follows:

[0031] 1. Isolation and culture of primary synovial FLS:

[0032] 1) Turn on the ultraviolet lamp in the cell culture hood for 30 minutes for disinfection and sterilization.

[0033] 2) Take out the fresh synovial tissue from the sterile surgical bag and place it in a sterile petri dish.

[0034] 3) Add sterile PBS buffer to the sterile petri dish, wash the synovial tissue to remove blood, etc., and repeat this step 2 - 3 times.

[0035] 4) Use a pipette to suck out the excess PBS, and use sterile ophthalmic scissors to cut the synovial tissue into small pieces as small as possible. Transfer it to a 50 mL centrifuge tube when it appears paste - like or in a mass.

[0036] 5) Centrifuge at 350 g for 5 minutes to remove the upper - layer adipose tissue. Repeat step 4 until there is almost no adipose tissue and the synovial tissue is small enough.

[0037] 6) Prepare the digestion solution, add 10 - 15 mL to the centrifuge tube, and then add a 1:100 penicillin - streptomycin mixture and type II collagenase at a concentration of 0.4%.

[0038] 7) Add the digestion solution to the precipitated tissue after the last centrifugation, mix well, and place it in an incubator at 37°C to start digestion. Shake the centrifuge tube once every 2 hours during the digestion process, and digest for about 6 - 8 hours.

[0039] 8) Place a 100 - μm cell sieve on a 50 mL centrifuge tube, filter the digested digestion solution, and centrifuge the filtrate at 350 g for 5 minutes.

[0040] 9) Discard the supernatant, resuspend the cells with DMEM medium containing 10% fetal bovine serum, and transfer them to a cell culture flask for culture.

[0041] 10) After culturing for one day, FLS will compete with impurity cells for adhesion. The impurity cells will float, and the impurity cells can be removed by changing the medium. The adherent cells are FLS.

[0042] 2. RNA extraction:

[0043] 1) Collect FLS cells, add 1 mL of Trizol to the cell pellet, and pipette to mix well.

[0044] 2) Place it on ice for 20 minutes, add 200 μL of chloroform, cover the lid and shake vigorously for 1 minute, then place it on ice for 15 minutes.

[0045] 3) Pre - cool the centrifuge to 4°C in advance and centrifuge at 12000 rpm for 20 minutes.

[0046] 4) After centrifugation, the mixture is divided into upper and lower phases. The upper layer is a colorless aqueous phase containing RNA, and the lower layer is an organic phase. Carefully take out the EP tube, and use a 200 - μL pipette to carefully aspirate the upper aqueous phase into another enzyme - free EP tube.

[0047] 5) Add an equal amount of isopropanol solution, invert the tube up and down to mix well, and let it stand overnight at -20°C.

[0048] 6) The next day, pre-cool the centrifuge to 4°C in advance and centrifuge at 12,000 rpm for 20 minutes.

[0049] 7) Discard the supernatant, add 1 mL of 75% anhydrous ethanol (prepared with DEPC water) to the precipitate, invert and mix to wash the precipitate, and centrifuge at 8,000 rpm for 5 minutes. Repeat the above steps once.

[0050] 8) Use a 10 μL pipette to carefully aspirate the residual ethanol in the EP tube, invert the EP tube on the filter paper, and air-dry it at room temperature.

[0051] 9) After the sample is air-dried, add 35 μL of DEPC water to dissolve the RNA.

[0052] 10) Concentration detection: Use Nanodrop to detect the concentration and purity of the extracted RNA.

[0053] 11) RNA integrity detection: Prepare a 1% agarose gel, aspirate 1 μL of RNA for electrophoresis, at 100 V for 15 minutes. Place the gel on a UV transilluminator to observe the RNA electrophoresis results: more 28s than 18s, and almost no visible 5s indicates good RNA integrity and no degradation.

[0054] 3. Reverse transcription:

[0055] 1) Remove genomic DNA: Prepare the following mixture in an RNase-free centrifuge tube

[0056]

[0057] Gently pipette the system to mix well, and react at 42°C on a PCR for 2 minutes.

