钙网织蛋白通过调节c-FLIP表达促进类风湿关节炎滑膜增生的分子机制研究

发布时间：2017-12-28 01:01

  本文关键词：钙网织蛋白通过调节c-FLIP表达促进类风湿关节炎滑膜增生的分子机制研究　出处：《天津医科大学》2016年硕士论文　论文类型：学位论文

  更多相关文章： 类风湿关节炎 钙网织蛋白 细胞凋亡 细胞源性 FLICE 抑制蛋白 NF-κB信号通路

【摘要】：目的类风湿关节炎(Rheumatoid arthritis,RA)是一种以滑膜组织增生及慢性关节滑膜炎症浸润为主要病理特征的系统性自身免疫病,关节滑膜成纤维细胞的异常增殖及凋亡减少是滑膜组织增生的重要发病机制之一。已有研究报道钙网织蛋白(Calreticulin,CRT)可能与RA的发病有关。本课题组前期研究亦发现,CRT在RA患者血清、滑膜及关节液中均呈现高表达,血清CRT表达量与RA患者的疾病活动度评分DAS28正相关,且CRT可通过NO途径参与RA滑膜血管翳的形成。有研究报道CRT可抑制胞外死亡受体途径之一的Fas-Fas L介导的T细胞凋亡进而参与RA的发病机制。本课题拟探讨CRT通过调节抗凋亡蛋白c-FLIP表达进而抑制胞外死亡受体途径介导的RA滑膜成纤维细胞凋亡,进而参与RA滑膜增生及炎症浸润的发病机制,为临床RA滑膜炎症的靶向治疗提供新的理论依据和实验基础。方法1.于2014年1月至2015年5月收集行关节镜及膝关节置换术患者的标本进行实验研究。关节滑液标本包括:RA患者32例,OA患者37例。滑膜组织标本包括:RA患者14例,OA患者19例。2.采用酶联免疫吸附试验(ELISA)检测RA及OA患者关节滑液中CRT的含量。3.采用免疫组织化学方法分析CRT和c-FLIP蛋白在RA及OA滑膜组织中的表达和定位并对结果进行半定量分析;同时对CRT及c-FLIP的表达量进行相关性分析。4.采用联合酶消化法分离RA及OA滑膜成纤维细胞并进行培养,观察分析RA及OA滑膜成纤维细胞在体外生长的特性。5.采用CCK-8试验分析CRT对RA滑膜成纤维细胞增殖能力的影响。6.采用流式细胞技术分析CRT对TRAIL诱导的RA滑膜成纤维细胞凋亡的影响。7.采用细胞免疫荧光方法检测RA滑膜成纤维细胞中CRT及c-FLIP蛋白的表达及细胞定位。8.采用免疫印迹法(Western Blot)及实时荧光定量PCR(q RT-PCR)检测RA滑膜成纤维细胞中c-FLIP蛋白及m RNA的表达,分析外源性CRT对滑膜成纤维细胞表达c-FLIP的影响并评估CRT介导的c-FLIP表达在NF-κB信号通路活化中的作用。9.采用SPSS 20.0软件包进行统计学分析。计量资料以(?)±s表示。分别采用t检验、SNK-q检验、Pearson线性相关性分析等进行相关的统计学处理。检验水准为双侧α=0.05。结果1.ELISA结果显示,RA患者关节滑液中CRT含量[(7.2±3.0)ng/ml]显著高于OA患者[(3.8±0.6)ng/ml](t=6.724,P0.01)。2.免疫组织化学结果显示,RA关节滑膜组织CRT和c-FLIP均呈高表达,滑膜组织的衬里层和衬里下层、炎性细胞及血管周围有较多阳性染色。相较而言,OA滑膜中CRT和c-FLIP仅在衬里层及血管周围有极少量的表达。RA滑膜组织中CRT的表达量与c-FLIP呈正相关。3.两组刚贴壁的细胞呈圆形,后逐渐延伸为长梭形或纺锤形。RA患者滑膜成纤维细胞原代细胞在接种后12~24h即开始贴壁,4~5天后铺满瓶底,OA患者滑膜成纤维细胞原代细胞贴壁生长缓慢,接种后24~48h开始贴壁,直至7~8天后达到融合状态。传代后细胞生长变快,RA组细胞每2~3天即可传代1次,而OA组细胞传代需培养3~4天。4.CCK-8结果显示,CRT具有促进RA滑膜成纤维细胞增殖的作用。5.CRT对凋亡诱导剂TRAIL诱导的RA滑膜成纤维细胞凋亡具有抑制作用。6.qRT-PCR、Western Blot以及细胞免疫荧光检测结果均表明,与OA患者的滑膜成纤维细胞相比,RA患者滑膜成纤维细胞中CRT和c-FLIP的m RNA及蛋白表达水平均明显升高。7.q RT-PCR和Western Blot的检测结果显示,不同剂量的人重组CRT作用后,RA滑膜成纤维细胞中c-FLIP的m RNA表达水平和蛋白表达水平均升高,且c-FLIP表达水平的升高呈CRT浓度依赖性。8.在CRT的作用下,RA滑膜成纤维细胞中p-NF-κB表达水平明显升高,而总NF-κB蛋白表达水平没有明显的改变。应用NF-κB信号通路抑制剂后,CRT上调RA滑膜成纤维细胞中c-FLIP表达的作用被抑制。结论1.CRT与c-FLIP在RA患者滑膜组织中的表达量均升高,且CRT与c-FLIP在RA患者滑膜组织中表达量呈正相关2.体外实验中发现,RA滑膜成纤维细胞中,CRT与c-FLIP均呈高表达,CRT可上调RA滑膜成纤维细胞c-FLIP的表达,且具有剂量依赖性。3.CRT可能通过调节RA滑膜成纤维细胞中抗凋亡蛋白c-FLIP的表达激活NF-κB通路进而参与RA滑膜组织增生及炎症持续过程。本课题研究结果提示RA患者滑膜成纤维细胞CRT及抗凋亡蛋白c-FLIP表达增加,且CRT可能通过调控c-FLIP的表达使得滑膜成纤维细胞凋亡减少,且可能通过对c-FLIP的调节激活NF-κB信号通路,进而促进RA滑膜组织增生及炎症反应,参与RA病理机制。
[Abstract]:The purpose of rheumatoid arthritis (Rheumatoid arthritis RA) is a kind of system with synovial hyperplasia and chronic synovitis infiltration were the main pathological features of autoimmune disease, abnormal proliferation and apoptosis of synovial fibroblasts into the weight reduction is one of the pathogenesis of hyperplasia of the synovial tissue to. It has been reported that Calreticulin (CRT) may be associated with the pathogenesis of RA. Our research group also found that CRT was highly expressed in serum, synovial fluid and synovial fluid of RA patients. The expression level of serum CRT was positively correlated with the disease activity score DAS28 of RA patients, and CRT could