类风湿关节炎患者外周血单个核细胞中c-FLIP与外源性凋亡途径的相关性分析

Expression of c-FLIP in peripheral blood mononuclear cells of patients with rheumatoid arthritis and its relation with extrinsic apoptotic pathway

目的:分析类风湿关节炎患者外周血单个核细胞(PBMC)中Fas相关死亡区样白介素1转换酶抑制蛋白(c-FLIP)与外源性凋亡途径相关蛋白死亡结构域蛋白(FADD)、caspase-8表达水平变化的相关性。方法:选择2014年1月至2015年6月福建医科大学附属漳州市医院类风湿关节炎患者60例,按照类风湿关节炎疾病活动指数将患者分为低、中、高活动度组(分别为22、20和18例)。同时选择健康体检者25名作为健康对照组。采用实时定量RT-PCR和蛋白质印迹法分别检测PBMC中c-FLIP、FADD和caspase-8的mRNA和蛋白表达水平,并采用Pearson检验对c-FLIP与FADD、caspase-8表达的相关性进行分析。结果:类风湿关节炎患者PBMC中c-FLIP、FADD和caspase-8的mRNA表达量均高于健康对照组(均P <0. 05)。其中,中活动度组PBMC中FADD和caspase-8的mRNA表达量高于低活动度组(均P <0. 05),c-FLIP的mRNA表达量与低活动度组相近(P>0. 05);高活动度组PBMC中c-FLIP的mRNA表达量高于中、低活动度组(均P <0. 05),caspase-8的mRNA表达量低于中、低活动度组(均P <0. 05),FADD的mRNA表达量高于低活动度组(P <0. 05)。类风湿关节炎患者PBMC中c-FLIP mRNA水平与FADD mRNA水平呈正相关(r=0. 323,P <0. 05),与caspase-8mRNA水平呈负相关(r=-1. 104,P <0. 05)。中活动度组c-FLIP和FADD蛋白表达量高于健康对照组、低活动度组和高活动度组(P <0. 05或P <0. 01);中、高活动度组caspase-8蛋白表达量高于健康对照组和低活动度组(P <0. 05或P <0. 01),且高活动度组caspase-8蛋白表达量高于中活动度组(P <0. 05)。结论:类风湿关节炎患者PBMC中凋亡相关蛋白c-FLIP可能参与了外源性凋亡途径,并可为类风湿关节炎病情评估提供参考。

Objective: To investigate the expression of apoptosis related protein cellular Fas associated death domain like interleukin 1 converting enzyme inhibitory protein( c-FLIP) in peripheral blood mononuclear cells( PBMCs) of patients with rheumatoid arthritis and its relation with extrinsic apoptotic pathway. Methods: Sixty patients with rheumatoid arthritis were collected from Zhangzhou Affiliated Hospital of Fujian Medical University during January 2014 and June 2015,including 22 patients with low activities( DAS28 < 3. 2),20 patients with middle activities( 3. 2≤DAS28≤5. 1),and 18 patients with high activities( DAS28 > 5. 1). And 25 healthy controls were also collected. The mRNA and protein expression levels of c-FLIP and the extrinsic apoptotic pathway related proteins Fas-associated protein with death domain( FADD),caspase-8 in PBMCs were detected by real-time RT-PCR and Western blot,respectively. Correlations between c-FLIP and FADD, caspase-8 in PBMCs were analyzed by pearson test. Results: mRNA expression levels of c-FLIP,FADD and caspase-8 in PBMCs of patients with rheumatoid arthritis were all higher than those of healthy controls( all P < 0. 05). mRNA expression levels of FADD and caspase-8 in patients with middle activities were significantly higher than those in patients with low activities( all P < 0. 05),but the mRNA expression level of c-FLIP was not significantly higher than that in patients with low activities. mRNA expression level of c-FLIP in patients with high activities was higher than those in patients with middle or low activities( all P <0. 05),while the mRNA expression level of caspase-8 was lower than those in patients with middle or low activities( all P < 0. 05). mRNA expression level of FADD in patients with high activities was higher than those in patients with low activities( P <0. 05). Pearson analysis showed that there was a positive correlation between c-FLIP and FADD mRNA expression( r = 0. 323,P < 0. 05),and negative correlation between c-FLIP and caspase-8 mRNA expression( r =-1. 104,P < 0. 05). The protein expression levels of c-FLIP and FADD in patients with middle activities were significantly higher than those in control group and patients with low or high activities( P < 0. 05 or 0. 01). The protein expression levels of caspase-8 in patients with middle and high activities were significantly higher than those in control group and patients with low activities( P < 0. 05 or P < 0. 01),and the protein expression level of caspase-8 in patients with high activities was higher than that in patients with middle activities( P <0. 05). Conclusion: c-FLIP may be involved in the extrinsic apoptotic pathway in rheumatoid arthritis,and can provide reference for the evaluation of disease activities.