Application of TRADD related reagent in rheumatoid arthritis related products

Abstract

The invention relates to the technical field of biological medicine, in particular to application of a TRADD related reagent in rheumatoid arthritis related products. The invention finds that a TNFR1 signal channel is obviously enhanced in RA synovial tissues, and the expressions of TNFR1, TRAF2, TRADD and p65 are higher than those of normal synovial tissues, so that the invention provides the application of the reagent for detecting the expression of TRADD in the preparation of detection products for rheumatoid arthritis.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the application of TRADD-related reagents in products related to rheumatoid arthritis. Background Art

[0002] Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by severe synovitis leading to joint deformity and even loss of function. Fibroblast-like synoviocytes (FLSs) are one of the major cell types in the inflamed synovial tissue of RA patients and are key effector cells in the pathogenesis of RA. The tumor-like proliferation of FLSs is widely considered to underlie the pathology of RA. Therefore, understanding the mechanisms of abnormal FLSs proliferation and preventing their proliferation are key strategies for inhibiting RA synovitis and effectively controlling symptoms.

[0003] Tumor necrosis factor (TNF) receptor type 1-associated death domain protein (TRADD) is a multifunctional scaffold protein that plays a central role in transmitting TNF receptor type 1 (TNFR1) signals, mediating various downstream signaling pathways, and regulating cellular functions, including cell survival and apoptosis. Upon stimulation by TNF-α, the TRADD N-terminal domain interacts with the C-terminal domain of TNF receptor-associated factor 2 (TRAF2), recruiting TRAF2 to TNFR1. This activates downstream pathways, including nuclear factor-κB (NF-κB), c-Jun N-terminal kinase (JNK), and mitogen-activated protein kinase (MAPK), triggering proinflammatory and proproliferative responses. Numerous studies have reported the key role of TRADD in regulating cell survival and apoptosis; however, the function of TRADD in RA remains unclear. Summary of the Invention

[0004] To solve the above problems, the present invention provides the use of TRADD-related reagents in rheumatoid arthritis-related products.

[0005] Use of a reagent for detecting TRADD expression in preparing a detection product for rheumatoid arthritis. The amino acid sequence of TRADD is: maagqngheewvgsaylfvessldkvvlsdayahpqqkvavyralqaalaesggspdvlqmlkihrsdpqlivqlrfcgrqpcgrflrayregalraalqrslaaalaqhsvplqlelragaerldalladeerclscilaqqpdrlrdeelaeledalrnlkcgsgarggdgevasaplqppvpslsevkpppppppaqtflfqgqpvvnrplslkdqqtfarsvglkwrkvgrslqrgcralrdpaldslayeyereglyeqafqllrrfvqaegrratlqrlvealeeneltslaedllgltdpnggla, recorded as SEQ ID NO. 4.

[0006] Preferably, the reagent for detecting TRADD expression is a reagent used for detecting TRADD expression in a sample in immunoblotting, immunohistochemistry or fluorescent quantitative PCR technology.

[0007] Preferably, the sample is synovial tissue.

[0008] Preferably, the reagent comprises a TRADD antibody.

[0009] Preferably, the reagent comprises a BCA kit.

[0010] Preferably, the detection product is a kit, which includes the reagent for detecting TRADD expression.

[0011] Use of an agent for inhibiting TRADD expression in the preparation of a therapeutic product for rheumatoid arthritis.

[0012] Preferably, the agent for inhibiting TRADD expression includes an agent used in CRISPR / Cas9 technology, an agent used in shRNA technology, or an agent used in siRNA technology.

[0013] Preferably, the reagent used in the CRISPR / Cas9 technology includes any one of the sequences shown in SEQ ID NO.1 to SEQ ID NO.3.

[0014] Preferably, the reagents used in the CRISPR / Cas9 technology also include lentiviral vectors.