[0058] 2) Reverse transcription reaction: Directly add 5×HiScript III qRT SuperMix to the reaction tube in step 1

[0059]

[0060] Gently pipette the system to mix well, react at 37°C on a PCR instrument for 15 minutes and then at 85°C for 5 seconds. The product can be immediately used for qPCR reaction or stored at -20°C.

[0061] 4. Fluorescent quantitative PCR

[0062] 1) Primer design and synthesis: The primers were designed by oneself using primer5 software and synthesized by Beijing Qingke Gene Technology Co., Ltd. The sequences are as follows

[0063]

[0064] 2) qPCR system:

[0065]

[0066]

[0067] Mix the system gently by pipetting in the dark, and perform reaction quantification on a fluorescence quantitative PCR instrument. Conditions: pre-denaturation at 95°C for 30 seconds, denaturation at 95°C for 10 seconds, annealing and extension at 60°C for 30 seconds, repeat the denaturation-annealing-extension cycle 35 times, and use the instrument's default melting curve acquisition program.

[0068] The results are as Figure 1 shown, the level of NFATc2 in synovial fibroblasts of patients with rheumatoid arthritis is significantly higher than that of healthy people.

[0069] II. Detect the level of NFATc2 in synovial fibroblasts by Western blot. The specific process is as follows:

[0070] 1) Protein extraction: Wash the intervened FLS cells twice with PBS, then add 200 μL of RIPA protein lysate and pipette gently to mix. Lyse on ice for 30 minutes, centrifuge at 4°C and 12,000 g for 20 minutes, and take the supernatant for long-term storage at -80°C.

[0071] 2) Protein quantification: Dilute the 2 mg / mL BSA standard with PBS to a final concentration of 0.5 mg / mL; add the standard to 96-well plates in volumes of 0 μL, 1 μL, 2 μL, 4 μL, 8 μL, 12 μL, 16 μL, and 20 μL in sequence, and make up to 20 μL with PBS in each well; add 19 μL of PBS and 1 μL of protein sample to the sample wells; mix solution A and solution B at a ratio of 1:50, add 200 μL to each well, and react in an incubator at 37°C for 30 minutes. Measure the OD value at 562 nm with an enzyme-linked immunosorbent assay (ELISA) reader, and calculate the protein concentration in the sample according to the standard curve.

[0072] 3) Protein denaturation: Determine the loading volume according to the protein concentration calculated above, dilute all samples with PBS to the same concentration to ensure the same loading volume. Mix the diluted protein sample with 5× Loading Buffer at a volume ratio of 4:1, seal with a sealing film, and heat in a metal bath at 100°C for 10 minutes.

[0073] 4) Electrophoresis: Add the calculated protein and marker to the loading wells, perform electrophoresis at a constant voltage of 80 V for 30 minutes, then change to a constant voltage of 120 V until the bromophenol blue runs to the bottom of the gel, and then stop.

[0074] 5) Transfer membrane: Cut the gel strip of the target size, cover it with the PVDF membrane activated by methanol, and place it in the transfer apparatus in the order of filter paper - gel - PVDF membrane - filter paper. Put the transfer apparatus into a box filled with crushed ice and transfer the membrane at a constant current of 250 mA.

[0075] 6) Blocking: Put the PVDF membrane into the 5% skim milk blocking solution and block it on a shaker at room temperature for 1 hour.

[0076] 7) Primary antibody incubation: Wash the blocked PVDF membrane to remove the blocking solution, transfer it to the antibody dilution solution containing the primary antibody, and incubate it on a shaker at 4°C overnight (12 - 16 hours).

[0077] 8) Washing: The next day, transfer the incubated PVDF membrane to TBST and wash it on a shaker at room temperature for 10 minutes each time, for a total of three times.

[0078] 9) Secondary antibody incubation: Prepare the corresponding secondary antibody, transfer the washed PVDF membrane to the antibody dilution solution containing the secondary antibody, and incubate it on a shaker at room temperature for 1 hour.

[0079] 10) Washing: Transfer the incubated PVDF membrane to TBST and wash it on a shaker at room temperature for 10 minutes each time, for a total of three times.

[0080] 11) Exposure: Prepare the exposure solution and perform exposure imaging on the exposure instrument.

[0081] 12) Gray scale analysis: Use ImageJ software to perform gray scale analysis on the image.

