RICTOR对类风湿关节炎成纤维样滑膜细胞活力的影响  

Effect of RICTOR expression on cell viability in rheumatoid arthritis fibroblast-like synoviocytes

作　　者：郭欣[1] 胡爱玲[2] 方霖楷[1] 刘岩[1] 潘云峰[1] 

机构地区：[1]中山大学附属第三医院风湿免疫科,广东广州510630 [2]中山大学附属第三医院护理部,广东广州510630

出　　处：《中国病理生理杂志》2013年第3期526-530,共5页Chinese Journal of Pathophysiology

基　　金：广东省科技计划(No.2012B031800363)

摘　　要：目的:通过siRNA介导的RNA干扰技术沉默类风湿关节炎(RA)成纤维样滑膜细胞(FLS)mTORC2的特异组成蛋白RICTOR的表达,观察其对细胞活力的影响。方法:组织块法培养RA-FLS。应用阳离子脂质体转染的方法,把化学合成的特异性RICTOR siRNA转染RA-FLS,并以转染非特异性siRNA作为阴性对照。利用荧光定量PCR法分析转染24 h后细胞RICTOR mRNA表达水平的变化;Western blotting法分析转染48和72 h后细胞RICTOR蛋白表达水平的变化;以噻唑蓝(MTT)比色法检测转染成纤维样滑膜细胞不同时间(24、48和72h)RICTOR siRNA对细胞活力的影响。结果:荧光定量PCR结果显示特异性RICTOR siRNA转染组与对照组相比,细胞中RICTOR的mRNA表达水平显著下调,24 h干扰效率达78.3%±63.71%(P<0.01)。Western blotting结果显示与对照组相比,RICTOR siRNA转染组48 h和72 h后RICTOR蛋白表达水平明显降低,沉默效率分别为92.48%±6.14%和98.57%±1.40%(均P<0.01)。MTT结果显示,早期(24和48 h)RICTOR siRNA转染组与阴性对照组细胞存活率比较无显著差异;72 h后,RICTOR siRNA转染组与阴性对照相比,细胞活力明显降低,抑制率为90.14%±1.90%(P<0.01)。结论:转染特异性RICTOR siRNA可降低RA-FLS的活力,提示mTORC2可能与RA-FLS的生长有关。AIM ： To investigate the effects of RICTOR expression on the viability of rheumatoid arthritis fibro- blast-like synoviocytes （RA-FLS）. METHODS： RA-FLS were obtained by tissue culture. Chemically synthesized double- stranded siRNAs targeting RICTOR gene were transfected into RA-FLS by cationic liposome. The nonspecific siRNA was al- so transfected into the negative control cells. The mRNA expression of RICTOR was detected by RT-qPCR after transfection for 24 h. Western blotting was used to evaluate the inhibitory effects of siRNAs on RICTOR expression after transfection for 48 h and 72 h. The cell viability was examined by MTT assay. RESULTS： Compared with control group, the mRNA ex- pression of RICTOR significantly decreased by 78.36% ± 3.71% after the transfection of RICTOR siRNA for 24 h. The protein level of RICTOR was also obviously lower in RICTOR siRNA transfection group than that in control group after transfection for 48 h （ decreased by 92.48% ± 6.14% ） and 72 h （ decreased by 98.57% ± 1.40% ）. Knock-down of R/C- TOR in RA-FLS for 72 h markedly decreased the cell viability. CONCLUSION： Transfection of RICTOR siRNA reduces the viability of RA-FLS, indicating that mTORC2 may be required for the survival of RA-FLS.

关 键 词：RICTOR蛋白 关节炎 类风湿 成纤维样滑膜细胞 RNA干扰 

分 类 号：R593.22[医药卫生—内科学]