RNA干扰特异性沉默Bcl—xl对类风湿关节炎滑膜细胞Bcl—xl Bax Caspase-3表达的影响  

The effect of siRNA targeted against Bcl-xl on the expression of apoptosis relevant factors Bcl-xl, Bax, Caspase-3

作　　者：高薇[1] 冯欣[1] 杨镇嘉[1] 李云霞[1] 任立冰[1] 

机构地区：[1]辽宁医学院附属第一医院风湿免疫科,锦州121001

出　　处：《中华风湿病学杂志》2013年第1期37-40,F0003,共5页Chinese Journal of Rheumatology

基　　金：辽宁省教育厅高等学校科研计划（2008392）

摘　　要：目的探讨RNA干扰特异性沉默Bcl—xl基因后对类风湿关节炎（RA）滑膜细胞增殖及凋亡相关因子Bcl-xl、Bax、天冬氨酸特异性半胱氨酸蛋白酶（Caspase．3）表达的影响。方法采用组织块胶原酶消化法进行人RA关节滑膜细胞原代培养及传代，利用基因库提供的Bcl—xlcDNA序列构建靶向Bcl-xl基因的siRNA质粒，同时合成一个错义序列作为阴性对照。Lipofectaminew2000转染滑膜细胞，分别于转染后24、48、72h应用四甲基偶氧唑蓝（MYr）法评价RNA干扰沉默Bcl-xl后对滑膜细胞增殖的影响；应用蛋白印迹法观察转染后不同时间滑膜细胞凋亡相关因子Bcl—xl、Bax、Caspase．3的蛋白表达情况。多样本均数比较采用单因素方差分析，两两比较采用LSD．t或Tamhane’s严检验对结果进行统计。结果MTT实验发现RNA干扰特异性沉默Bcl—xl后可明显抑制滑膜细胞增殖，其中在转染后48h时其抑制作用最强，随着时间的延长这种抑制作用逐渐减弱，但与空白对照组及阴性对照组比较仍差异有统计学意义（P〈0．05）；蛋白印迹法检泓结果显示与空白及阴性对照组相比siRNA转染后Bcl-xl蛋白表达明显下降（P〈0．01），其中在转染后48h时表达最少，与之相反Bax，Caspase-3蛋白表达则在转染后明显增加。结论RNA干扰特异性沉默Bcl-xl可以明显下调RA滑膜细胞抗凋亡因子Bcl-xl的表达，活化Caspase-3蛋白，从而对抑制滑膜细胞的过度增殖发挥重要作用。Objective To investigate the effect of siRNA targeted against Bcl-xl on cell proliferation of rheumatoid arthritis （RA） and the effect on expression of apoptosis relevant factors Bcl-xl, Bax, Caspase-3. Methods Human RA syuovial cells were cukured and passed by tissue block collagenase digestion method. The siRNA plasmid targeting Bcl-xl gene was constructed by Bcl-xl cDNA sequence provided by gene banks, while single missense sequences were used as negative controls. LipofectamineTM 2000 was used to transfect synovial cells. The effect of Bel-xl silencing on proliferation of synovial cells was evaluated by MTT at 24, 48, 72 hours after transfeetion. The expressions of Bcl-xl, Bax, Caspase-3 protein, synovial apoptosisrel- ated factors were determined by Western blotting after transfeeted at different time points. The average of multiple-sample average was analyzed by single-factor X2 test or LSD-t and Tamhane＇s T2 test was used for two- two comparison. Results MTT result s showed that RNA interference that specifically silent Bcl-xl could obviously suppress the proliferation of sliding film cells, which was most eveident at 48 hour.~. This inhibition was gradually weakened with prolonged time, but when compared was the control group, differences was signi- ficant （P〈O.05）. Western blotting results displayed that when compared to the control group, Bcl-xl protein expression obviously declined after transfection P〈0.01, which was the least at 48 h time point. Bax, Caspase- 3 protein expression were obviously increased when compared to the coutrol group. Conclusion The expression of Bcl-xl, a RA synovial cell anti-apoptotic factor, is significantly reduced by specific RNA interference silencing Bcl-xl, which may play an important role in inhibiting the excessive proliferation of synovial ceils.

关 键 词：关节炎 类风湿 RNA干扰 细胞凋亡 Bcl—xl 

分 类 号：R394[医药卫生—医学遗传学]