中文English
ISSN 1001-5256 (Print)
ISSN 2097-3497 (Online)
CN 22-1108/R
Issue 4
Apr.  2012

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

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  • Published Date: 2012-04-20
  • 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.

     

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