[0082] The results are as Figure 2 shown. The level of NFATc2 in synovial fibroblasts of rheumatoid arthritis patients is significantly higher than that of normal people.

[0083] III. Detect the expression level of NFATc2 in synovial fibroblasts by immunofluorescence experiment

[0084] 1) Preparation of cell slides: Count the digested cells, then place the sterile cell slides into the cell well plate, inoculate the counted cells into the cell well plate, and culture them.

[0085] 2) Fixation: Take out the cell well plate, aspirate all the culture medium, wash it once with PBS, aspirate all the PBS, and add 4% paraformaldehyde to fix for 20 minutes.

[0086] 3) Washing: Aspirate all the fixing solution, add an appropriate amount of PBS, and wash it on a shaker for 5 minutes, for a total of three times.

[0087] 4) Membrane permeabilization: Prepare 0.1% TritonX - 100 as the membrane permeabilization solution, add it to the well plate, permeabilize for 10 minutes, and then wash it three times with PBS for 5 minutes each time.

[0088] 5) Blocking: Add 5% BSA and block for 30 minutes.

[0089] 6) Primary antibody incubation: Add the primary antibody prepared with the blocking solution and incubate overnight in a humidified chamber at 4°C.

[0090] 7) Rewarming: Take out the humidified chamber and restore to room temperature.

[0091] 8) Washing: Wash three times on a shaker with PBST for 5 minutes each time.

[0092] 9) Secondary antibody incubation: Add the fluorescent secondary antibody prepared with the blocking solution and incubate for 1 h in the dark in a humidified chamber at room temperature.

[0093] 10) Washing: Wash three times on a shaker in the dark with PBST for 5 minutes each time.

[0094] 11) Nuclear counterstaining and mounting: Drop the mounting medium containing DAPI on the glass slide. Carefully pick up the coverslip with forceps, place it cell-side down, and gently cover it. Surround it with neutral resin to fix it and prevent the coverslip from slipping.

[0095] 12) Imaging: Perform fluorescence imaging using a laser confocal microscope.

[0096] The results are as Figure 3 shown. The level of NFATc2 in synovial fibroblasts of patients with rheumatoid arthritis is significantly higher than that in normal individuals.

[0097] Example 2 Detection of the level of NFATc2 in synovial tissues of patients with rheumatoid arthritis:

[0098] Detect the expression level of NFATc2 in synovial tissues by immunohistochemistry. The specific procedure is as follows:

[0099] 1) Deparaffinization and rehydration: Place the synovial tissue sections in fresh xylene for overnight deparaffinization, and then transfer them to 100% alcohol, 95% alcohol, 85% alcohol, 75% alcohol, 50% alcohol, and distilled water for gradient hydration, 5 minutes for each step.

[0100] 2) Antigen retrieval: Use the microwave retrieval method. Immerse the sections in 0.01 M sodium citrate buffer at pH 6.0, heat in a microwave oven at medium-high power for 8 minutes. Stop heating when uniform small bubbles appear on the surface of the sections and maintain the temperature for 5 minutes. Take out the beaker and let it cool naturally to room temperature.

[0101] 3) Removal of endogenous peroxidase: Drop 15 μL of 3% H 2 0 2 onto the tissue on the sections and incubate in the dark at room temperature for 10 minutes.

[0102] 4) Blocking: Wash three times with PBST for 5 minutes each time, dry the moisture, and add 15 μL of 5% BSA to the tissue on the section and block at room temperature for 1 hour.

[0103] 5) Primary antibody incubation: Dry the blocking solution, and add 15 μL of the diluted primary antibody to the tissue on the section. Incubate overnight in a wet box at 4°C.

[0104] 6) Rewarming: The next day, take out the wet box and restore to room temperature for 30 minutes.

[0105] 7) Washing: Wash three times with PBST for 5 minutes each time.

[0106] 8) Secondary antibody incubation: Dry the moisture, add 15 μL of the diluted secondary antibody to the tissue on the section, and incubate in the dark at room temperature for 30 minutes.

[0107] 9) Washing: Wash three times with PBST for 5 minutes each time.

[0108] 10) Color development: Dry the moisture, add 15 μL of DAB color development solution (prepared freshly) to the tissue on the section, and react in the dark for 10 minutes.

