Objective       To construct a lentiviral RNA interference (RNAi) vector targeting rat TGFβ1

and determine its effect on silencing TGFβ1 gene and the expression and secretion of alpha-1 type Ⅰ collagen (Col1α1) in the rat hepatic stellate cell line HSC-T6.       Methods        Three RNAi sites of TGFβ1 gene were chosen according to its mRNA sequence. Three pairs small interference RNA (siRNA) of the 3 RNAi sites were synthesized. Three pair of siRNA were transfected into HSC-T6 cells to identify the optimal siRNA sequence by reverse transcription polymerase chain reaction (RT-PCR). Then the shRNA oligonucleotide targeting the optimal siRNA sequence was designed

synthesized and annealed for double-strand formation. The annealed oligonucleotide fragment was subcloned into pGreenPuro plasmid to form pGreenPuro/TGFβ1 shRNA lentiviral vector. After identified by restriction enzyme digestion and sequencing

the pGreenPuro/TGFβ1 shRNA plasmid was transfected into 293T cells to produce lentiviral particles. The lentiviral vector particles were transfected into HSC-T6 cells

and the expression of green fluorescent protein (GFP)

TGFβ1 and Col1α1 in the transfected cells were observed under inverted microscope and assayed by RT-PCR and Western blotting

respectively.       Results        The optimal siRNA sequence was confirmed. Digestion and sequencing identified that lentiviral vector pGreenPuro/TGFβ1 shRNA had been constructed successfully. In 48 h after the lentiviral vector transfection

no expression of  TGFβ1 and mild expression of Col1α1 was seen in HSC-T6 cells by RT-PCR. Western blotting showed mild expression of the 2 molecule at the same time point.       Conclusion        The lentiviral RNAi vector targeting rat TGFβ1 is constructed successfully. This vector can effectively silence TGFβ1 and inhibit the expression and secretion of Col1α1 in HSC-T6 cells.