类风湿关节炎滑膜成纤维细胞粘着斑激酶与p53对细胞增殖的研究

发布时间：2018-08-14 13:17

【摘要】：背景:类风湿关节炎(RA)以慢性和侵蚀性滑膜炎为主要特点的一种自身免疫病,可影响多个关节导致其破坏、变形,并最终造成身体残疾,给患者带来极大的痛苦。RA患病率为0.5%~1%,大量研究表明成纤维样滑膜细胞(FLS)的异常增生是RA发病的关键。粘着斑激酶(FAK)是一种蛋白激酶,参与细胞内的信号转导。在肿瘤研究领域,FAK能通过其N-末端的FERM结构域与p53结合,进而抑制细胞凋亡,即FAK可通过抑制p53基因的表达来阻止肿瘤细胞凋亡从而导致肿瘤的发生。研究发现RA患者FLS与肿瘤细胞有许多相似的特性,如进行性增生、迁移和侵袭,其机制可能同肿瘤细胞无限增殖及转移的机制相似。我科前期研究表明,FAK在RA滑膜组织中表达量较正常滑膜组织显著升高,且通过抗风湿治疗后,可抑制FAK表达,促进FLS凋亡,这表明FAK在FLS的过度增生过程中起重要作用。还有研究表明,p53缺失可能参与RA滑膜组织增生这一病理过程。那么在RA的发病过程中,FAK是否可与p53相互作用,使p53表达减少导致FLS过度增殖,从而参与RA的发病机制,值得研究。目的:1、通过体外研究FAK、p53在RA成纤维滑膜细胞中的表达,探讨RA的发病机制;2、探讨FAK、p53对RA成纤维滑膜细胞增殖的影响,为治疗RA提供新靶点。方法:1、取确诊的RA患者滑膜组织进行体外培养;2、对体外培养的RA患者滑膜细胞进行不同处理,分别加入TNF-α(10ng/ml)及不同浓度的蛋白酶抑制剂(MG-132)(1μM、5μM、10μM、20μM),48h后RT-PCR检测各组FLS上FAK及p53 m RNA的水平;3、体外培养的滑膜细胞分别加入不同浓度的蛋白酶抑制剂(MG-132)(1μM、5μM、10μM、20μM),分别于处理后的24h、48h、72h、96h进行细胞增殖试验;4、采用SPSS 17.0统计软件分析。结果:1、TNF-α作用后滑膜细胞的FAK m RNA水平较对照组增高,差异有统计学意义(P0.05);TNF-α作用后p53 m RNA水平与对照组比较有所降低,差异无统计学意义(P0.05);2、MG-132作用后滑膜细胞的p53 m RNA水平较对照组增高,差异有统计学意义(P0.05),以浓度为5μM时p53 m RNA水平最高;MG-132作用后FAK m RNA水平较对照组有所降低,差异无统计学意义(P0.05);3、不同浓度的MG-132作用后,FLS的增殖情况与对照组比较受到抑制,差异有统计学意义(P0.05)。结论:1、粘着斑激酶在体外经TNF-α作用后其m RNA水平增高,经MG-132作用后其m RNA水平略有降低,粘着斑激酶参与类风湿关节炎发病过程;2、p53在体外经MG-132作用后其m RNA水平增高,p53可抑制类风湿关节炎成纤维滑膜细胞的过度增殖,在类风湿关节炎发病机制中有重要作用。
[Abstract]:Background: rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic and erosive synovitis. It affects multiple joints and results in destruction, deformation, and ultimately physical disability. The prevalence of RA was 0.5%. A large number of studies showed that the abnormal proliferation of (FLS) in fibroblast synoviocytes was the key to the pathogenesis of RA. Focal adhesion kinase (FAK) is a protein kinase involved in intracellular signal transduction. In the field of tumor research, FAK can bind to p53 through its N-terminal FERM domain, which can inhibit the apoptosis of tumor cells. That is, FAK can inhibit the apoptosis of tumor cells by inhibiting the expression of p53 gene, leading to tumorigenesis. It has been found that FLS in RA patients has many similar characteristics with tumor cells, such as progressive proliferation, migration and invasion, and its mechanism may be similar to that of infinite proliferation and metastasis of tumor cells. Our previous study showed that the expression of FAK in RA synovial tissue was significantly higher than that in normal synovial tissue, and FAK could inhibit the expression of FAK and promote the apoptosis of FLS after anti-rheumatism treatment, which indicated that FAK played an important role in the process of FLS hyperproliferation. It has also been suggested that p53 deficiency may be involved in RA synovial hyperplasia. Therefore, it is worth studying whether FAK can interact with p53 in the pathogenesis of RA, which can reduce the expression of p53 and lead to excessive proliferation of FLS, and thus participate in the pathogenesis of RA. Objective to investigate the expression of FAKP p53 in RA fibroblast synoviocytes in vitro, to explore the pathogenesis of RA and the effect of FAKN p53 on the proliferation of fibroblast synoviocytes, and to provide a new target for the treatment of RA. Methods the synovial tissue of the confirmed RA patients was cultured in vitro, and the synovial cells of the RA patients were treated in different ways. TNF- 伪 (10ng/ml) and different concentrations of protease inhibitor (MG-132) (1 渭 M5 渭 MU 10 渭 MU 20 渭 M) were added to RT-PCR for 48 h. The levels of FAK and p53 m RNA on FLS were detected by RT-PCR. The cultured synovial cells were treated with different concentrations of MG-132 (1 渭 M5 渭 M10 渭 M10 渭 M20 渭 M),) respectively at 24 h, 48 h, 72 h, 96 h after treatment with TNF- 伪 (TNF- 伪) and different concentrations of protease inhibitor (MG-132). Cell proliferation test 4 was analyzed by SPSS 17.0 software. Results the level of FAK m RNA in synovial cells treated with TNF- 伪 was significantly higher than that in control group (P0.05), and the level of p53 m RNA decreased after TNF- 伪 treatment. There was no significant difference (P0.05) the level of p53 m RNA in synovial cells treated with MG-132 was significantly higher than that in control group (P0.05). The highest level of p53 m RNA was found in 5 渭 M and the level of FAK m RNA decreased after MG-132 treatment. There was no significant difference (P0.05), the proliferation of MG-132 was inhibited after different concentrations of MG-132 compared with the control group, the difference was statistically significant (P0.05). Conclusion the level of m RNA increased after TNF- 伪 treatment in vitro, and the level of m RNA decreased slightly after treatment with MG-132. Adhesion kinase participates in the pathogenesis of rheumatoid arthritis (RA) and its m RNA level increases after treated with MG-132 in vitro. P53 can inhibit the excessive proliferation of fibroblasts from rheumatoid arthritis (RA) and play an important role in the pathogenesis of rheumatoid arthritis (RA).
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R593.22

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