[0109] 11) Counterstaining: Dry the moisture, add 15 μL of hematoxylin solution to the tissue on the section and stain for 5 - 10 minutes.

[0110] 12) Differentiation and blueing: Wash three times with PBST for 5 minutes each time, add 1% hydrochloric acid ethanol to the tissue for 2 - 3 seconds, and then quickly rinse with running water for 15 minutes.

[0111] 13) Dehydration and mounting: Subsequently, transfer to 50% alcohol, 75% alcohol, 85% alcohol, 95% alcohol, 100% alcohol for gradient dehydration, 5 minutes for each step. Dry the section and mount it with neutral balsam, cover with a coverslip, avoiding air bubbles.

[0112] 14) Photographing: Use an upright microscope for photographing.

[0113] The results are as Figure 4 shown, the level of NFATc2 in the synovial tissue of rheumatoid arthritis patients is significantly higher than that of normal people.

[0114] Example 3 Effect of NFATc2 on the migration and invasion ability of synovial fibroblasts:

[0115] 1. Cell transfection

[0116] The NFATc2 - knockdown shRNA plasmid was designed and constructed by Beijing Tsingke Biotechnology Co., Ltd.

[0117] 1) Digest RA - FLS cells in the logarithmic growth phase, inoculate them in a 6 - well plate, and after they adhere and grow to 90% - 95%, perform transfection.

[0118] 2) Dilute solution B with Opti-MEM medium and mix gently.

[0119] 3) Use Opti-MEM medium to dilute the plasmid DNA to obtain a DNA premix, then add solution A and gently mix to obtain diluted DNA.

[0120] 4) Add the diluted DNA to the diluted solution B (1:1 ratio) and incubate at room temperature for 5 minutes.

[0121] 5) Add the DNA-liposome complex dropwise to the cells and mix gently.

[0122] 6) 37°C, 5% CO 2 The cells were cultured in an incubator for 48 hours, and the knockdown efficiency was detected by WB.

[0123] The results are as follows Figure 5 As shown, shNFATc2-3 could significantly reduce the expression of NFATc2.

[0124] 2. Transwell invasion assay

[0125] 1) The night before the experiment, take out the Matrigel matrix gel from -80℃ and freeze and thaw at 4℃. Pre-cool the sterile pipette tips, transwell chambers, cell well plates and ice boxes in advance.

[0126] 2) Place the transwell chamber in a 24-well plate, mix the matrix gel and serum-free culture medium at a ratio of 1:8, spread 60 μL of the diluted matrix gel in the transwell chamber, and place the plate in a cell culture incubator for 3 hours to allow it to solidify.

[0127] 3) The RA-FLS from different treatment groups were digested and counted and inoculated into transwell chambers (5×10 4 cells / well).

[0128] 4) Add 500 μL of DMEM medium containing 10% FBS to the lower chamber plate and incubate at 37°C with 5% CO 2 Incubate for 24 h.

[0129] 5) Take out the transwell chamber, absorb the culture medium, and gently wipe off the remaining cells on the upper layer of the chamber with a dry cotton swab.

[0130] The sections were fixed with 4% paraformaldehyde for 10 min and washed three times with PBS.

[0131] 6) Then fix with methanol for 30 minutes, wash three times with PBS, stain with 0.1% crystal violet solution for 30 minutes, and wash three times with PBS.

[0132] 7) Photograph under an inverted microscope, and randomly select five fields of view for photographing and cell counting.

[0133] The results are as Figure 6 shown. After the level of NFATc2 in synovial fibroblasts decreases, the number of cell migration and invasion significantly decreases. This indicates that NFATc2 has a promoting effect on the migration and invasion ability of synovial fibroblasts.

[0134] 3. Scratch migration assay

[0135] 1) Seed the RA-FLS of different treatment groups that have been digested and counted into a six-well plate (1×10 5 cells / well).

[0136] 2) When the cells reach 95% confluence, use a 10-μL pipette tip to scratch along the diameter of the well.

[0137] 3) Wash the scratched-off cells 3 times with sterile PBS. After sucking out all the PBS solution, add serum-free DMEM solution.

[0138] 4) Culture in an incubator at 37°C, and take pictures under an inverted microscope at the same field of view at 0 h and 24 h respectively.

