Effects Of Antisense TGFβRⅡ And TIMP-1 Eukrayotic Expressing Plasmids On Rat Hepatic Fibrosis

Background: Hepatic fibrosis is a prosthetic response to chronic liver injury. The mechanism of hepatic fibrosis is the imbalance of synthesis and degradation of extracellular matrix (ECM) by regulation of many kinds of cell and cytokine. In the formation of liver fibrosis and cirrhosis, transforming growth factorβ(TGFβ), especially TGFβ1 play a pivotal role in the regulation of the production, degradation and accumulation of ECM. Secreted as a latent precursor, TGFβis activated at sites of injury. Signaling by TGFβoccurs through a family of transmember and ser/thr kinase receptors. Both components of the receptor complex, known as receptor (TβRI) and receptorⅡ(TβRⅡ) are essential for signal transduction .Excessive a c cumulation of ECM in the fibrosis liver, caused not only by increased synthesis but also by decreased degradation of ECM due to the activity of MMPsregulating ECM degradation was inhibited by their specific inhibitors, called TIMPs. At present, gene therapy for liver fibrosis targeting TGFβand TIMP-1 separately, inhibiting the expression by blocking the TGFβsignal transduction, and inhibiting the expression of TIMP-1, enhance active of interstitial collagenase. But the therapeutic researches which target antisense TβRII and TIMP-1 as therapeutic tool have not been reported till now. Our present experiments were performed to construct antisense TβRII eukaryotic expressing plasmids. We aimed to test the hypothesis that the introduction of these two exogenous plasmids into rat models induced liver fibrosis may halt the progression of liver fibrosis.Method:After consult literature,choose and synthesize the proper nested primers of TβRⅡ,RT-Nest-PCR and gene recombinant techniques were used to construct the fragments of TβRⅡ.After purification,The PCR products of TβRⅡand eukrayotic expressing plasmid pcDNA3.1(+) were cut and TβRⅡwere inserted in reverse direction into pcDNA3.1(+),and then transferred into JM-109 strain,and positive clone was selected.By using enzyme-cutting and DNA autosequencing,the successful construction of antisense TβRⅡeukrayotic expressing plasmid were proved. Ninety female SD rats were randomly distributed into 6 groups: 15 in normal control group,15 in antisense TβRⅡand antisense TIMP-1 transfection as treatment group,15 in antisense TβRⅡtransfection as treatment group ; 15 in antisense TIMP-1 transfection as treatment group;15 in pcDNA3.1(+) transfection as treatment control group;15 in experimental liver fbrosis model . The treatment groups were induced by composite factor injering CCl4 and alcohol and hypsi-fat hypsi-cholesterol;Among the antisense groups,the recombinant plasmid and...