转染靶向RhoA基因小干扰RNA对高糖刺激下人肾小球系膜细胞RhoA/ROCK信号通路表达的影响  

Effects of inhibiting RhoA by Stealth RNA on high glucose-induced RhoA/ROCK signaling pathway in human mesangial cells

作　　者：马东蔚[1] 王秋月[1] 陈启光[2] 吴丹[3] 王娇[1] 侯鲁鲁[1] 

机构地区：[1]中国医科大学附属第一医院内分泌科,沈阳110001 [2]中国医科大学附属第一医院泌尿外科 [3]沈阳市二四二医院干诊科

出　　处：《中华医学杂志》2011年第20期1417-1421,共5页National Medical Journal of China

基　　金：辽宁省教育厅2008年度高等学校科研项目（2008800）;辽宁省医学高峰建设工程重点科研项目（200938）

摘　　要：目的 观察小G蛋白（RhoA）小干扰RNA（Stealth RNA^TM siRNA）对高糖刺激下的人肾小球系膜细胞（HMC）炎症反应及纤维化的影响,探讨RhoA/ROCK信号通路在糖尿病肾病发生发展中的作用.方法 传代培养的HMC同步化后分组,LipofectaminerTM2000脂质体转染抑制RhoA表达的Stealth RNA或无关的siRNA,用荧光倒置显微镜观察各组转染效率,利用实时定量RT-PCR、ELISA技术检测RhoA、ROCK-I、纤维连接蛋白（FN）、结缔组织生长因子（CTGF）、肿瘤坏死因子-α（TNF-α）的表达情况.结果 （1）RhoA-siRNA转染可抑制高糖刺激的HMC中P＆oA、ROCK-I、CTGF mRNA的表达水平,各mRNA的表达较高糖组少26%-60%（均P〈0.05）.（2）RhoA-siRNA转染HMC后继续培养48 h,FN、CTGF和TNF-α仪的蛋白表达水平较高糖组明显少[FN：（1.99±0.04）mg/L比（4.31±0.13）ms/L,CTGF：（4.98-1-0.17）mg/L比（6.06±0.09）ms/L;TNF-α：（61.17±2.59）ns/L比（91.76±2.27）ng/L;均P〈0.05],几乎使FN和CTGF下降到正常糖培养HMC的水平.结论 通过RNA干扰技术抑制RhoA的表达,可减少高糖刺激下HMC中FN、CTGF、TNF-α的积聚,降低细胞外基质的蓄积,减少肾小球的纤维化和炎症反应,为糖尿病肾病的防治提供新的干预靶点.Objective To observe the effect of small interference RNA（Steahh RNAiTM siRNA）of RhoA on the inflammatory response and fibrosis in human mesangial cell（HMC）and explore the role of RhoA/ROCK signaling pathway in the process of diabetic nepropathy.Methods Synchronized HMC were divided into several groups.LipofectamineTM2000 was employed to transfect RhoA-siRNA and RhoA-negative siRNA into the above cells.RhoA-siRNA could inhibit the expression of RhoA.The expressions of RhoA,ROCK-I,fibronectin（FN）,connective tissue growth factor（CTGF）and tumor necrosis factor-alpha（TNF-α）were detected by real-time reverse transeription-polymerase chain reaction（RT.PCR）and ELISA（enzyme-linked immunosorbent assay）.Resuits （1）The expressions of RhoA,ROCK-I and CTGF mRNA were inhibited by RhoA siRNA transfection in high glucose-induced HMC.The expression of each mRNA was reduced 26%-60%as compared with the high glucose-induced group（P〈0.05）.（2）After RhoA siRNA transfection and culturing with high glucose for 48 h.FN, the secretions of CTGF and TNF-αsignificantly declined[FN：（1.99±0.04）mg/L vs（4.31±0.13）mg/L,CTGF：（4.98±0.17）ms/L vs（6.06±0.09）mg/L;TNF-α[：（61.17±2.59）ng/L vs（91.76±2.27）ng/L,all P〈0.05].The levels of FN and CTGF almost decreased to those of normal glucose-induced HMC.Conclusions The levels of FN,CTGF and TNF-αin higsh glucose-induced HMC may be lowered by inhibiting RhoA through RNA interference and reducing the accumulation of extracellular matrix, glomerular fibrosis and inflammation.Thus it provides a new intervention target for the prevention of diabetic nephropathy.

关 键 词：ρA GTP结合蛋白质 RNA干扰 肾小球系膜细胞 RhoA/ROCK信号通路 

分 类 号：R587.2[医药卫生—内分泌]