RICTOR在肿瘤坏死因子-α参与的类风湿关节炎成纤维样滑膜细胞活化中的作用  

Role of RICTOR on the tumor necrosis factor-α associated cell activation in rheumatoid arthritis fibrobast-like synoviocytes

作　　者：郭欣[1] 潘云峰[2] 方霖楷[2] 郭兴华[2] 刘岩[2] 吴云婷 

机构地区：[1]广东省第二人民医院风湿免疫科,广州510630 [2]中山大学附属第三医院风湿免疫科

出　　处：《中华风湿病学杂志》2015年第1期42-45,共4页Chinese Journal of Rheumatology

基　　金：广东省科技计划（2012B031800363）

摘　　要：目的 初步探讨RICTOR在RA-成纤维样滑膜细胞（FLS）活化机制中的作用.方法 收集3例RA患者滑膜组织,应用组织块法培养FLS.分别以浓度为10、20 ng/ml的TNF-α处理RA-FLS,利用蛋白印迹法检测RICTOR的蛋白表达水平.应用阳离子脂质体把特异性RICTOR siRNA转染RA-FLS后,再以10 ng/ml的TNF-α处理RA-FLS,使用蛋白印迹法检测RICTOR的蛋白表达水平,四甲基偶氮唑盐（MTT）法检测RA-FLS活性抑制率.统计学处理采用单因素方差分析及LSD-t检验.结果 TNF-α处理RA-FLS 48 h后,RICTOR的蛋白表达较阴性对照组明显升高（均P＜0.05）.转染RICTOR siRNA后,RICTOR蛋白表达：对照组＜TNF 10 ng/ml组＜TNF 20 ng/ml组,吸光度相对值依次为0.35±0.06、0.60±0.09、1.10±0.12,组间两两比较差异有统计学意义（均P＜0.05）.转染RICTOR siRNA后,阴性对照组、RICTOR（-）/TNF-α（-）组、RICTOR （-）/TNF-α（＋）组RICTOR蛋白吸光度相对值分别为0.4984±0.1401、0.0128±0.002 0、0.0425±0.0273,MTT检测阴性对照组、RICTOR（-）/TNF-α（-）组、RICTOR（-）/TNF-α（＋）组、RICTOR（＋）/TNF-α（＋）组细胞活性抑制率分别为（0.7±2.0）％、（13.3±4.5）％、（15.5±4.9）％、（-7.7±5.2）％,显示无论是否加入TNF-α刺激,RA-FLS中RICTOR的蛋白表达水平均较转染阴性对照siRNA组明显下降,且RA-FLS活性抑制率较转染阴性对照siRNA组明显增高（均P＜0.05）.结论 沉默RICTOR所引起的RA-FLS活性抑制不能被TNF-α逆转,提示RICTOR可能在TNF-α参与的RA-FLS增殖活化机制中起着重要作用.Objective To study the role of RICTOR on rheumatoid arthritis-fibrobast-like synoviocytes （RA-FLS） activation.Methods FLS were isolated from the primary synovial tissues,which were obtained during joint replacement surgery or arthroscopy from three patients with RA.RA-FLS were stimulated with TNF-α at the dose of 10 ng/ml and 20 ng/ml for 48 h.The expression of RICTOR was detected by western blotting.Chemically synthesized RICTOR gene targeted for double-stranded siRNAs were transfected into RA-FLS by cationic liposome.After being transfected with RICTOR siRNA for 48 h,RA-FLS was treated with or without TNF-α for 48 h.The expression of RICTOR was evaluated by western blotting,and the cell viability was analyzed by methylthiazoltetrazolium （MTT） assay.The data were analyzed using analysis of variance （ANOVA） and LDL-t test.Results The expression of RICTOR protein was significantly higher in the TNF-α stimulated group （at the dose of 10 ng/ml and 20 ng/ml for 48 h） than that in the control group （bothP＜0.05）,while the mean change of RICTOR/GAPDH ratios of band optical density x＋s was 0.35±0.06 for the control group,0.60±0.09 for the TNF 10 ng/ml group and 1.10±0.12 for the TNF 20 ng/ml group.Moreover,the expression of RICTOR protein was obviously decreased in RICTOR siRNA transfection groupthan that in control after being trans-fected for 96 h （both P＜0.05）,and ratios of control group,RICTOR （-）/TNF-α（-） group and RICTOR（-）/TNF-α（＋） group was 0.498 4±0.140 1,0.012 8±0.002 0,0.042 5±0.027 3respectively.After the silence of RICTOR,the cell viability decreased in RA-FLS,no matter with or without TNF-α for 48 h later （both P＜0.05）.Conclusion These results indicat that RICTOR might play an important role in the TNF-α associated activation of RA-FLS.

关 键 词：关节炎 类风湿 成纤维样滑膜细胞 肿瘤坏死因子-Α RICTOR 

分 类 号：R593.22[医药卫生—内科学]