中华医学杂志
中華醫學雜誌
중화의학잡지
National Medical Journal of China
2011年
20期
1417-1421
,共5页
马东蔚%王秋月%陈启光%吴丹%王娇%侯鲁鲁
馬東蔚%王鞦月%陳啟光%吳丹%王嬌%侯魯魯
마동위%왕추월%진계광%오단%왕교%후로로
ρA GTP结合蛋白质%RNA干扰%肾小球系膜细胞%RhoA/ROCK信号通路
ρA GTP結閤蛋白質%RNA榦擾%腎小毬繫膜細胞%RhoA/ROCK信號通路
ρA GTP결합단백질%RNA간우%신소구계막세포%RhoA/ROCK신호통로
rhoA GTP-binding protein%RNA interference%Mesangial cells%RhoA/ROCK signaling pathway
目的 观察小G蛋白(RhoA)小干扰RNA(Stealth RNAm 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.
目的 觀察小G蛋白(RhoA)小榦擾RNA(Stealth RNAm siRNA)對高糖刺激下的人腎小毬繫膜細胞(HMC)炎癥反應及纖維化的影響,探討RhoA/ROCK信號通路在糖尿病腎病髮生髮展中的作用.方法 傳代培養的HMC同步化後分組,LipofectaminerTM2000脂質體轉染抑製RhoA錶達的Stealth RNA或無關的siRNA,用熒光倒置顯微鏡觀察各組轉染效率,利用實時定量RT-PCR、ELISA技術檢測RhoA、ROCK-I、纖維連接蛋白(FN)、結締組織生長因子(CTGF)、腫瘤壞死因子-α(TNF-α)的錶達情況.結果 (1)RhoA-siRNA轉染可抑製高糖刺激的HMC中P&oA、ROCK-I、CTGF mRNA的錶達水平,各mRNA的錶達較高糖組少26%-60%(均P<0.05).(2)RhoA-siRNA轉染HMC後繼續培養48 h,FN、CTGF和TNF-α儀的蛋白錶達水平較高糖組明顯少[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],幾乎使FN和CTGF下降到正常糖培養HMC的水平.結論 通過RNA榦擾技術抑製RhoA的錶達,可減少高糖刺激下HMC中FN、CTGF、TNF-α的積聚,降低細胞外基質的蓄積,減少腎小毬的纖維化和炎癥反應,為糖尿病腎病的防治提供新的榦預靶點.
목적 관찰소G단백(RhoA)소간우RNA(Stealth RNAm siRNA)대고당자격하적인신소구계막세포(HMC)염증반응급섬유화적영향,탐토RhoA/ROCK신호통로재당뇨병신병발생발전중적작용.방법 전대배양적HMC동보화후분조,LipofectaminerTM2000지질체전염억제RhoA표체적Stealth RNA혹무관적siRNA,용형광도치현미경관찰각조전염효솔,이용실시정량RT-PCR、ELISA기술검측RhoA、ROCK-I、섬유련접단백(FN)、결체조직생장인자(CTGF)、종류배사인자-α(TNF-α)적표체정황.결과 (1)RhoA-siRNA전염가억제고당자격적HMC중P&oA、ROCK-I、CTGF mRNA적표체수평,각mRNA적표체교고당조소26%-60%(균P<0.05).(2)RhoA-siRNA전염HMC후계속배양48 h,FN、CTGF화TNF-α의적단백표체수평교고당조명현소[FN:(1.99±0.04)mg/L비(4.31±0.13)ms/L,CTGF:(4.98-1-0.17)mg/L비(6.06±0.09)ms/L;TNF-α:(61.17±2.59)ns/L비(91.76±2.27)ng/L;균P<0.05],궤호사FN화CTGF하강도정상당배양HMC적수평.결론 통과RNA간우기술억제RhoA적표체,가감소고당자격하HMC중FN、CTGF、TNF-α적적취,강저세포외기질적축적,감소신소구적섬유화화염증반응,위당뇨병신병적방치제공신적간예파점.
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.
