Effect of exogenous Smad7 gene transfected hepatic stellate cells on mRNA expression of transforming growth factor beta 1, collagen Ⅰ and collagen Ⅲ

Xiayan YANG; Yong YANG; Yong ZHENG; Rui LI; Ting ZHOU

ABSTRACT

BACKGROUND:

Smad7 is a major repressible protein in transforming growth factor β(TGF-β1) signal transduction pathway,which possess antifibrotic effects.

OBJECTIVE:

To construct rat Smad7 eukaryotic vector and to observe the mRNA expression level of TGF-β1, collagen Ⅰ and collagen Ⅲ in rat hepatic stellate cells (HSC)-T6 cell.DESIGN, TIME AND

SETTING:

The gene recombination and cytology observation experiment was performed at the First Affiliated Hospital of Shihezi University School of Medicine.MATERIALS pcDNA3.1 (+) plasmid was reserved in the laboratory. E coil DH5α was presented by Key Laboratory of Xinjiang Endemic and Ethnic Diseases, Shihezi University School of Medicine. The HSC T6 cell was provided by Cancer Institute and Hospital, Chinese Academy of Medical Sciences.

METHODS:

Rat Smad7 cDNA was cloned into eukaryotic plasmid pcDNA3.1(+) to construct Smad7/pcDNA3.1(+) plasmid and transfected it into HSC-T6 ceils by Lipofectmine 2000. The experiment was divided into normal control, empty vector and Smad7 transfected groups, and the positive cells were selected by G418.MAIN OUTCOME

MEASURES:

The levels of Smad7, TGF-β1, collagen Ⅰ and Ⅲ mRNA was detected by reverse transcriptase polymerase chain reaction, respectively.

RESULTS:

Smad7 eukaryotic vector was successfully constructed and confirmed by endonuilease digestion and sequencing. Compared to the control and empty vector groups, Smad7 mRNA expression was significantly higher in Smad7 transfected group (P < 0.01 ); and TGF-β1 and collagen Ⅰ mRNA expression was notably reduced (P < 0.01). There was no statistically significant difference of the change of collagen Ⅲ mRNA expression among the three groups (P>0.05). The difference of Smad7, TGF-β1,collagen Ⅰ and Ⅲ mRNA expression had no statistically significant between control and empty vector groups (P_(all) > 0.05)

CONCLUSION:

Smad7 eukaryotic expression vector is successfully constructed. The Smad7 gene can effectively expressed in transfected HSC-T6 cell, and decrease mRNA expressions of TGF-β1 and collagen Ⅰ.