中华肾脏病杂志
中華腎髒病雜誌
중화신장병잡지
2012年
8期
616-621
,共6页
谭若芸%方奕%苏卫芳%杨俊伟%张炜%顾民
譚若蕓%方奕%囌衛芳%楊俊偉%張煒%顧民
담약예%방혁%소위방%양준위%장위%고민
肝细胞生长因子%纤维化%转化生长因子β1%Smad泛素化调节因子2%上皮细胞-间充质细胞转分化
肝細胞生長因子%纖維化%轉化生長因子β1%Smad汎素化調節因子2%上皮細胞-間充質細胞轉分化
간세포생장인자%섬유화%전화생장인자β1%Smad범소화조절인자2%상피세포-간충질세포전분화
Hepatocyte growth factor%Fibrosis%Transforming growth factor beta1%Smurf2%Epithelial-mesenchymal transition
目的 通过观察肝细胞生长因子( HGF)对正常大鼠肾上皮细胞(NRK-52E)转化生长因子β1( TGF-β1)诱导的Smad泛素化调节因子2(Smurf2)表达的影响,探讨HGF拮抗肾小管上皮细胞-间充质细胞转分化(EMT)的分子机制.方法 以NRK-52E细胞为研究对象,给予TGF-β1(5μg/L)处理0～24 h；或部分细胞经HGF(20 μg/L)预处理30 min后,再予TGF-β1(5μg/L)处理1h或48 h；另外部分细胞予以Smurf2质粒表达载体或Smurf2 siRNA 转染24 h后,再予HGF处理24 h.应用Western印迹及间接免疫荧光染色方法检测Smurf2、SnoN、E钙黏蛋白(E-cadherin)、α平滑肌肌动蛋白(α-SMA)和纤连蛋白(FN)的表达.结果 与对照组相比,TGF-β1处理后迅速上调NRK-52E细胞中Smurf2蛋白表达(P＜0.01)；同时显著诱导FN和α-SMA蛋白表达,并下调E-cadherin表达.而予HGF预处理细胞后,可快速抑制TGF-β1诱导的Smurf2表达上调(P＜0.01)；并逆转TGF-β1介导的SnoN(P＜0.01)、E-cadherin(P＜0.05)、α-SMA(P＜0.01)和FN(P＜0.01)表达变化.此外,在NRK-52E细胞中过表达Smurf2蛋白可部分抑制HGF诱导的SnoN蛋白上调；而抑制Smurf2表达则可进一步促进HGF诱导的SnoN蛋白表达.结论 在肾小管上皮细胞中,HGF可能通过下调Smurf2表达抑制SnoN蛋白发生泛素-蛋白酶体依赖性降解,进而拮抗TGF-β1介导EMT形成.
目的 通過觀察肝細胞生長因子( HGF)對正常大鼠腎上皮細胞(NRK-52E)轉化生長因子β1( TGF-β1)誘導的Smad汎素化調節因子2(Smurf2)錶達的影響,探討HGF拮抗腎小管上皮細胞-間充質細胞轉分化(EMT)的分子機製.方法 以NRK-52E細胞為研究對象,給予TGF-β1(5μg/L)處理0～24 h；或部分細胞經HGF(20 μg/L)預處理30 min後,再予TGF-β1(5μg/L)處理1h或48 h；另外部分細胞予以Smurf2質粒錶達載體或Smurf2 siRNA 轉染24 h後,再予HGF處理24 h.應用Western印跡及間接免疫熒光染色方法檢測Smurf2、SnoN、E鈣黏蛋白(E-cadherin)、α平滑肌肌動蛋白(α-SMA)和纖連蛋白(FN)的錶達.結果 與對照組相比,TGF-β1處理後迅速上調NRK-52E細胞中Smurf2蛋白錶達(P＜0.01)；同時顯著誘導FN和α-SMA蛋白錶達,併下調E-cadherin錶達.而予HGF預處理細胞後,可快速抑製TGF-β1誘導的Smurf2錶達上調(P＜0.01)；併逆轉TGF-β1介導的SnoN(P＜0.01)、E-cadherin(P＜0.05)、α-SMA(P＜0.01)和FN(P＜0.01)錶達變化.此外,在NRK-52E細胞中過錶達Smurf2蛋白可部分抑製HGF誘導的SnoN蛋白上調；而抑製Smurf2錶達則可進一步促進HGF誘導的SnoN蛋白錶達.結論 在腎小管上皮細胞中,HGF可能通過下調Smurf2錶達抑製SnoN蛋白髮生汎素-蛋白酶體依賴性降解,進而拮抗TGF-β1介導EMT形成.
