Cloning Of Rat TGF-β₃ Gene And Its Effect On TGF-β₁,Type I Collagen Synthesis Of Hepatic Stellate Cells

Part I Cloning of TGF-β₃ gene and its expression in hepatic stellate cellsObjective To clone rat transforming growth factor (TGF)-β₃ cDNA, and to investigate TGF-β₃ gene expression in transfected rat hepatic stellate cell line (HSC-T6).Methods One amplification fragment (1.2Kb) was obtained from the rat osteoblasts by RT-PCR method and cloned into expression vector pcDNA3.1(+) (5.4Kb), which was named pcDNA3.1(+)-TGF-β₃ (6.6Kb). The cloned gene was confirmed to code for rat TGF-β₃ by restriction enzyme analysis. HSC-T6 was transfected with pcDNA3.1(+)-TGF-β₃ plasmid by LipofictamineTM 2000 mediated gene transfer. The transient expression was detected by real-time quantitative PCR method 24h,48h,72h later.Resultsâ‘ The recombinant eukaryotic expression vector for TGF-β₃ was digested with NheI and XbaI ,and the electrophoresis of the digested products showed two fragment: 1.2 kb fragment and 5.4 kb fragment. The sequence of TGF-β₃ DNA fragment was identical to that published in GenBank;â‘¡The TGF-β₃ gene transfected HSC-T6 showed prominently elevated mRNA expression of TGF-β₃ 24h,48h,72h later, and the expression of TGF-β3 mRNA increased significantly 48h later(P<0.05). Conclusion The recombinant eukaryotic expression vector for TGF-β₃ was constructed correctly, TGF-β₃ gene could be expressed effectively in transfected HSC-T6 by pcDNA 3.1(+)-TGF-β₃ plasmid 48h later, which provided the experimental basis of TGF-β₃ transgenic therapy for fibrosis diseases.Partâ…¡Effect of TGF-β₃ on TGF-β1,type I collagen synthesis of hepatic stellate cellsObjective : To observe the effects of transforming growth factor (TGF)-β₃ gene transfer on TGF-β1 and type I collagen synthesis of hepatic stellate cell line (HSC-T6). Methods: Construct TGF-β1 expression plasmid (pcDNA3.1(+)-TGF-β1). The recombinant expression plasmid pcDNA3.1(+)-TGF-β1 was transfected into cultured HSC-T6. Positive colnes were selected by G418, the expression of TGF-β1 was detected by real-time quantitative PCR and western blot one month later. The positive clones were transfected with the recombinant expression plasmid pcDNA3.1(+)-TGF-β₃, the expression of TGF-β1 mRNA and type I collagen mRNA were detected by real-time quantitative PCR, the expression of TGF-β1 protein and type I collagen protein were detected by western blot 48h later.Results: Higher mRNA and protein level of TGF-β1 were observed in positive colnes which were transfected with recombinant expression plasmid pcDNA3.1(+)-TGF-β1 (P<0.05), type I collagen mRNA and protein increased significantly in positive clones (P<0.05). TGF-β1 protein,type I collagen mRNA and protein decreased significantly in clones which were transfected with recombinant expression plasmid pcDNA3.1(+)-TGF-β3 (P<0.05), but the expression of TGF-β1 mRNA had no obvious difference (P>0.05).Conclusion: TGF-β₃ could inhibit synthesis of type I collagen through decreasing the synthesis of TGF-β1 protein after TGF-β₃ gene was transfected into HSC-T6 which highly expressed TGF-β1, TGF-β₃ might be a effective cell factor which could inhibit the development of liver fibrosis.