Inhibitory Effect Of T-614on TNF-α Production In Human Fibroblast-like Synoviocytes Of RA

Rheumatoid arthritis (RA) is a kind of aggressive arthritis as the main performance of systemic autoimmune diseases. This happens to women, male and female ratio is about1:3. RA can occur at any age,and30to50years old is the peak age of the disease. Incidence of RA in China mainland area is0.2to0.4%. Because the etiology and pathogenesis of this disease is not completely clear, at present which is still lack of clinical cure and effective measures to prevent the disease. Treatment goal of RA is to relieve joint symptoms, remit disease progression, prevent and reduce the destruction of the joints, protect function of joints, maximize the patient’s quality of life. We should emphasize the principle of early diagnosis and early treatment, drug combination and individualized treatment. Treatments include general treatment, drug therapy, surgical treatment, among them drug treatment is the most important. T-614,a novel immunomodulator, can inhibit the production of inflammation factors,attenuate joint inflammation and articular damage. At present, the research for T-614in inflammation factors inhibition mainly stay in the stage of animal models,while solid evidence about inhibition of inflammation of the RA patients’synovial cells in vitro has been minimal until now.Part1Screen patients into groups and collect specimensObjective:To screen active rheumatoid arthritis patients into groups and collect synovial membrane samples.Methods:1. Screened patients into groups:A. the standards of included cases(1) Diagnosis accord with2010ACR/EULAR classification criteria.(2) Age18years old or moreB.the standards of eliminated cases(1) Does not comply with the above diagnostic criteria.(2) Ready to pregnant women, pregnant women or lactating women.(3) Serious heart, liver and kidney dysfunction (more than1.5times that of the upper limit of normal). (4) Person who has a history of alcoholism.(5) Have a malignant tumor.(6) The patients with a history of allergies for T-614.(7) Participate in any of the clinical drug trial.2. Collected specimens:Collected synovial tissue of patients complying with the above standard and doing synovial membrane resection.Results:1. Selected5patients complying with the standards into the groups.2.Acquired the synovial tissue of the above patients’pathological changes.Conclusions:Screened the active rheumatoid arthritis patients into groups successfully and acquire the synovial tissue of the RA patients’pathological changes.Part2In vitro synovial cell culture and identificationObjective:To research culture method of fibroblast-like synovial cells in vitro for RA patients and identification method.Methods:1. With in vitro culture method by type I collagen enzyme, synovial cell tissue specimens of5patients with rheumatoid arthritis (RA) were done primary culture and subculture, morphology and proliferation of fibroblast-like synovial cells cultured was observed.2.When primary cells were subcultured to three or four generations,with cell immunohistochemical staining method, expression level of Vimentin and CD68was detected, synovial cell types were identified.Results:1. Cultured synovial cells of5RA patients successfully.2. Synovial cells showed Vimentin staining positive, CD68staining negative.Conclusions:Fibroblast-like synovial cells of RA patients were acquired successfully.Part3T-614inhibits the production of TNF-aObjective:To research the role of T-614to TNF-aMethods: 1. The cells could be divided into four groups, which were the control group, MTX group, T-614group and the two drugs combination group,respectively. With protein immunoblot method (western blot),TNF-a protein expression levels of the four groups in the cells were detected.2.For the above four groups, by the method of enzyme-linked immunosorbent (ELISA),we detected secretion level of extracellular TNF-a in the4groups.Results:1.Western blot test results showed that TNF-a expression level from high to low,in turn,is the control group, MTX group, T-614group and the two drugs combination group.2. ELISA detection results showed that TNF-a secretion level from high to low, in turn, is the control group, MTX group, T-614group, the two drugs combination group.Conclusions:T-614inhibits the production of TNF-a effectively,the inhibitory effect of which was combined with MTX is more significant.