Effects Of Antisense TβRI,TβRⅡ Eukaryotic Expressing Plasmid And That Cooperate With Antisense TIMP-1 Eukaryotic Expressing Plasmid On Rat Liver Fibrosis

Background: In China the high incidence of liver fibrosis is an essential link of cirrhosis. The current studies show Liver fibrosis is a kind of reversible disease, so the key to treat cirrhosis is to prevent and delay the occurrence of liver fibrosis.Studies find that the cells of liver fibrosis are the most important factor in transforming growth factorβ(transforming growth factorβ, TGF-β), the signal transduction pathway to the MFA has been stated: First, the combination of the activation TGF-βand type II TGF -βreceptor (TβRⅠI) has a kinase activation due to phosphorylation. Type I, II receptor complex will be formed by recombining TβRⅠI with the combination of TGF-βwith type I TGF-βreceptor (TβRⅠ), which phosphorylates TβRⅠto make it have kinase activation. Finally, the activated TβRⅠactivates the special receptor Smads (R-Smads) 0f phosphorylation. Thereby the transformation of hepatic stellate cells (HSC) and the synthetic and degradation of ECM are regulated. That is , the role of TGF-βmust be based on the transmembrane receptor on the cell membrane TβRⅠand TβRⅠI. Therefore, inhibiting the expression of TβRⅠcan be inferred to affect TGF-βsignal transduction in theory, and thus inhibit TGF-βto promote fibrosis.In normal liver, the MMP (matrixmetalloprotei-nases, MMPs) and MMP inhibitor Organization (tissue inhibitor of metallopropteinases, TIMPs) are in a state of dynamic equilibrium to maintain the balance between the formation of collagen and its degradation. When damaged factors act on the liver, activate HSC, break the balance beween MMPs and TIMPs, degradation of collagen fibers will be reduced, then a lot of collagen in the liver tissue will be deposited. the main TIMPs in the liver is TIMP-1 and TIMP-2, TIMP-1 expresses more significantly. MMPs in the human is mainly MMP-1, in the rodents is MMP-13. The binding of TIMP-1 and the specifics of MMP-1/MMP-13 can inhibit its activity. It can be inferred that inhibiting the expression o TIMP-1 will reduce the effect of TIMP-1 to MMP-1/MMP-13, increase the degradation of ECM, reduce ECM deposition, delay the occurrence and development of liver fibrosisThis study aims to mensurate the rat's tissue, TβRⅡand TIMP-1, the con tent of MMP-13 mRNA content and expression of its Protein in the experimental models on the basis of application of molecular biology detection technology RT-PCR and western-bloting (Western blot), and to analyze the collected data scientifically and verify this issue from the perspective of biological molecules. The cooperation role of anti-transforming growth factor receptor and anti-protease inhibitor-matrix can intervene the occurrence of liver fibrosis and the development of Liver fibrosis effectively. The ultimate goal is to find effective ways to prevent and delay the chronic liver to hardening of the liver defibrillators disease with gene therapy and to lay the foundation of clinical application for future human gene therapy of liver fibrosis.Material and methods:Take experimental rat's liver preserved below the low-temperature of -80℃: 12 cases of the normal control group, 12 cases of the model control group, 13 cases of P group, 13 cases of TIMP-1 antisense expression vector eukaryotic group, 13 cases of anti-sense therapy group TβRⅠ, 13 cases of anti-sense TβRⅡgroup, 13 cases of anti-sense TβRⅠand anti-sense TIMP-1 treatment group, 13 cases of antisense TβRⅡtreatment group and anti-TIMP-1-treated group.1. Use semi-quantitative RT-PCR detection to test TβRⅠmRNA, TβRⅡmRNA, TIMP-1mRNA, MMP-13 mRNA in liver tissue.2. Detect the expression of TβRⅠ, TβRⅡ, TIMP-1, MMP-13 protein in rat liver with the technology of western blot.3. Analyze the statistics and verify the proof systematically and scientifically.Results:Compared with the control group and air plasmid group, TβRⅠ, TβRⅡand the content and protein expression of TIMP-1 mRNA are dropped in the treatment group of anti-sense TβRⅠ, treatment group of anti-sense TβRⅠand anti-sense TIMP-1, the anti-TIMP-1-treated group, treatment group of anti-sense TβRⅡ, treatment group of anti-sense TβRⅡand anti-sense TIMP-1. The comparison of every group has statistical significance (P <0.05).TβRⅠ, TβRⅡand the content and protein expression of TIMP-1 mRNA have significant differences (P <0.05) between the control group and the experimental treatment group, anti-sense TβRⅠ+ anti-TIMP-1-treated group has more statistical significance than the anti-sense therapy group of TβRⅠ, Anti-TIMP-1-treated group (P <0.05); anti-sense TβRⅡ+ anti-TIMP-1-treated group has more statistical Significance than the anti-sense TβRⅡtreatment group and the anti-TIMP-1-treated group (P <0.05).TβRⅠ, TβRⅡand content and protein expression of TIMP-1 mRNA haven't significant difference between model control group and the air plasmid control group (P> 0.05).Conclusion:1. Extracell matrix (ECM) is reduced, the development of liver fibrosis is lowered by anti-sense TβRⅠ, TβRⅡeukaryotic cells blocking the acting access of TGF-β1. Experiments also show the existence of TGF-βsignaling pathway.2.The MMP-13 inhibits the TIMP-1, outside of cells (ECM) degradation increases, inhibit the process of liver fiber by anti-sense TβRⅠ, TβRⅡeukaryotic cells blocking TGF-β1 acting channel,3.Extracell matrix (ECM) increases by the inhibition of TIMP-1 to MMP-13 through antagonism of anti-sense TIMP-1 eukaryotic cells.4. Two recombinant synergies can generate stack effect to achieve the goal to delay the effective suppression of liver fibrosis. The ultimate goal is to seek a way to delay and prevent all kinds of chronic liver disease to become cirrhosis.