|
The purpose of liver fibrosis (hepatic fibrosis) is a cirrhosis pre pathological process, from a variety of causes, in China's major viral hepatitis B, alcoholic liver fibrosis as its etiology is also on the rise in recent years, for hepatic fibrosis of combat no exact effective means. The major pathological processes of hepatic fibrosis is the extracellular matrix (extra cell matrim, ECM) deposition, and collagen type I is the main component of the ECM, 5-10% of the total protein of the normal liver collagen, wherein the collagen type I protein of the total collagen protein 40-50%, 40-50% of the total collagen type III collagen, liver fibrosis, collagen can be 50% of the total protein or more, in which the proportion of type I collagen to total collagen liter type III collagen is reduced to 20-30% to 60-70%. The visible hepatic fibrosis is the result of an excessive increase in type I collagen in the liver. Has now been proved that liver fibrosis is a reversible pathological process, this study design siRNA against rat collagen type I alpha 1 chain gene (COL1A1) and construct the recombinant plasmid simultaneous detection of the inhibition of the expression of type I collagen effect, provide a new target for gene therapy of liver fibrosis. Materials and methods, experimental materials eukaryotic expression vector pGPU6/GFP/Neo E. coli DH5α, plasmid extraction kit, restriction endonuclease BamH Ⅰ Bbs Ⅰ, Pst Ⅰ, T4 ligase, Taq enzyme, Lipofectamine 2000 G418, COL1A1 rabbit polyclonal antibody, β-actin rabbit anti-human polyclonal antibody, horseradish peroxidase-conjugated goat anti-rabbit secondary antibody, T6 rat hepatic stellate cells, experimental methods, screening siRNA sequences and build COL1A1 cDNA sequence, recombinant plasmids obtained from the NCBI website rats design three different sites for the COL1A1 gene siRNA sequences, the synthetic double-stranded DNA (double-stranded DNA) connected to with the U6 promoter pGPU6/GFP/Neo carrier on were named pGPU6/GFP/Neo-shRNA-A pGPU6/GFP/Neo-shRNA-B, pGPU6/GFP/Neo-shRNA-C. 2, cells transfected by liposome-mediated recombinant plasmid was transfected into rat hepatic stellate cell line HSC-T6 cells. Stabilized by G418 screening monoclonal expanding culture after 2 weeks. 3, the inhibitory effect was detected by Western blot (Western blotting) was used to detect expression of type Ⅰ collagen. 4, statistical processing of experimental data (?) ± s, the SPSS13.0 software analysis data, the comparison between groups using multiple sets of single-factor analysis of variance (ANOVA), P lt; 0.05 difference was statistically significant. Experimental results, the choice of the siRNA against the COL1A1 gene sequence of COL1A1-A: 5'-GAGTATGGAAGCGAAGGTTCC-3 ', 3'-CTCATACCTTCGCTTCCAAGG-5'. Of COL1A1-B: 5'-ACAAGGTGACAGAGGCATAAA-3 ', 3'-TGTTCCACTGTCTCCGTATTT-5 3 '; COL1A1-C: 5'-TGATGGTTCTCCTGGCAAAGA-3', 3'-ACTACCAAGAGGACCGTTTCT-5'2, recombinant plasmids by BamH Ⅰ, Pst Ⅰ ??single enzyme, positive plasmids were sequenced and analyzed the result confirmed targeting COL1A1 mRNA exactly recombinant plasmid shRNA sequence design, build successful. 3, Western blotting confirmed the recombinant plasmid inhibition rate of hepatic stellate cells COL1A1 protein respectively and for 26.93D.86%, 66.29%. Conclusion We successfully constructed a 3 for rat COL1A1 plasmid vector, the expression of type I collagen produced inhibition, especially pGPU6/GFP/Neo-shRNA-C effect, so as to provide a new treatment of liver fibrosis way.
|