Smad3 shRNA抑制TGFβ1对HSC-T6细胞活化的抗肝纤维化作用  

ShRNA targeted to rat Smad3 decreases the profibrosis role of TGFβ1 in HSC-T6 cells

作　　者：郑素军[1] 邢欣悦[1] 武聚山[1] 王世美[1] 张莹[1] 韩源平[1] 俞豪[2] 陈煜[1] 刘梅[1] 段钟平[1] 

机构地区：[1]首都医科大学附属北京佑安医院人工肝中心,北京100069 [2]首都医科大学基础医学院细胞生物学系,北京100069

出　　处：《临床肝胆病杂志》2012年第4期289-295,共7页Journal of Clinical Hepatology

基　　金：国家自然科学基金资助项目(30800979);北京自然科学基金资助项目(7102085);北京市科技新星资助项目(2007B055);北京市卫生系统高层次卫生人才培养计划(2011-3-083);北京市教育委员会科技发展计划项目(KM201010025024)

摘　　要：目的观察Smad3 shRNA对TGFβ1作用于大鼠HSC-T6细胞增殖、细胞周期以及分泌肝纤维化胶原的影响。方法Smad3 shRNA慢病毒感染HSC-T6,应用流式细胞仪检测其感染率,Real-time PCR检测对Smad3 mRNA表达的抑制作用;将HSC-T6对照、HSC-T6+TGFβ1(10 ng/ml)、Smad3 shRNA以及Smad3 shRNA+TGFβ1(10 ng/ml)这4组细胞进行如下检测:(1)MTT检测细胞增殖;(2)流式细胞仪检测TGFβ1作用24、36 h细胞周期分布;(3)Real-time PCR检测24、36 h时细胞周期、增殖及肝纤维化相关基因mRNA的表达变化;(4)ELISA检测24、36 h培养液上清中胶原蛋白(COL)Ⅰ、COLⅢ及α-SMA的含量。结果Smad3 shRNA慢病毒感染HSC-T6细胞至96 h,感染率为71.3%,对Smad3 mRNA的表达抑制率为50%。(1)MTT结果显示,TGFβ1促进HSC-T6增殖;与相应未感染Smad3 shRNA病毒组相比,Smad3 shRNA组以及Smad3 shRNA+TGFβ1组细胞增殖能力均下降。(2)细胞周期检测发现,24、36 h时,Smad3 shRNA+TGFβ1组与Smad3 shRNA组相比,细胞周期分布差异无统计学意义(P>0.05);(3)Real-time PCR显示,Smad3 shRNA+TGFβ1组较HSC-T6+TGFβ1组,Smad3、原癌基因(c-myc)、细胞周期依赖性激酶-2(CDK-2)、细胞周期素E(cyclin E)、表皮生长因子(EGF)、核因子-κB(NF-κB)、金属蛋白酶组织抑制剂-1(TIMP-1)、纤溶酶原激活物抑制剂-1(PAI-1)、α平滑肌肌动蛋白(α-SMA)、COLⅠ及基质金属蛋白酶(MMP)14等基因的mRNA在24、36 h表达均下调,HGF mRNA及COLⅢmRNA在24 h时上调、36 h时下调,Bcl-2 mRNA、MMP1 mRNA及MMP9 mRNA在24 h和36 h两个时段均表达上调。(4)ELISA检测发现,同一时间点比较,分别在24、36 h,TGFβ1可促进HSC-T6细胞COLⅠ(P=0.00,P=0.02)、α-SMA(P=0.00,P=0.01)的分泌;与HSC-T6+TGFβ1组相比,Smad3 shRNA+TGFβ1组COLⅠ于36 h分泌减少(P=0.00)、而α-SMA在24、36 h两个时间点分泌减少(P=0.00)。结论 Smad3 shRNA抑制了TGFβ1对HSC-T6的活化,显示了抗肝纤维化作用。Objective To investigate the effects of shRNA targeted to Smad3 on the cell proliferation,cell cycle and the collagen secretion induced by TGFβ1 in rat HSC-T6 cell line.Methods After infecting HSC-T6 with Smad3 shRNA lentivirus,flow cytometry was used to detect the infection rate,and fluorescence quantitative Real-time PCR was used to detect its inhibition of Smad3 mRNA expression.The cells were divided to four groups： HSC-T6 cell control group,HSC-T6＋TGFβ1（10 ng/ml）group,Smad3 shRNA group and Smad3 shRNA＋TGFβ1（10 ng/ml）group.The detections were as follows：（1）MTT was used to test the cell proliferation;（2）Flow cytometry was performed to measure the cell cycle;（3）Real-time PCR was used to quantify the mRNA expression of genes related to cell cycle,cell proliferation and liver fibrosis;（4） Collagen-I,Collagen-Ⅲ,and α-SMA secreted by HSC-T6 cells were detected by ELISA.Results The infection rate is 71.3% after Smad3 shRNA lentivirus infected HSC-T6 for 96 hrs,and the Smad3 mRNA expression inhibirory rate is 50%.（1）MTT assay revealed that TGFβ1 treatment promote cell proliferation.However,cell proliferation decreased in both Smad3 shRNA group and Smad3 shRNA＋TGFβ1 group.（2）Results from flow cytometry showed that,at 24 and 36 hrs,in comparison to Smad3 shRNA group,Smad3 shRNA ＋TGFβ1 group have no significant change in cell cycle distribution（P0.05）.（3）Real-time PCR results showed that,compared to HSC-T6＋TGFβ1 group,Smad3 shRNA＋TGFβ1 group decreased the mRNA expression of Smad3,c-myc,cdk2,cyclin E,EGF,NF-κB,TIMP1,PAI-1,α-SMA,Collagen I and MMP14.Conversely,the mRNA expression of Bcl-2,MMP1,MMP9 increased after TGFβ1 treatment for 24hours and 36hours.HGF mRNA and Collagen Ⅲ mRNA increased at 24hrs but decreased at 36hrs.（4） ELISA showed that TGFβ1 can promote HSC-T6 cell COL I secretion（P=0.00,P=0.02,respectively） and α-SMA secretion（P=0.00,P=0.01,respectively）after TGFβ1 treatment for 24 hours and 36hours.Compared to the HSC-T6＋TGF

关 键 词：SMAD3 shRNA 慢病毒载体 肝硬化 转化生长因子β1 肝星状细胞 

分 类 号：R575.2[医药卫生—消化系统]