[0015] Compared with the prior art, the present invention is beneficial in that: The present invention found that the TNFR1 signaling pathway was significantly enhanced in RA synovial tissue, and the expressions of TNFR1, TRAF2, TRADD and p65 were all higher than those in normal synovial tissue. Therefore, the use of a reagent for detecting TRADD expression in the preparation of a detection product for rheumatoid arthritis was proposed.

[0016] The present invention successfully constructed a TRADD-knockout MH7A cell line using CRISPR / CAS9 technology. TRADD deficiency impedes the formation of TNFR1 complex I, leading to inhibition of phosphorylation of the NF-κB, JNK, and p38 pathways, and reduced p65 nuclear translocation, thereby suppressing TNF-α-induced MH7A cell proliferation and migration. Based on this, the present invention provides the use of an agent that inhibits TRADD expression in the preparation of a therapeutic product for rheumatoid arthritis.

[0017] The present invention explores the function of TRADD in RA FLSs and its mechanism of action involving the TNFR1 signaling pathway. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 These are the immunoblotting results of Example 1 of the present application, wherein A is the expression result of TNFR1, TRADD, TRAF2 and p65 in synovial tissue of RA patients, B is the statistical result of TNFR1 expression, C is the statistical result of TRAF2 expression, D is the statistical result of TRADD expression, and E is the statistical result of p65 expression.

[0019] Figure 2 These are the immunohistochemical results of Example 2 of the present application, wherein A is the expression result (1) and statistical result (2) of TNFR1 in the synovial tissue of RA patients, B is the expression result (3) and statistical result (4) of TRADD, C is the expression result (5) and statistical result (6) of TRAF2, and D is the expression result (7) and statistical result (8) of p65.

[0020] Figure 3 This is a schematic diagram of the results of Example 3 of the present application using CRISPR / Cas9 technology to detect the knockout efficiency of TRADD in MH7A cells, wherein A is a fluorescence graph, and B is an expression graph (1) and a statistical graph (2).

[0021] Figure 4 This is a schematic diagram of Example 4 of the present application using high-content cell imaging technology to detect the effect of TRADD deficiency on MH7A cell proliferation, wherein A is the imaging result and B is the proliferation statistical result.

[0022] Figure 5This is a schematic diagram of the results of Transwell assay for the effect of TRADD deficiency on MH7A cell migration in Example 5 of the present application, wherein A is a micrograph and B is the migration statistics result.

[0023] Figure 6 This is a schematic diagram of the results of immunoprecipitation detection of the effect of TRADD deficiency on the formation of TNFR1 complex I in Example 6 of the present application, wherein A is a representative immunoblot image of RIPK1, TRADD and TRAF2 binding to TNFR1 in cells, B is an analysis diagram of RIPK1 binding to TNFR1, C is an analysis diagram of TRADD binding to TNFR1, and D is an analysis diagram of TRAF2 binding to TNFR1.

[0024] Figure 7 This is a schematic diagram of the results of immunoblotting to detect the effect of TRADD deficiency on the phosphorylation expression of NF-κB pathway members (IKKα / β, IκBα and p65) in Example 1 of the present application, wherein A is a representative immunoblot image of the total expression and phosphorylation levels of IKKα / β, IκBα and p65 in cells, B is an analysis graph of the phosphorylation expression of IKKα / β in cells, C is an analysis graph of the phosphorylation expression of IκBα in cells, and D is an analysis graph of the phosphorylation expression of p65 in cells.

[0025] Figure 8 This is a schematic diagram of the results of immunoblotting to detect the effect of TRADD deficiency on JNK and p38 phosphorylation expression in Example 1 of the present application, wherein A is a representative immunoblot image of the total expression and phosphorylation levels of JNK and p38 in cells, B is an analysis diagram of JNK phosphorylation expression in cells, and C is an analysis diagram of p38 phosphorylation expression in cells.

[0026] Figure 9 This is a schematic diagram of the results of immunoblotting to detect the effect of TRADD deficiency on p65 expression in the cell nucleus in Example 7 of the present application, wherein A is a representative immunoblot image of p65 expression in the cytoplasm and cell nucleus, and B is an expression analysis diagram of p65 in the cell nucleus.

