Inhibition Of CTGF Expression In Rat Hepatic Stellate Cellular By RNA Interference

Hepatic fibrosis is a major complication of chronic liver disease such as viral hepatitis or alcoholic liver disease, it is serious to human health, and a variety of cytokines play important part in liver fibrosis. This study aims to design plasmid expressing siRNA that target connective tissue growth factor and control plasmid, and transfer them into the cultured rat hepatic stellate cells, then use Real-time PCR to detect the expressing effect of CTGFmRNA, which contribute to the gene therapy of hepatic fibrosis.Materials and Methods1. Materials:(1) Hepatic stellate cellular-T6 (HSC-T6), Beijing Golden zijing biological products co., Ltd. purchased.(2) Primary reagents:DMEM high sugar company GiBco culture (United States), fetal bovine serum (American Hyclone company), trypsin (Invitrogen Corporation); primers sequences (Takara); pGCsi plasmid (with green fluorescent protein, neomycin, H1 promoter) (Shanghai Jikai gene chemical technology co., Ltd.); Lipofectamine2000(invitrogen Corporation); plasmid small extraction Kit (days of root biochemical technology co., Ltd.); the enzyme(Labsystems Dragon company); RNAiso Plus(Takara); Real-time PCR Kit (Takara).2. Methods:(1) Construct CTGFshRNA recombinant plasmid with pGCsi vector,and select effective suppression sequences by transfecting it into hepatic stellate cell.(2) Transfect 4 recombinant plasmids into HSC-T6, set up a blank control group, 24 hours later, analyze the cell transfection efficiency.(3) Finded out the effective suppression of CTGF expression sequence. 3. Statistical analysis:SPSS 13.0 software for analysis of variance.Resultsl.The CTGFshRNAs had been successfully designed and inserted into pGCsi plasmid.2.pGCsi-CTGFshRNA was successfully transfected into the hepatic stellate cells.3.Compared with the controls,the expression of CTGFmRNA of pGCsi-CTGFshRNAl and pGCsi-CTGFshRNA2 groups dramatically decline, the expression of CTGFmRNA of pGCsi-CTGFshRNA3 group and nonspecific shRNA group were inapparently down-regulate.Conclusion1.Construct pGCsi plasmid expression vector successfully.2.Recombination plasmid was successfully transfected into the hepatic stellate cells.3.Succeeded in Screening pGCsi-CTGFshRNA1 and pGCsi-CTGFshRNA2 sequence which could suppress expression of CTGF effectively.And pGCsi-CTGF-shRNA could inhibit the expression of CTGF in hepatic stellate cells.