Effect Of TGF-β₁siRNA Against Transforming Growth Factor-β₁ On Biological Character Of Activated HSCs

Objective Live fibrosis is a common sequel to diverse liver injuries .In the formation of live fibrosis and cirrhosis ,many cytokines produce marked effects on fibrogensis .Molecular mechanisms involved in fibrogensis reveal that transforming growth factor- β1 play a pivotal role in HSC activation,degradation and accumulation of extracellular matrix. RNA interference(RNAi) is a biological phenomena to silence target genes and successfully utilized to down regulate some endogenous gene expression. we aim to design pSilencer2.1-U6 plasmid expressing small interfering RNAs(siRNA) that target TGF- β1, and to investigate the effect of small interfering RNA targeting transforming growth factor-β1 on collagen and activation signing in hepatic stellate cells (HSC).Methods (1)Rat Transforming growth factor- β1 cDNA sequence was obtained from NCBI website. The three sites of RNAi action were selected in Transforming growth factor- β1 cDNA through online design of Ambion company. The corresponding double-stranded DNA was used to construct pSilencer -U6 plasmid, which could transcribe small interference RNA ,namely siTGFA, siTGFB and siTGFC; (2)HSC-T6 cells were transfected with a green fluorescent protein (GFP) -labeled siRNA to assess transfection efficiency ; (3)To get most effective and optimal dosage siRNA, the three plasmids were transfected into HSC-T6 cells with liposome metafectene with 1μg, 2μg, 4μg 6ug.8ug siRNA respectively, and untreated HSC T6 were used as control; (4)The most effective and optimal dosage siRNA was transfected into HSC-T6.Total RNA and protein of the cells, after their incubation with siRNA for 1, 3 and 5 days, were extracted, and the supernatants were collected; (5)The expressions of TGF- β1, Smad3 ,Smad7 ,type Ⅰ and Ⅲ collagen mRNA were detected by reverse transcnption-polymerase chain reaction (RT-PCR); (6)Concentration of hyaluronic acid ,type Ⅳ collagen and type Ⅲ pro-collagen in the supernatants were determined by radioimmunoassay.; (7)The protein expressions of a-SMA and TGF- β1 were assessed by Western blot and expressions of I collagen was also assessed by immunohistochemistry...