participate in the formation of RA synovial pannus through NO pathway. It is reported that CRT can inhibit the apoptosis of T cells mediated by Fas-Fas L, one of the extracellular death receptor pathways, and then participate in the pathogenesis of RA. This paper intends to discuss the CRT by regulating the expression of the anti apoptotic protein c-FLIP and inhibit extracellular death receptor pathway mediated by RA synovial fibroblast apoptosis, which involved in the pathogenesis of inflammatory infiltration and hyperplasia of synovial RA, RA synovitis for clinical targeted therapy provides a new theoretical basis and experimental basis. Methods from January 2014 to May 2015 1. from arthroscopic knee replacement surgery patients were studied. Joint synovial fluid specimens included 32 patients with RA and 37 patients with OA. The synovial tissue specimens included 14 patients with RA and 19 patients with OA. 2. the content of CRT in synovial fluid of RA and OA patients was detected by enzyme-linked immunosorbent assay (ELISA). 3. immunohistochemical method was used to analyze the expression and location of CRT and c-FLIP protein in RA and OA synovial tissues, and semi quantitative analysis of the results. At the same time, the correlation analysis between CRT and c-FLIP expression was performed. 4. RA and OA synovial fibroblasts were isolated and cultured by combined enzyme digestion. The characteristics of the growth of RA and OA synovial fibroblasts in vitro were observed and analyzed. 5. the effects of CRT on the proliferation of RA synovial fibroblasts were analyzed by CCK-8 test. 6. the effect of CRT on the apoptosis of RA synovial fibroblasts induced by TRAIL was analyzed by flow cytometry. 7. the expression of CRT and c-FLIP protein in RA synovial fibroblasts and the cell location were detected by cell immunofluorescence. 8. by Western blot (Western Blot) and real-time quantitative PCR (Q RT-PCR) detection of RA synovial fibroblasts expressing c-FLIP protein and m RNA, the analysis of exogenous CRT on synovial fibroblasts on the expression of c-FLIP and CRT mediated c-FLIP expression evaluation in NF- B signaling pathway in the activation effect. 9. the SPSS 20 software package was used for statistical analysis. The measurement data are (?) (?) + s. T test, SNK-q test, Pearson linear correlation analysis were used to carry out the related statistical treatment. The test level was bilateral alpha =0.05. Results the results of 1.ELISA showed that the content of CRT in synovial fluid of RA patients [(7.2 + 3) ng/ml] was significantly higher than that of OA patients [(3.8 + 0.6) ng/ml] (t=6.724, P0.01). 