목적 통과관찰간세포생장인자( HGF)대정상대서신상피세포(NRK-52E)전화생장인자β1( TGF-β1)유도적Smad범소화조절인자2(Smurf2)표체적영향,탐토HGF길항신소관상피세포-간충질세포전분화(EMT)적분자궤제.방법 이NRK-52E세포위연구대상,급여TGF-β1(5μg/L)처리0～24 h；혹부분세포경HGF(20 μg/L)예처리30 min후,재여TGF-β1(5μg/L)처리1h혹48 h；령외부분세포여이Smurf2질립표체재체혹Smurf2 siRNA 전염24 h후,재여HGF처리24 h.응용Western인적급간접면역형광염색방법검측Smurf2、SnoN、E개점단백(E-cadherin)、α평활기기동단백(α-SMA)화섬련단백(FN)적표체.결과 여대조조상비,TGF-β1처리후신속상조NRK-52E세포중Smurf2단백표체(P＜0.01)；동시현저유도FN화α-SMA단백표체,병하조E-cadherin표체.이여HGF예처리세포후,가쾌속억제TGF-β1유도적Smurf2표체상조(P＜0.01)；병역전TGF-β1개도적SnoN(P＜0.01)、E-cadherin(P＜0.05)、α-SMA(P＜0.01)화FN(P＜0.01)표체변화.차외,재NRK-52E세포중과표체Smurf2단백가부분억제HGF유도적SnoN단백상조；이억제Smurf2표체칙가진일보촉진HGF유도적SnoN단백표체.결론 재신소관상피세포중,HGF가능통과하조Smurf2표체억제SnoN단백발생범소-단백매체의뢰성강해,진이길항TGF-β1개도EMT형성.
Objective To investigate the possible mechanism that hepatocyte growth factor (HGF) inhibits renal tubular epithelial-mesenchymal transition (EMT),and to determine whether Smurf2 expression induced by TGF-β1 can be reversed by HGF in normal rat kidney epithelial cells (NRK-52E).Methods Using rat NRK-52E cell line as an in vitro system,NRK-52E cells were incubated with 5 μg/L TGF-β1 for 0-24 h.Part of cells were pretreated with 20 μg/L HGF for 30 min or not,then incubated with or without 5 μg/L TGF-β1 for 1 h or 48 h.The other cells were transfected with pFlag-Smurf2 or Smurf2 siRNA for 24 h,then treated with or without 20 μg/L HGF for 24 h.The expressions of Smurf2,SnoN,E-cadherin,alpha-smooth muscle actin (α-SMA) and fibronectin (FN) were detected by Western blotting and indirect immunofluorescence staining assays.Results Compared to normal control,TGF-β1 could rapidly induce Smurf2 protein expression in a short time (P＜0.01).Meanwhile,the expressions of FN and α-SMA were significantly induced,and the expression of E-cadherin was reduced in NRK-52E cells by TGF-β1.In contrast,in the NRK-52E cells pretreated with HGF,HGF could obviously inhibit Smurf2 expression induced by TGF-β1,and reversed the down-regulation of SnoN (P＜0.01) and E-cadherin (P＜0.05),the up-regulation of α-SMA (P＜0.01) and FN (P＜0.01) induced by TGF-β1.Moreover,overexpression of Smurf2 in NRK-52E cells could partly inhibit the up-regulation of SnoN protein by HGF,while down-regulation of Smurf2 could up-regulate the expression of SnoN induced by HGF.Conclusions HGF can abolish EMT induced by TGF-β1 in renal tubular epithelial cells through down-regulating Smurf2 expression and suppressing ubiquitin-proteasome dependent degradation of SnoN.