[0027] Figure 10 This is a schematic diagram of the results of immunofluorescence detection of the effect of TRADD deficiency on the distribution of p65 in the cell nucleus in Example 8 of the present application, wherein A is a representative immunofluorescence image of p65 localization in cells, and B is a distribution analysis diagram of p65 in 200 randomly selected cells.

[0028] Figure 11 Map of the lentiviral vector for the purpose.

[0029] Figure 12 A map of the basic carrier. DETAILED DESCRIPTION

[0030] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited by the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are all conventional methods unless otherwise specified.

[0031] GraphPad Prism 8.0.0 software was used for statistics and analysis. t Differences between two groups were compared using the sham test, and differences between multiple groups were compared using one-way analysis of variance (ANOVA). Differences between multiple groups with two variables were compared using two-way analysis of variance (ANOVA) followed by Tukey's multiple comparisons test. Statistical results are presented as mean ± standard deviation (SD). P <0.05 was considered statistically significant.

[0032] In the present invention, TRADD + / + -MH7A indicates the presence of TRADD in MH7A cells. - / - -MH7A indicates that TRADD is absent in MH7A cells Example 1 Immunoblotting was used to detect the expression of TNFR1, TRADD, TRAF2 and p65 in synovial tissues of RA patients Synovial tissue was washed with pre-chilled PBS, excess fat was removed, and residual liquid was blotted with filter paper. Approximately 100 mg of tissue was weighed and placed in a 1 mL tissue homogenizer. 1 mL of lysis buffer (RIPA:PMSF:phosphatase inhibitor = 99:1:1) was added and repeatedly ground on ice until no visible solids were visible. The supernatant was transferred to a pre-chilled EP tube. After ultrasonication, the tube was frozen and thawed three times at -80°C. Centrifuge at 12,000 g for 10 min at 4°C, retain the supernatant, and quantify protein using a BCA kit. The supernatant was then used for immunoblotting to detect TNFR1, TRADD, TRAF2, and p65 expression.

[0033] The results are as follows Figure 1 As shown in Figure 4, the expressions of TNFR1, TRADD, TRAF2, and p65 in the synovial tissue of RA patients were significantly increased compared with those in normal synovial tissue (Figure AD, ±s, n=5),* P <0.05.

[0034] Example 2 Immunohistochemistry was used to detect the expression of TNFR1, TRADD, TRAF2 and p65 in synovial tissues of RA patients Place synovial tissue sections in a 60°C oven for 2-3 hours. Hydrate sections by sequentially soaking in xylene I (10 minutes), xylene II (10 minutes), 100% anhydrous ethanol I (5 minutes), 100% anhydrous ethanol II (5 minutes), 95% ethanol (3 minutes), 85% ethanol (3 minutes), 70% ethanol (3 minutes), and distilled water (5 minutes). Permeabilize sections with 0.5% Triton-X 100 for 30 minutes at room temperature. Cold-retrieve sections with EDTA antigen retrieval solution for 10 minutes at room temperature. Incubate sections with 3% peroxidase for 10 minutes at room temperature. Apply diluted primary antibodies against TNFR1, TRADD, TRAF2, and p65 (1:200) and incubate at 4°C overnight. Wash sections with PBS and add an appropriate amount of reaction enhancement solution for 20 minutes at room temperature. Sections were incubated with an appropriate amount of enhanced enzyme-labeled goat anti-mouse / rabbit IgG polymer at room temperature for 20 minutes. DAB solution was then added to the sections and incubated at room temperature for 1 minute. Sections were rinsed with double-distilled water and stained with hematoxylin solution at room temperature for 2 minutes. Sections were dehydrated, evaporated with xylene, and mounted with neutral resin. Primary antibodies against TNFR1, TRADD, TRAF2, and p65 were all from Proteintech, with catalog numbers: TNFR1: 21574-1-AP; TRADD: 15468-1-AP; TRAF2: 67315-1-Ig; and p65: 10745-1-AP.

