Research Of Smad-7 Unites Timp-lsiRNA Expression Plasmids On Rat Liver Fibrosis

Objectives:The purpose of the study was to copy the liver fibrosis in the rat model, through the intraperitoneal injection of Lipofectmine 2000 embedding Smad-7 and TIMP-1siRNA plasmid analyze each two plasmids and joint action on the influence of rat liver fibrosis.Material and Methods:1. The model rats with hepatic fibrosis induced with carbon tetrachloride The experimental rats were given 40% carbon tetrachloride subcutaneous injection. The first injection does was 5ml/kg. The other was 3ml/kg every four days for 12 times. In regard of feeding, we used 20% ethanol solution as the only drink liquids, at the same time we used the high-fat and high-cholesterol as the diet. Establish normal controls rats, subcutaneous injecting 0.9% saline solution, same with the experimental group, use the standard dose feeding method.2. Observation of result Applicate HE dyeing and VG dyeing to observe liver tissue, classificate pathological morphology change of liver fibrosis; Use immunohistochemical staining to observe typeⅢcollagen expression in the liver tissue; Adopt Western blot to detect type I collagen expression, using RT-PCR to test Smad-2 mRNA,TIMP-1mRNA, with the expression ofβ-actin mRNA as internal control and the image analysis technology testing the strength of the above indexes expression. Experimental results data was analysed by SPSS 13.0 software. Kruskal-Wallis was used to determine the significance of difference among the six groups. Ridit test was used to describing the significance of pathological difference among the six groups. P< 0.05 shows a statistically significant difference.Results:1. The typeⅢcollagen expression by immunohistochemistry testing:typeⅢcollagen expression in each treatment group is decreased than liver fibrosis control group, the difference was statistically significant(P<0.05). And compared with each single groups, type III collagen expression of the joint treatment group reduced obviously, the difference was statistically significant(P<0.01).2. Western blot to detect typeⅠcollagen protein expression:compared with each single treatment group, the typeⅠcollagen expression in model control group is decreased, the difference was statistically significant (P<0.05), and the joint treatment group is reduced obviously, the difference was statistically significant (P<0.01).3. RT-PCR detect the expression of Smad-2mRNA and TIMP-1mRNA:Smad-2mRNA expression in each treatment group is decreased than liver fibrosis control group, the difference was statistically significant(P<0.05). And compared with each single groups, Smad-2mRNA expression of the joint treatment group reduced obviously, the difference was statistically significant(P<0.01).TIMP-1mRNA expression in each treatment group is decreased than liver fibrosis control group, the difference was statistically significant (P<0.05). And compared with each single groups, TIMP-1mRNA expression of the joint treatment group reduced obviously, the difference was statistically significant(P<0.01).Conclusion:1. TIMP-1siRNA/pcDNA3.1(+) eukaryotic expressing plasmid and Smad-7/pcDNA3.1 (+) eukaryotic expressing plasmid all can inhibit the protein of typeⅠ,Ⅲcollagen expression, and the joint group is most obvious2. Blocking the MMPs-TIMPs system and TGF-β/Smad-7 signaling pathway together is stronger than one single blocking for the effect of anti-fibrosis, confirm furtherly the occurrence and development of liver fibrosis is a process that a variety of pathways participated in.