本文關(guān)鍵詞：鈣網(wǎng)織蛋白通過調(diào)節(jié)c-FLIP表達促進類風濕關(guān)節(jié)炎滑膜增生的分子機制研究　出處：《天津醫(yī)科大學》2016年碩士論文　論文類型：學位論文

  更多相關(guān)文章： 類風濕關(guān)節(jié)炎 鈣網(wǎng)織蛋白 細胞凋亡 細胞源性 FLICE 抑制蛋白 NF-κB信號通路

【摘要】：目的類風濕關(guān)節(jié)炎(Rheumatoid arthritis,RA)是一種以滑膜組織增生及慢性關(guān)節(jié)滑膜炎癥浸潤為主要病理特征的系統(tǒng)性自身免疫病,關(guān)節(jié)滑膜成纖維細胞的異常增殖及凋亡減少是滑膜組織增生的重要發(fā)病機制之一。已有研究報道鈣網(wǎng)織蛋白(Calreticulin,CRT)可能與RA的發(fā)病有關(guān)。本課題組前期研究亦發(fā)現(xiàn),CRT在RA患者血清、滑膜及關(guān)節(jié)液中均呈現(xiàn)高表達,血清CRT表達量與RA患者的疾病活動度評分DAS28正相關(guān),且CRT可通過NO途徑參與RA滑膜血管翳的形成。有研究報道CRT可抑制胞外死亡受體途徑之一的Fas-Fas L介導的T細胞凋亡進而參與RA的發(fā)病機制。本課題擬探討CRT通過調(diào)節(jié)抗凋亡蛋白c-FLIP表達進而抑制胞外死亡受體途徑介導的RA滑膜成纖維細胞凋亡,進而參與RA滑膜增生及炎癥浸潤的發(fā)病機制,為臨床RA滑膜炎癥的靶向治療提供新的理論依據(jù)和實驗基礎。方法1.于2014年1月至2015年5月收集行關(guān)節(jié)鏡及膝關(guān)節(jié)置換術(shù)患者的標本進行實驗研究。關(guān)節(jié)滑液標本包括:RA患者32例,OA患者37例。滑膜組織標本包括:RA患者14例,OA患者19例。2.采用酶聯(lián)免疫吸附試驗(ELISA)檢測RA及OA患者關(guān)節(jié)滑液中CRT的含量。3.采用免疫組織化學方法分析CRT和c-FLIP蛋白在RA及OA滑膜組織中的表達和定位并對結(jié)果進行半定量分析;同時對CRT及c-FLIP的表達量進行相關(guān)性分析。4.采用聯(lián)合酶消化法分離RA及OA滑膜成纖維細胞并進行培養(yǎng),觀察分析RA及OA滑膜成纖維細胞在體外生長的特性。5.采用CCK-8試驗分析CRT對RA滑膜成纖維細胞增殖能力的影響。6.采用流式細胞技術(shù)分析CRT對TRAIL誘導的RA滑膜成纖維細胞凋亡的影響。7.采用細胞免疫熒光方法檢測RA滑膜成纖維細胞中CRT及c-FLIP蛋白的表達及細胞定位。8.采用免疫印跡法(Western Blot)及實時熒光定量PCR(q RT-PCR)檢測RA滑膜成纖維細胞中c-FLIP蛋白及m RNA的表達,分析外源性CRT對滑膜成纖維細胞表達c-FLIP的影響并評估CRT介導的c-FLIP表達在NF-κB信號通路活化中的作用。9.采用SPSS 20.0軟件包進行統(tǒng)計學分析。計量資料以(?)±s表示。分別采用t檢驗、SNK-q檢驗、Pearson線性相關(guān)性分析等進行相關(guān)的統(tǒng)計學處理。檢驗水準為雙側(cè)α=0.05。結(jié)果1.ELISA結(jié)果顯示,RA患者關(guān)節(jié)滑液中CRT含量[(7.2±3.0)ng/ml]顯著高于OA患者[(3.8±0.6)ng/ml](t=6.724,P0.01)。2.免疫組織化學結(jié)果顯示,RA關(guān)節(jié)滑膜組織CRT和c-FLIP均呈高表達,滑膜組織的襯里層和襯里下層、炎性細胞及血管周圍有較多陽性染色。相較而言,OA滑膜中CRT和c-FLIP僅在襯里層及血管周圍有極少量的表達。RA滑膜組織中CRT的表達量與c-FLIP呈正相關(guān)。3.兩組剛貼壁的細胞呈圓形,后逐漸延伸為長梭形或紡錘形。RA患者滑膜成纖維細胞原代細胞在接種后12~24h即開始貼壁,4~5天后鋪滿瓶底,OA患者滑膜成纖維細胞原代細胞貼壁生長緩慢,接種后24~48h開始貼壁,直至7~8天后達到融合狀態(tài)。傳代后細胞生長變快,RA組細胞每2~3天即可傳代1次,而OA組細胞傳代需培養(yǎng)3~4天。4.CCK-8結(jié)果顯示,CRT具有促進RA滑膜成纖維細胞增殖的作用。5.CRT對凋亡誘導劑TRAIL誘導的RA滑膜成纖維細胞凋亡具有抑制作用。6.qRT-PCR、Western Blot以及細胞免疫熒光檢測結(jié)果均表明,與OA患者的滑膜成纖維細胞相比,RA患者滑膜成纖維細胞中CRT和c-FLIP的m RNA及蛋白表達水平均明顯升高。7.q RT-PCR和Western Blot的檢測結(jié)果顯示,不同劑量的人重組CRT作用后,RA滑膜成纖維細胞中c-FLIP的m RNA表達水平和蛋白表達水平均升高,且c-FLIP表達水平的升高呈CRT濃度依賴性。8.在CRT的作用下,RA滑膜成纖維細胞中p-NF-κB表達水平明顯升高,而總NF-κB蛋白表達水平?jīng)]有明顯的改變。應用NF-κB信號通路抑制劑后,CRT上調(diào)RA滑膜成纖維細胞中c-FLIP表達的作用被抑制。結(jié)論1.CRT與c-FLIP在RA患者滑膜組織中的表達量均升高,且CRT與c-FLIP在RA患者滑膜組織中表達量呈正相關(guān)2.體外實驗中發(fā)現(xiàn),RA滑膜成纖維細胞中,CRT與c-FLIP均呈高表達,CRT可上調(diào)RA滑膜成纖維細胞c-FLIP的表達,且具有劑量依賴性。3.CRT可能通過調(diào)節(jié)RA滑膜成纖維細胞中抗凋亡蛋白c-FLIP的表達激活NF-κB通路進而參與RA滑膜組織增生及炎癥持續(xù)過程。本課題研究結(jié)果提示RA患者滑膜成纖維細胞CRT及抗凋亡蛋白c-FLIP表達增加,且CRT可能通過調(diào)控c-FLIP的表達使得滑膜成纖維細胞凋亡減少,且可能通過對c-FLIP的調(diào)節(jié)激活NF-κB信號通路,進而促進RA滑膜組織增生及炎癥反應,參與RA病理機制。
[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.
【學位授予單位】：天津醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R593.22
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本文編號：1343903