Effects Of Antisense TIMP-1 Eukrayotic Expressing Plasmids On Rat Liver Fibrosis

Background: Liver fibrosis is characteristic of the unbalance of degradation and synthesis of extracellular matrix (ECM). Matrix metalloproteinases and their inhibitors---tissue inhibitor of matrix metalloproteinases (MMPs and TIMPs) play an important role in the balance of degradation and synthesis of ECM. In the system the most important pair of balance factors are the interstitial collagenase MMP-1 (humman)/MMP-13 (Rat) and TIMP-1. In normal liver the two factors keep in equilibrium state. Once the balance is broken, elevating TIMP-1 expression may contribute to deposition of ECM in liver by inhibiting the degrading activity of MMP-1/MMP-13, which causes the hepatic fibrosis. The common view point to liver fibrosis is elevating TIMP-1 expression can inhibit the degrading activity of MMPs, which cause the ultra-deposition of ECM. Our present experiment were performed to construct antisense TIMP-1 eukraotic expressing plasmids and transfect in vivo . We aimed to test the effect of the exogenous plasmid and effectiveness of gene therapy. Methods:1. Construction of antisense TIMP-1 eukraotic expressing plasmids Trizol reagent was used to extract total RNA in rat liver. The proper nested primers of TIMP-1 were choosed and synthesized. RT-nest-PCR and gene recombinant techniques were used to construct the fragment of TIMP-1. After purification, the PCR products of TIMP-1 and eukrayotic expressing plasmid PCDNA3.1(+) were cut and TIMP-1 were inserted reversely into PCDNA3.1(+),and then transferred into JM-109 strain .2.Indentification of antisense TIMP-1 eukrayotic expressing plasmids Enzyme-cutting and PCR indentification and DNA autosequencing were used to prove the successful construction of antisense TIMP-1 eukrayotic expressing plasmid.3. Duplication and treatment of liver fibrosis model Sixty-four female SD rats were randomly distributed into two groups: 12 as normal control group(N), the left induced by CCl₄ to make model: 40% CCl₄ 0.3 ml/100g subcutaneously was injected once every 5 days, double in the first dosage; 15% ethanol was the only drink; corn flour and lard were fed. Two weeks later, 49 rats left by CCl₄ induced group were randomly distributed into three groups: 19 in experimental liver fibrosis model induced by CCl₄ as disease control group(C), 19 in PCDNA3.1(+) transfection as treatment control group(P),11 in antisense TIMP-1 transfection as treatment group(AT). C and P group were kept on subcutaneously injecting CCl₄, 50ug of...