Construction Of Eukaryotic Expression Vector For Rat Smad7 And Studying Its Anti-fibrosis In Hepatic Stellate Cell Line HSC-T6

Objectives:Hepatic fibrosis (HF) is the common pathology basis and features of many kinds of chronic liver disease, it's characterized by deposition of the extracellular matrix(ECM). Transforming growth factorβ(TGF-β) is the most important promoting factor of the development of hepatic fibrosis.ECM deposition is the key medium and the activation of hepatic stellate cells (HSC) is the central link. Smad belongs to signal transduction protein mediating TGF-βfrom the membrane into the cell nucleus. Smad7 can regulate TGF-βsignal transduction negatively. This study constructed and identified rat Smad7/pcDNA3.1(+) eukaryotic expressing plasmid, and transfected it into HSC-T6 cell by Lipofectmine 2000, the purpose is observing whether the extraneous Smad7 gene can transfect HSC-T6 effectively as a anti-fibrosis factor. It provides the experimental foundation for further studying that transfection of this plasmid alone or combined with other plasmids to block the TGF-β/smad signaling in different steps and hepatic fibrosis.Methods:Design specific primers according to rat Smad7 genome sequence.TRIzol assay was performed to obtain the total RNA from the rat liver tissues. The fragment of Smad7 cDNA was obtained by RT-PCR. Smad7/pcDNA3.1 (+) recombinant plasmid was constructed as molecular cloning techniques, and the positive clones were confirmed by double digestion and sequencing. Transfected Smad7/pcDNA3.1(+) plasmid into HSC-T6 cells by Lipofectmine2000. The position and expression of Smad7 andα-SMA in HSC was detected using immunocytochemical staining. The mRNA and protein of Smad7 was assayed by RT-PCR and Western-blot respectively. The expression ofα-SMA was performed using Western-blot, and the level of TGF-β1,collagenⅠandⅢmRNA by RT-PCR.Results: Smad7/pcDNA3.1(+) eukaryotic expressing plasmid was constructed successfully. And the expression of Smad7 andα-SMA located at the cell cytoplasm, and the positiveα-SMA showed the activation of HSC-T6 cells. Smad7 mRNA and protein expression was significantly higher in Smad7 transfected group than control and empty vector group 1.053±0.009(P=0.009)and 0.083±0.016(P=0.020),α-SMA protein, TGF-β1 and collagenⅠmRNA expression was notably reduced in Smad7 transfected group than control and empty vector group 0.518±0.085,0.961±0.013,0.592±0.096 (P=0.032, 0.000,0.004,respectively).There was no statistically significant difference of the change of collagenⅢmRNA expression among the three group(sP=0.950).The difference of Smad7 mRNA and protein,α-SMA protein, TGF-β1, collagenⅠandⅢmRNA expression weren't statistically significant between control and empty vector group(P=0.944, 0.664, 0.612, 0.595, 0.967, 0.950, respectively).Conclusion:1. Smad7 /pcDNA3.1(+) eukaryotic expressing plasmid is constructed successfully.2. The expression of Smad7 andα-SMA located at the cell cytoplasm and the positiveα-SMA showd the activation of HSC-T6 cells.3. Smad7 eukaryotic expression vector can express Smad7 effectively after transfected into rat HSC-T6 cells, Smad7 mRNA and protein expression was significantly higher in Smad7 transfected group.4. The expressionα-SMA protein was notably reduced because of exogenous Smad7 gene, made the number of the actived HSC-T6 cells reduce. It may reduce the deposition of the extracellular matrix for Smad7 overexpression.5. Overexpression of Smad7 can effect the mRNA expression level of TGF-β1,collagenⅠ, it maybe has activity of anti-fibrosis after transfected into rat HSC-T6 cells through inhibiting TGF-β/Smad signaling.