Smad3 shRNA抑制TGFβ1对HSC-T6细胞活化的抗肝纤维化作用

郑素军; 邢欣悦; 武聚山; 王世美; 张莹; 韩源平; 俞豪; 陈煜; 刘梅; 段钟平

Smad3 shRNA抑制TGFβ1对HSC-T6细胞活化的抗肝纤维化作用

1. 首都医科大学附属北京佑安医院人工肝中心
2. 首都医科大学基础医学院细胞生物学系

基金项目:

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)；

详细信息

中图分类号: R575.2
计量
- 文章访问数: 3299
- HTML全文浏览量: 36
- PDF下载量: 742
- 被引次数: 0
出版历程
- 出版日期: 2012-04-20

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

Research funding:

摘要

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+TGF...
- Smad3 shRNA,慢病毒载体 /
- 肝硬化 /
- 转化生长因子β1 /
- 肝星状细胞
Abstract: 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 (P>0.05) . (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+TGFβ1 group, Smad3 shRNA+TGFβ1 group decreased the secretion of COL I (P=0.00) at 36 hrs, and α-SMA (P=0.00, P=0.00, respectively) at 24hrs and 36hrs.Conclusion Smad3 shRNA inhibited the activation effect of TGFβ1 on HSC-T6, showing the prower of anti-hepatic fibrosis.
- Smad3 shRNA /
- lentiviral vector /
- liver cirrhosis /
- transforming growth factor-β

HTML全文

参考文献(19)

[1]Brand ao DF, Ramalho LN, Ramalho FS, et al.Liver cirrhosis and hepatic stellate cells[J].Acta Cir Bras, 2006, 21 (Suppl 1) :54-57.

[2]Liu C, Gaca MD, Swenson ES, et al.Smads2and3are dif-ferentially activated by transforming growth factor-β (TGF-β) in quiescent and activated hepatic stellate cells[J].J Biol Chem, 2003, 278 (13) :11721-11728.

[3]Schnabl B, Kweon YO, Frederick JP, et al.The role of Smad3in mediating mouse hepatic stellate cell activation[J].Hepatology, 2001, 34 (1) :89-100.

[4]郑素军, 夏云, 任红, 等.端粒酶逆转录酶小干扰RNA对肝癌细胞的体外抑制作用[J].中华肝脏病杂志, 2004, 12 (9) :530-533.

[5]George J, Tsutsumi M.siRNA-mediated knockdown of con-nective tissue growth factor prevents N-nitrosodimethylamine-induced hepatic fibrosis in rats[J].Gene Ther, 2007, 14 (10) :790-803.

[6]Jiang C, Tan T, Yi XP, et al.Lentivirus-mediated shRNA targeting XIAP and survivin inhibit SW1990pancreatic cancer cell proliferation in vitro and in vivo[J].Mol Med Report, 2011, 4 (4) :667-674.

[7]陈鹏, 郑素军, 王世美, 等.靶向大鼠smad3基因的siRNA筛选及其shRNA重组慢病毒的构建[J].临床肝胆病杂志, 2011, 27 (5) :94-98.

[8]Subeq YM, Ke CY, Lin NT, et al.Valsartan decreases TGFβ1 production and protects against chlorhexidine digluconate-induced liver peritoneal fibrosis in rats[J].Cytokine, 2011, 53 (2) :223-230.

[9]Cheng K, Yang N, Mahato RI.TGF-beta1gene silencing for treating liver fibrosis[J].Mol Pharm, 2009, 6 (3) :772-779.

[10]Bissell DM, Roulot D, George J.Transforming growth factor beta and the liver[J].Hepatology, 2001, 34 (5) :859-867.

[11]张玉霞, 刘铭球.细胞周期调控研究进展[J].国外医学遗传学分册, 2001, 24 (5) :262-266.

[12] 柳洋, 文继舫, 郑长黎.TGF-β与细胞周期调控的研究进展[J].国外医学.生理、病理科学与临床分册, 2003, 23 (5) :541-544.

[13]Saile B, Knittel T, Matthes N, et al.CD95/CD95L-mediated apoptosis of the hepatic stellate cell proliferation during hepatic tissue repair[J].Am J Pathol, 1997, 151 (5) :1265-1272.

[14]Cui D, Zhang S, Ma J, et al.Short interfering RNA target-ting NF-kappa B induces apoptosis of hepatic stellate cells and attenuates extracellular matrix production[J].Dig Liver Dis, 2010, 42 (11) :813-817.

[15]Lin J, Chen A.Activation of peroxisome proliferator-activa-ted receptor-gamma by curcumin blocks the signaling path-ways for PDGF and EGF in hepatic stellate cells[J].Lab In-vest, 2008, 88 (5) :529-540.

[16]Chang YZ, Yang L, Yang CQ, et al.Migration of hepatic stellate cells in fibrotic microenvironment of diseased liver model[J].Hepatobiliary Pancreat Dis Int, 2008, 7 (4) :401-405.

[17]Liu X, Hu H, Yin JQ.Therapeutic strategies against TGF-b signaling pathway in hepatic brosis[J].Liver Int, 2006, 26 (1) :8-22.

[18]Inagaki Y, Higashi K, Kushida M, et al.Hepatocyte growth factor suppresses profibrogenic signal transduction via nucle-ar export of Smad3with galectin-7[J].Gastroenterology, 2008, 134 (4) :1180-1190.

[19]Lee WJ, Park SE, Rah DK.Effects of hepatocyte growth factor on collagen synthesis and matrix metalloproteinase production in keloids[J].J Korean Med Sci, 2011, 26 (8) :1081-1086.

施引文献

资源附件(0)

访问统计