[0139] The results are as Figure 7 shown. After the level of NFATc2 in synovial fibroblasts decreases, the cell migration ability significantly weakens. This indicates that NFATc2 has a promoting effect on the migration ability of synovial fibroblasts.

[0140] Example 4 Effect of inhibiting NFATc2 level on arthritis:

[0141] Inhibit the level of NFATc2 in an animal model of arthritis, and observe the disease condition of arthritis and detect inflammatory indicators:

[0142] 1. Construction and sampling of CIA model

[0143] 1) Collagen preparation: Dissolve bovine type II collagen dry powder with 0.05 M acetic acid to prepare a concentration of 4 mg / mL, and fully dissolve it overnight at 4°C.

[0144] 2) Homogenization and emulsification: In a laminar flow hood, add complete Freund's adjuvant and collagen solution to the bottle in a volume ratio of 1:1 with a sterile pipette tip and mix well. Place the bottle on ice, and vertically insert a high-speed homogenizer into the mixture in the bottle. Homogenize at 20,000 rpm for 2 minutes, stop for 5 minutes, and repeat this process two to three times until the emulsified droplets do not disperse in water for a long time, indicating successful emulsification.

[0145] 3) Primary immunization: Anesthetize DBA / 1 mice by intraperitoneal injection of 1% sodium pentobarbital. Aspirate the emulsifier with a 1 mL sterile disposable syringe and insert the needle 3 cm from the base of the mouse tail. Subcutaneously inject 100 μL of the emulsion into each mouse.

[0146] 4) Secondary immunization: On the 21st day after primary immunization, replace the complete Freund's adjuvant with an incomplete Freund's adjuvant mixed and emulsified with collagen (the emulsification procedure is the same as in step 2). After the mice are anesthetized, insert the needle 2 cm from the base of the tail, avoiding the needle holes of the first immunization. Subcutaneously inject 100 μL of the emulsion into each mouse.

[0147] 5) Clinical scoring: Observe the degree of joint swelling in mice within 28 days after primary immunization and count the score every three days. Each limb is scored from 0 to 4 points, with a total of 0 to 16 points. The scoring system is defined as 0 = no signs of erythema and swelling, 1 = erythema and mild swelling limited to the tarsus or ankle joint, 2 = erythema and mild swelling extending from the ankle joint to the tarsus, 3 = erythema and moderate swelling extending from the ankle joint to the metatarsophalangeal joint, 4 = erythema and severe swelling including the ankle joint, foot, toes, or limb ankylosis. Repeated measures analysis of variance (ANOVA) is used to test for significance.

[0148] 6) Intervention: On the day of the second immunization, inject shRNA (slow virus-coated, 2×10 8 TU / mL) into the joints of the mice weekly.

[0149] 7) Photographing of mouse joints: Take pictures of the four limbs of the mice and group and save the records before sacrificing the mice.

[0150] 8) Sampling of joints: Sacrifice the mice, peel off the skin of the mouse legs, separate the mouse legs from the pelvis along the greater trochanter, separate the muscles on the mouse legs and fix them on the sponge foam, soak the separated hind feet of the mice in 4% paraformaldehyde solution for fixation, and change to the decalcifying solution after 24 hours, and update the decalcifying solution every 3 days.

[0151] The results are as Figure 8 shown. The joints of CIA mice (arthritis mice) showed obvious swelling. Knockdown of the level of NFATc2 in synovial fibroblasts of joints could relieve joint swelling in mice. And as Figure 9 shown, the clinical scoring of joints showed that after reducing the level of NFATc2, the joint score could be decreased, indicating that the joint damage in mice was relieved.

[0152] 2. H&E staining

[0153] 1) Paraffin sections of mouse joints: Place the sections in fresh xylene for overnight dewaxing, and then transfer them to 100% alcohol, 95% alcohol, 85% alcohol, 75% alcohol, 50% alcohol, and distilled water for gradient hydration, 5 minutes for each step. Then start the staining procedure.

[0154] 2) Nuclear staining: Slowly drip hematoxylin staining solution onto the tissue sample and stain for 10 minutes.

[0155] 3) Differentiation and blueing: Differentiate with 1% hydrochloric acid ethanol for 1 - 2 seconds, and rinse with tap water for 15 minutes for blueing.

