Inhibition effect on TGFβRⅡ expression by TGFβRⅡ-siRNA expression vector in hepatic stellate cells / 中国医师杂志

ABSTRACT

Objective To construct transforming growth factor β receptor Ⅱ (TGFβRⅡ) siRNA expression vector, and investigate the inhibitory effect on TGFβRⅡ in hepatic stellate cells (HSC-T6) ,thus offer a preliminary study for RNAi therapy in liver fibrosis. Methods The two sites of RNAi action were selected in TGFβRⅡ cDNA through online primer design software of Ambion company. The corresponding double-stranded DNA sequence was constructed into pSilencer-U6 plasmid, which could transcribe small interference RNA, the plasmid was transfected into HSC-T6 cells with Lipofectamine 2000. The expression of TGFβRⅡ mRNA and protein were detected by reverse transcription polymerase chain reaction (RT-PCR) and western blotting. Results Expression siRNA plasmids of siRNA-a and siRNA-b that target TGFβRⅡ were successfully constructed. Compared with control group, the expression of TGFβRⅡ mRNA was reduced by 0. 89 ± 0. 06 and 0. 25 ± 0. 03 in two siRNA groups, and expression of TGFβRⅡ protein reduced to 0. 86± 0. 05 and 0. 23 ± 0. 02, respectively. TGFβRⅡ expression was inhibited at mRNA and protein levels after transfected with the siRNA-b plasmid( P <0. 01 ).. There was no difference in the TGFβRⅡ expression after transfected with the siRNA-a plasmid( P > 0. 05). Conclusion TGFβRⅡ-siRNA can effective inhibit expression of TGFβRⅡ in HSC-T6.