2. immunohistochemical results showed that CRT and c-FLIP in RA synovial tissues were highly expressed. There were more positive staining in the lining layer, lining layer, inflammatory cells and perivascular tissues of synovial tissues. In comparison, the CRT and c-FLIP in the OA synovial membrane only have a very small amount of expression around the lining and around the blood vessels. The expression of CRT in RA synovial tissue was positively correlated with c-FLIP. 3. the two groups of rigid adherent cells were round, and then extended to long spindle or spindle shaped. The synovial fibroblast primary cells in RA patients began to adhere to the 12~24h after inoculation, and then filled the bottom of the bottle after 4~5 days. The synovial fibroblast primary cells of OA patients adhered slowly and grew slowly. After inoculation, 24~48h began to adhere to the wall, and reached the fusion state until 7~8 days later. After the passage of the cells, the cells grew faster, and the cells of group RA could be passed 1 times per 2~3 day, and the cells of group OA needed to be cultured for 3~4 days. 4.CCK-8 results showed that CRT could promote the proliferation of RA synovial fibroblasts. 5.CRT inhibits the apoptosis of RA synovial fibroblasts induced by apoptosis inducer TRAIL. The results of 6.qRT-PCR, Western Blot and cellular immunofluorescence showed that compared with synovial fibroblasts of OA patients, the m RNA and protein expression levels of CRT and c-FLIP in synovial fibroblasts of RA patients increased significantly. The detection results of 7.q RT-PCR and Western Blot showed that the m RNA expression level and protein expression level of c-FLIP in RA synovial fibroblasts increased after different doses of recombinant human CRT, and the expression level of c-FLIP increased in a concentration dependent manner. 8. under the action of CRT, the expression level of p-NF- kappa B in RA synovial fibroblasts was significantly increased, but the total expression level of NF- kappa B protein was not significantly changed. The effect of CRT on the expression of c-FLIP in RA synovial fibroblasts was inhibited by the use of NF- kappa B signaling pathway inhibitors. Conclusion 1.CRT and c-FLIP expression in synovial tissue of patients with RA were increased, and the CRT and c-FLIP in the synovial tissue of RA patients was positively related to the amount of 2. found expression in vitro, RA synovial fibroblasts, high expression of CRT and c-FLIP showed that CRT can upregulate the expression of fibroblast RA synovial c-FLIP, and in a dose dependent manner. 3.CRT may activate NF- kappa B pathway by regulating the expression of anti apoptotic protein c-FLIP in RA synovial fibroblasts, and then participate in RA synovial tissue hyperplasia and inflammatory process. The results of this study suggest that RA synovial fibroblasts CRT and anti apoptotic protein c-FLIP expression increased, and CRT could regulate the expression of c-FLIP makes synovial fibroblasts apoptosis, and possibly through regulation of c-FLIP activation of NF- B signaling pathway, thereby promoting proliferation and inflammatory reaction in RA synovial tissue, involved in the pathological mechanism of RA.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R593.22
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