Inhibition Of COL1A1 Expression By RNA Interference In Rat Hepatic Stellate Cell

ObjectiveHepatic fibrosis is the early pathological states of liver cirrhosis.It can be caused by a series of reasons,and in China it is mainly caused by HBV hepatisis,then the prevalence of alcoholic fibrosis has ascended.there hasn't been any exact valid method to prevent it.Extra cell matrim(ECM) deposition is the main pathological change of liver fibrosis,and its primary ingredient is collagen typeⅠ.Collagen protein accounts for 5 to 10 percent of total protein in normal liver,and both collagen typeⅠandⅢaccount for 40-50 percent of collagen protein individually.In hepatic fibrosis,collagen protein can account for 50 percent or even more,and in this situation collagen type I will increase to 60-70 percent,while collagen typeⅢwill decrease to 20-30 percent. It can be concluded that hepatic fibrosis results from the excessive increase of collagen typeⅠ.It has been proved that hepatic fibrosis is an reversible pathological change. Then in this study we construct an expression plasmid of shRNA targeted against rat procollagen,typeⅠalpha 1(COL1A1) and investigate its effects on collagen typeⅠprotein expression of rat hepatic stellate cell.Materials and MethodsMaterialspGPU6/GFP/Neo vector,E.coli DH5α,Plasmid purification Kit,BamHI, BbsⅠ,PstⅠ,T4 DNA ligase,Taq enzyme,Rat hepatic stellate cell,Rabbit anti-COL1A1 polyclonal antibody,Rabbit anti -P-actin antibody,HRP-banding secondary antibodyMethods1.Screen siRNA sequence and construct recombinant plasmidRat procollagen type I alpha 1 cDNA sequence was obtained from NCBI website, and selected three sites as siRNA sequence.The corresponding double-stranded DNA was cloned into pGPU6/GFP/Neo vector with U6 promotor,namely pGPU6/GFP/Neo-shRNA-A,pGPU6/GFP/Neo-shRNA-B and pGPU6/GFP/Neo-shRNA-C.2.Tranfect HSCThe COL1A1 shRNA plasmid was transfected into HSC via lipofectamine 2000. Transfected HSC was selected by G418 and selected monoclone cell line to culture on a large scale.3.Examine inhibition effectsWestern Blotting detected collagen TypeⅠprotein expression.4.Statistica analysisData was expressed by(?)±s and was statistically analyzed by ANOVA.Values of P<0.05 were considered significant.Results1.Selected siRNA sequence targeted Rat COL1A1 gene:2.Sequence analysis confirm that three recombinant plasmid are identical to designed shRNA sequence;3.Western blotting results show that inhibition ratios of collagen tupeⅠprotein in transfected HSC are 26.93%,44.86%and 66.29%respectively.Conclusion1.We succeed in constructing three recombinant plasmid targeted COL1A1 gene;2.Three recombinant plasmids can effectively inhibit the typeⅠcollagen protein expression;especially pGPU6/GFP/Neo-shRNA-C.It provides the new methods for the treatment of liver fibrosis.