[0035] The results are as follows Figure 2 As shown in Figure 4, the expressions of TNFR1, TRAF2, TRADD, and p65 in RA patient synoviocytes were significantly increased compared with those in normal synoviocytes (Figures AD, ±s, n=5). * P <0.05,** P <0.01,*** P <0.001

[0036] Example 3 Detection of TRADD knockout efficiency in MH7A cells using CRISPR / Cas9 technology The target gene lentiviral vector (produced by Shanghai GeneChem Technology Co., Ltd.) was used to infect MH7A cells, knocking out TRADD in the cells and constructing MH7A cells that stably express the Cas-9 protein. Shanghai GeneChem Technology Co., Ltd. was commissioned to construct the target gene lentiviral vector. The target gene lentiviral vector information is as follows:

[0037] Vector name: GV708, map as Figure 11 As shown; Basic vector: Lenti-CAS9-puro plasmid, map as shown Figure 12 As shown; Element sequence: U6-sgRNA-EF1a-Cas9-FLAG-CMV-EGFP-P2A-puro; Control insert sequence: CGCTTCCGCGGCCCGTTCAA.

[0038] First, prepare 2 mL of cells with a density of 1.2 × 10 5 Take 86 μL / well of the cell suspension (100 μg / mL) and add it to 20 wells in a 96-well plate. Three wells are used as the control group. Remove the virus from the -80°C refrigerator and slowly thaw on ice. Dilute the virus to a titer of 1×10 in complete culture medium. 8 TU / mL, 5×10 7 TU / mL and 1×10 7 TU / mL, after dilution, each group has at least 35μL. Infect for 10 hours in a 37℃, 5% CO2 incubator, add 100 μL of complete medium to each well to maintain normal cell growth. After 72 hours of cell infection, remove the cells and observe the abundance of fluorescence on the cells under a fluorescence microscope. The infection efficiency of the virus is about 80%, and the infection conditions and infection multiplicity (MOI) corresponding to the group with good cell growth can be used as the basis for subsequent infection experiments. In addition, proteins were extracted from the blank control group, negative control group, and MH7A cell groups infected with the three sgRNA viruses. The sequences of the three sgRNAs are shown in Table 1. The knockout effect was detected by immunoblotting, and the immunoblot bands were analyzed and processed using Image J software. Using the blank control group as the benchmark, the TRADD expression and knockout efficiency between the groups were calculated.

[0039] Table 1 sgRNA sequences The results are as follows Figure 3 As shown, three sg-RNAs with different sequences were each selected at four MOI titers (1, 10, 50, 100). The corresponding virus volume was added to the well plate containing cells and infected for 72 hours. Figure 3As shown in Figure A, the higher the green fluorescence intensity, the higher the viral transfection efficiency. In addition, combined with the cell state, the transfection efficiency and cell state were best at MOI=50. MH7A cells were lysed at MOI=50, and the expression of TRADD was detected by immunoblotting. The results showed that compared with the normal group, the knockout efficiency of TRADD-sgRNA 11699 was the highest among the three viruses (Figure B, ±s, n=3). In the figure, TRADD-sgRNA 11699 is abbreviated as 699, TRADD-sgRNA 11700 is abbreviated as 700, and TRADD-sgRNA 11701 is abbreviated as 701. *** P <0.001.

[0040] Example 4 High-content cell imaging technique was used to detect the effect of TRADD deficiency on MH7A cell proliferation. TRADD + / + -MH7A or TRADD - / - -MH7A cells were digested and 100 μL of cell suspension was transferred to each well of a 96-well plate. The number of cells was approximately 3 × 10 3 Each well was cultured overnight in a 37°C, 5% CO2 incubator and transfected with the corresponding plasmid for 24 hours. The cells were then treated with TNF-α (20 ng / mL) for 24 hours. The cells were washed 1-2 times with PBS and fixed with 4% paraformaldehyde for 20 minutes. The cells were washed three times with PBS and stained with DAPI for 5 minutes. After washing with PBS, the cells were imaged using a high-content cytometry system and the number of cells in each well was counted.