[0156] 4) Cytoplasmic staining: Blot the surrounding moisture with filter paper, and slowly drip eosin staining solution and stain for 5 minutes.

[0157] 5) Dehydration and mounting: Then transfer to 50% alcohol, 75% alcohol, 85% alcohol, 95% alcohol, 100% alcohol for gradient dehydration, 5 minutes for each step. Dry the section and mount it with neutral balsam, cover with a cover slip, avoiding air bubbles.

[0158] 6) Photographing: Use an upright microscope for photographing.

[0159] 3. Safranin - O - fast green staining

[0160] 1) Deparaffinization and hydration: Place the section in fresh xylene for overnight deparaffinization, and then transfer to 100% alcohol, 95% alcohol, 85% alcohol, 75% alcohol, 50% alcohol, distilled water for gradient hydration, 5 minutes for each step.

[0161] 2) Wipe the moisture around the joint tissue, drip freshly prepared Weigert solution and stain for 3 minutes, then wash with water.

[0162] 3) Drip acidic differentiation solution and differentiate for 15 seconds, wash with distilled water for 10 minutes.

[0163] 4) Immerse in fast green solution for 5 minutes, quickly wash the section with weak acid solution for 10 seconds to remove residual fast green, and air dry.

[0164] 5) Immerse in safranin staining solution for 5 minutes.

[0165] 6) Dehydrate with 95% ethanol for 3 seconds, absolute ethanol for 3 seconds, absolute ethanol for 1 minute.

[0166] 7) Dry the slide, drip neutral resin for mounting.

[0167] 8) Use an upright microscope for photographing.

[0168] The results are as Figure 10 shown. H&E staining shows synovial tissue hyperplasia at the joints of CIA mice, infiltration of a large number of immune cells, and cartilage destruction. Safranin - O - fast green staining also shows joint cartilage destruction. After knocking down the level of NFATc2 in synovial fibroblasts of joints, joint destruction can be alleviated and arthritis symptoms can be improved.

[0169] The preferred embodiments of the present application have been described in detail above in conjunction with the accompanying drawings. However, the present application is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present application, various simple modifications can be made to the technical solutions of the present application, and these simple modifications all fall within the protection scope of the present application.

[0170] In addition, it should be noted that, in the above specific embodiments, the various specific technical features described can be combined in any suitable manner without conflict. To avoid unnecessary repetition, the present application will not separately describe various possible combination manners.

Claims

1. Use of NFATc2 as a biomarker for the preparation of products for the diagnosis, prevention or treatment of rheumatoid arthritis.

2. The use according to claim 1, characterized in that: The product for diagnosing rheumatoid arthritis includes a detection reagent for detecting the expression of NFATc2 in a biological sample. The expression level of NFATc2 in the synovial tissue and / or synovial fibroblasts of patients with rheumatoid arthritis is higher than that of normal people.

3. The use according to claim 1, characterized in that: Products for treating or preventing rheumatoid arthritis include drugs that inhibit the expression of NFATc2.

4. A diagnostic tool for rheumatoid arthritis, characterized in that: It comprises a NFATc2 detection reagent, and the NFATc2 detection reagent detects the expression amount of NFATc2.

5. The diagnostic tool for rheumatoid arthritis according to claim 4, characterized in that: The NFATc2 detection reagent is a NFATc2 nucleic acid quantitative detection reagent for detecting NFATc2 nucleic acid or a NFATc2 protein quantitative detection reagent for detecting NFATc2 protein.

6. The diagnostic tool for rheumatoid arthritis according to claim 5, characterized in that: The NFATc2 gene nucleic acid quantitative detection reagent includes at least one of a specific primer for the NFATc2 gene or transcript and a specific recognition probe for the NFATc2 gene or transcript; the NFATc2 protein quantitative detection reagent includes an antibody or antibody fragment that specifically binds to the NFATc2 protein.

7. The diagnostic tool for rheumatoid arthritis according to claim 5, characterized in that: The product is a test kit, a chip, a test paper or a high-throughput sequencing platform.

8. A pharmaceutical composition for preventing or treating rheumatoid arthritis, comprising an NFATc2 inhibitor.

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