Construction COL1A1-shRNA Expression PLasmid And Select Effective Sequence

AIMLiver fibrosis is a pathological changes of the chronic,liver disease,its essence is collagen synthesis increased,and degradation reduced,the disturbance of the two dynamic balance,resulting in liver ECM deposition,the main ingredient of ECM is collagen,Collagen typeⅠmainly.It has been shown that Collagen typeⅠis important to lead to the development of fibrosis.RNA interference(RNAi)is a purpose to achieve specific inhibition of gene expression,has successfully reduced the number of endogenous gene expression.Construction and selecting of the procollagen type 1 alpha 1(COL1Al)short hairpin RNA(shRNA)expression plasmid inhibit rat hepatic stellate cells(HSC)COL1AlmRNA expression.METHODSRat procollagen type 1 alpha 1 eDNA sequence was obtained from NCBI website. The three sites of RNAi action were selected in Procollagen type 1 alpha 1 cDNA through online design of the Whitehead Institute.The corresponding double-stranded DNA was used to construct pGPU6/GFP/Neo plasmid,which could transcribe small interference RNA,namely pGPU6/GFP/Neo-shRNA-A,pGPU6/GFP/Neo-shRNA-B,pGPU6/GFP/Neo-shRNA-C.HSC-T6 cells were transfected with a green fluorescent protein(GFP)-labeled siRNA to assess transfection efficiency.To get most effective and optimal dosage siRNA,the three plasmids were transfected into HSC-T6 cells with LipofectAMINE2000 with 1μg,2μ,g3μg,4μg siRNA respectively,and untreated HSC T6 were used as control.The most effective and optimal dosage siRNA was transfected into HSC-T6,after 24h transfection,we select stable expression cell line by G418 about 2wk.The expressions of COL1AlmRNA were detected by reverse transcription-polymerase chain reaction(RT PCR). RESULTSExpressing siRNA plasmids of the three sites that target COL1AlmRN were successfully construsted by Agarose electrophoresis and Sequence analysis.The transfection efficiency of 1μg,2μg,3μg,4μg were calculated respectively to be approximately 16.7%,20.3%,23.5%,22.3%,dose-dependent show that 2μg siRNA was optimal dosage in each groups.COL1AlmRNAlevels in HSC-T6 transfected with pGPU6/GFP/Neo-shRNA-A,pGPU6/GFP/Neo-shRNA-B,pGPU6/GFP/Neo-shRNA-C decreased by16.6%,63.3%,80.3%with 2μg siRNA plasmids respectively.CONCLUSIONConstructed pGPU6/GFP/Neo-shRNA-C expression plasmid can effectively inhibit the COL1AlmRNA expression.It provids the new methods and materials for the treatment of liver fibrosis.