[0041] The results are as follows Figure 4 As shown, TNF-α induced TRADD + / + -MH7A cells have a significant increase in number and strong proliferation capacity. - / - Compared with MH7A, TNF-α cannot promote TRADD - / - -MH7A cell proliferation capacity. + / + Compared with the +TNF-α group, TRADD - / - +TNF-α cells proliferate slowly ( ±s, n=3).*** P <0.001.

[0042] Example 5 Transwell assay to detect the effect of TRADD deficiency on MH7A cell migration The migration ability of MH7A cells was detected by Transwell plate method. + / +-MH7A or TRADD - / - -MH7A cells were trypsinized to prepare a cell suspension, and 200 μL of the cell suspension (approximately 1×10 cells) was added to the upper chamber of the Transwell chamber. 4 The cells were cultured for 24 hours. The cells were then washed twice with PBS and fixed with 0.25% crystal violet solution for 20 minutes. The cells were then washed three times with PBS. Five randomly selected fields of view were photographed under an inverted microscope. Cells were counted using Image J software, and migration rates were calculated. Migration rate = number of cells in the treated group / number of cells in the blank control group.

[0043] The results are as follows Figure 5 As shown, TNF-α induced TRADD + / + -MH7A cell migration was significantly enhanced. - / - Compared with MH7A, TNF-α cannot promote TRADD - / - -MH7A cell migration ability. + / + Compared with the +TNF-α group, TRADD - / - +TNF-α cells decreased in number and weakened in migration ability ( ±s, n=3). ** P <0.01,*** P <0.001.

[0044] Example 6 Immunoprecipitation assay to determine the effect of TRADD deficiency on the formation of TNFR1 complex I (1) TRADD + / + -MH7A or TRADD - / - -MH7A cells were plated in 10 cm culture dishes and stimulated with TNF-α (20 ng / mL) for various durations (0, 5, 15, and 30 min) when the cells reached approximately 90% confluency. Following stimulation, the cells were washed twice with PBS and lysed with 1 mL of IP lysis buffer at 4°C for 30 min on a shaker. The lysate was then collected and the protein lysate was disrupted using an ultrasonic cell disruptor.

[0045] (2) Mix IgG with Protein A / G plus-agarose and incubate at 4°C on a shaker for 2 h. Wash Protein A / G plus-agarose three times with IP wash buffer, discarding the supernatant each time; incubate Protein A / G plus-agarose with the protein sample at 4°C on a shaker for 2 h. Centrifuge at 4°C, 2000 rpm for 5 min, and retain the supernatant for subsequent use.

[0046] (3) Mix TNFR1 antibody with Protein A / G plus-agarose and incubate on a shaker at 4°C for 2 h. Wash Protein A / G plus-agarose three times with IP wash buffer, discarding the supernatant each time and retaining the Protein A / G plus-agarose.

[0047] (4) Combine the supernatant from step (2) and the Protein A / G plus-agarose from step (3) and incubate overnight at 4°C on a shaker. Wash three times with IP buffer and centrifuge at 2000 rpm for 5 min at 4°C.

[0048] (5) Add 32 μL IP lysis buffer and 8 μL protein loading buffer (5×) to the precipitated complex, boil at 100°C to denature the protein, and then perform immunoblotting.

