反义TIMP-1表达质粒对实验性肝纤维化的影响

Liver fibrosis represents the final common pathological outcome for the majority of chronic liver insults. Pathological accumulation of extracellular matrix (ECM) in cases of liver fibrosis reflects imbalance of production and degradation of matrix proteins, which is the main mechanism of hepatic fibrosis. Matrix metalloproteinases (MMPs) are a fam-ily of secreted zinc proteases which are capable of degrading collagen and other ECM components. Recent studies suggest that MMPs may participate in pathological respon-ses involved in liver fibrosis. Especially, interstitial collagenase (MMP-1 human, MMP-13 rat) play a key role in degrading collagen typeⅠ,Ⅲ(the main component of ECM). There were no significant changes in the expression of MMP-1during hepatic fibrosis；while exists great changes in enzymatic activity in the latten of fibrosis. Further study shows that MMP-1 is tightly regulated by tissue inhibitors of matrix metalloproteinases1 (TIMP-1). Through inhibiting the activity of MMP-1, TIMP-1 had stronger effects on decreasing the degradation of collagen Ⅰ, Ⅲ, enhancing the deposition of ECM of liver, and hasting the hepatic fibrosis.Purpose :We had used RT-Nest-PCR and gene recombinant techniques to construct the rat antisense TIMP-1 recombinant plasmids which can express in eucaryotic cells. The recombinant plasmid and the pcDNA3 empty plasmid were transfected in rHSCs by Effectene (QIAGEN) separately in order to inhibit expression of TIMP-1 gene, improving the activity of interstitial collagenase, enhancing the ECM degradation.Main content : Total RNAs were extracted from rat liver with Trizol (Life Tech, U.S.A) reagent. According to the full-length cDNA sequence that encoded rat TIMP-1, we designed special sense and antisense primers and obtained target sequence with the RT-NEST- PCR technique, adding several enzymatic site to this sequence from 5'end, which were linked into T4 vector with T4 DNA ligase and were transfected in JM-109 coli. After being selected by IPTG/X-gal, we constructed the subclone (PT/TIMP-1) and obtained the target sequence with restriction enzymes EcolRI and XhoI, the resulting inserts was subcloned into the plasmid pcDNA3 (Invitrogen CA) in a reversal direction and were tested by sequenced (PE377 Auto sequencer).In vitro, rHSCs were extracted from normal rat liver by pronase and collagenase digestion and purified by centrifugal elutriation, and were cultured on plastic dishes until they were activated to a myofibroblastic phenotype after 7-10 days. We have designed three groups, they are recombinant plasmid group, empty plasmid group and normal control group. After transfecting the HSCs with Effectene reagent, we have detected the expression of TIMP-1 in HSCs by Northern blot, Western blot, studied the activity of intersitial collagenase by Type I Collagenase Activity Assay (Chemicon ,Temecula CA ). AND quantifyed the type I，III collagens in conditioned media by western blot..In vivo, we have made the rat hepatic fibrosis model with pig serum. Also We have designed four groups, they are recombinant plasmid group, empty plasmid group, fibro-sis model group and normal control group. The recombinant plasmid group and empty plasmid group were injected with 100μg recombinant plasmids at 2,4,6,8 week separately through tail vein. After killing the rats at the end of 8th week, we detected the exogenous and TIMP-1 gene expression by Northern blot, RT-PCR and Western blot, studied the activity(latent and active) of intersitial collagenase by Type I Collagenase Activity Assay, quantified the turnover of the hydroxyproline and type I,III collagens via immuohisto-chemistry, and observed the liver pathologic changes by VG taining..Results and Data: The vitro experiment results show that the exogenous antisense TIMP-1 recombi-nant plasmid could express in rHSCs well, which have blocked the expression of TIMP-1 greatly at gene and protein level by Northern Blot and Western Blot, the ratio of TIMP-1/GAPDH was 0.67 , 2.41, and 2.97 separately at mRNA leve

慢性肝病时金属蛋白酶组织抑制因子-1（TIMP-1）异常增生抑制了胶原酶的活性，导致胶原等细胞外基质在肝内过度沉积，促进了肝纤维化的形成和发展。我们运用重组DNA技术构建匆錞IMP-1真核细胞表达质粒，并分别将其转化进Ito细胞和定向转移至肝脏，观察其对肝纤维化大鼠的体内外影响，为慢性肝病纤维化的治疗探寻新的更为有效的措施。