[0049] The results are as follows Figure 6 The following shows that in TRADD + / + In -MH7A cells, as the TNF-α stimulation time prolonged (0 min to 15 min), the co-expression of TNFR1, RIPK1, TRAF2, and TRADD was gradually upregulated. At around 15 min to 30 min, the co-expression was gradually downregulated. - / - In -MH7A cells, as the TNF-α stimulation time prolonged (0 min to 30 min), the co-expression of TNFR1 with RIPK1, TRAF2, and TRADD was not observed. + / + -MH7A and TRADD - / - Compared with MH7A cells, there was a significant difference in the co-expression of TNFR1 with RIPK1, TRAF2, and TRADD as the TNF-α stimulation time was prolonged (5 min to 15 min). That is, TRADD deficiency inhibited the formation of TNFR1 complex I induced by TNF-α in MH7A cells ( ±s, n=3). * P <0.05,** P <0.01,*** P <0.001.

[0050] Figure 7 In Example 1 of this application, immunoblotting was used to detect the effect of TRADD deficiency on the phosphorylation expression of NF-κB pathway members (IKKα / β, IκBα and p65). The results are as follows Figure 7 As shown, with TRADD - / - Compared with MH7A, after TNF-α stimulation, TRADD + / + -MH7A cells showed a significant increase in the phosphorylation levels of IKKα / β, IκBα, and p65 over time, while TRADD - / - -The phosphorylation levels of IKKα / β, IκBα, and p65 in MH7A cells did not change significantly over time (Figures AD, ±s, n=3). * P <0.05,** P <0.01,*** P <0.001.

[0051] Figure 8 In Example 1 of this application, immunoblotting was used to detect the effect of TRADD deficiency on the expression of JNK and p38 phosphorylation. The results are as follows Figure 8 As shown, with TRADD - / - Compared with MH7A, after TNF-α stimulation, TRADD + / + -The phosphorylation levels of JNK and p38 in MH7A cells increased significantly over time, while TRADD - / - -The phosphorylation levels of JNK and p38 in MH7A cells did not change significantly over time (Figures AC, ±s, n=3). * P <0.05,** P <0.01,*** P <0.001.

[0052] Example 7 Immunoblotting was used to detect the effect of TRADD deficiency on the expression of p65 in the nucleus Take the TRADD of the logarithmic growth period + / + -MH7A or TRADD - / - -MH7A cells were grown at 1×10 per well 6Plate cells in a 6-well plate. When the cell density reaches approximately 90% of the plate area, add TNF-α (20 ng / mL) for stimulation for 0, 15, 30, 60, and 90 minutes. Wash twice with PBS and dislodge the cells using a cell scraper or pipette them off with trypsin-free EDTA solution. Centrifuge at 3000 rpm for 5 minutes and save the pellet for later use. Avoid using trypsin to digest the cells, as this may degrade the target protein to be detected. Add 200 μL of Cytoplasmic Protein Extraction Reagent A (add PMSF before use) per 20 μL of cell pellet. Vortex vigorously for 5 seconds to completely resuspend the cell pellet and incubate at 4°C for 10-15 minutes. Add 10 μL of Cytoplasmic Protein Extraction Reagent B. Vortex vigorously at high speed for 5 seconds and incubate on ice for 1 minute. Vortex again at high speed for 5 seconds and centrifuge at 16,000 g for 5 minutes at 4°C. Transfer the supernatant to a pre-chilled EP tube; this is the cytoplasmic protein. A small volume of supernatant can be left on the precipitate to avoid sucking up the precipitate. Or wash the precipitate again with PBS. Add 50 μL of nuclear protein extraction reagent to the precipitate (add PMSF before use). Vortex vigorously at the highest speed for 15-30 seconds to thoroughly suspend the cell pellet. Place on ice and vortex vigorously again for 20 seconds every 1 minute for a total of 30 minutes. Centrifuge at 16000 g for 10 minutes at 4℃. Transfer the supernatant to a pre-cooled EP tube. The supernatant is the nuclear protein. Subsequently, use the cytoplasm and nuclear protein for immunoblotting.

[0053] The results are as follows Figure 9 As shown, TNF-α stimulation increased TRADD + / + -The expression of p65 in the nucleus of MH7A cells gradually increased. - / - - There was no significant change in p65 in the nucleus of MH7A cells ( ±s, n=3). * P <0.05,*** P <0.001.

[0054] Example 8 Immunofluorescence assay to detect the effect of TRADD deficiency on the distribution of p65 in the nucleus Place sterile round coverslips in a 24-well plate. + / + -MH7A or TRADD - / - -MH7A cells were digested and inoculated into the well plate, with approximately 5×10 4 / per well. Continue culturing for 48 h, then add TNF-α (20 ng / mL) and stimulate for 1 h. Gently wash the well plate with pre-cooled PBS, add 4% paraformaldehyde into the well plate, and fix at room temperature for 20 min. Add the permeabilization blocking solution and incubate at room temperature for 20 min. Add the p65 primary antibody (1:100) and incubate overnight at 4°C. Add the Alexa Fluor 647-Anti-rabbit fluorescent secondary antibody (1:200) and incubate at room temperature in the dark for 1 h. Add the DAPI solution to label the cell nuclei and incubate at room temperature in the dark for 5 min. Gently wash with the washing buffer 3×5 min. First, drop the anti-fluorescence quencher on the glass slide, then take out the coverslip and invert it onto the glass slide, and fix with nail polish. Observe the nuclear translocation of p65 under a Leica confocal microscope and take images. Randomly select 200 cells to count the subcellular localization of p65. Use Image J to analyze the fluorescence intensities of the cell nuclei and cytoplasm respectively. If the fluorescence intensity of the cell nucleus is significantly higher than that of the cytoplasm, such cells are "N>C"; if the fluorescence intensity of the cell nucleus is significantly lower than that of the cytoplasm, such cells are "N<C"; if the fluorescence intensities of the cell nucleus and cytoplasm are similar, such cells are "N=C".

[0055] The results are as Figure 10 shown, in TRADD + / + -MH7A, p65 is distributed in the cytoplasm, and in about 20% of the cells, p65 is mainly located in the cell nucleus; after TNF-α stimulation, in about 60% of the TRADD + / + -MH7A, p65 is mainly located in the cell nucleus; in TRADD - / - -MH7A, p65 is mainly distributed in the cytoplasm, and in about 16% of the cells, p65 is mostly located in the cell nucleus; after TNF-α stimulation, in about 23% of the TRADD - / - -MH7A, p65 is distributed in the cell nucleus. That is, the deletion of TRADD inhibits the nuclear distribution of p65 induced by TNF-α.

[0056] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that any value between the two endpoints of each numerical range and the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0057] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications to these embodiments once they know the basic creative concept. Therefore, the appended claims are intended to be construed to include the preferred embodiments and all changes and modifications falling within the scope of the present invention.

[0058] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. Application of reagents for detecting TRADD expression in the preparation of detection products for rheumatoid arthritis.

2. The use according to claim 1, characterized in that: The reagent for detecting TRADD expression is a reagent used for detecting TRADD expression in a sample in immunoblotting, immunohistochemistry or fluorescence quantitative PCR technology.

3. The use according to claim 2, characterized in that: The sample is synovial tissue.

4. The use according to claim 3, characterized in that: The reagents include TRADD antibodies.

5. The use according to claim 3, characterized in that: The reagents include a BCA kit.

6. The use according to claim 2, characterized in that: The detection product is a kit, which includes the reagent for detecting TRADD expression.

7. Use of the agent for inhibiting the expression of TRADD according to claim 1 in the preparation of a therapeutic product for rheumatoid arthritis.

8. The use according to claim 7, characterized in that: The reagents for inhibiting TRADD expression include reagents used in CRISPR / Cas9 technology, reagents used in shRNA technology, or reagents used in siRNA technology.

9. The use according to claim 8, characterized in that: The reagents used in the CRISPR / Cas9 technology include any one of the sequences shown in SEQ ID NO.1 to SEQ ID NO.

3.

10. The use according to claim 9, characterized in that: The reagents used in the CRISPR / Cas9 technology also include lentiviral vectors.

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