| Background: Liver fibrosis represents the final common pathological outcome for the majority of chronic liver insults. There is a growing consensus that fibrosis belongs to a reversible disease.Liver fibrosis is characterized by net accumulation of extracellular matrix(ECM), mainly type I,III collagens. Hepatic fibrosis is likely to result from an imbalance between the deposition and degradation of ECM molecules. Matrix degradation is catalyzes by the activity of family of Zn2+ -dependent enzymes, the matrix metalloproteinases (MMPs). The interstitial collagenase (MMP-1 human, MMP-13 rat) mainly degrade type I,III collagens, playing a key role in preventing excessive deposition of ECM. Further studies show that the activity of MMPs could be inhibited by tissue inhibitor of metalloproteinases (TIMPs), and TIMP-1 plays a important role in fibrotic development rather than other members. Elevated TIMP-1 expression may contribute to ECM deposition in liver by inhibiting the degrading activity of interstitial collagenase on type collagen I, III , which causes the hepatic fibrosis. Theoretically we can decrease even block the expression of TIMP-1 at mRNA level in rat liver, which may diminish the inhibitory effect of TIMP-1 on interstitial collagenase, and will be helpful to release the activity of interstitial collagenase, also, enhance the degradation of ECM.Hepatic stellate cell (HSC) are the main source of MMPs in liver. In response to liver injury, HSC undergo a phenotype transformation to a proliferating myofibroblast-like cell, which secreted MMPs, joining the ECM degradation process. At the meantime, HSC increased synthesis of TIMP-1 markedly.HSC play a prominent and centeral role in liver fibrosis. The active process of cultured HSC in vitro can mimic the liver fibrotic process in vivo. Our present experiments were performed to construct antisense TIMP-1 eukrayotic expressing plasmid and transfer this plasmid into activated HSC in vitro. We aimed to test the hypothesis that the introduction of this exogenous plasmid into rat HSC may block the expression of TIMP-1 and halt the progression of experimental liver fibrosis in vitro, which might provide novel method to chronic fibrosis.Methods: Nest primers were designed and synthesized according to rat TIMP-1 cDNA sequences (GENE Bank). RT-Nest-PCR and gene recombinant techniques were used to construct the fragments of TIMP-1, After separation, recovery and purification, the PCR products of TIMP-1 were connected with T vector and then transferred into JM-109 strain. PT/ TIMP-1 were successfully constructed after IPTG/X-gal screening. The purposed fragments were cut and inserted in reverse direction into eukrayotic expressing plasmid (pcDNA3), and then transferred into JM-109 strain again. By using enzyme-cutting identification and DNA autosequencing, the successful construction of antisense TIMP-1 eukrayotic expressing plasmid were proved. Rat HSC were extracted from normal rat liver by pronase and collagenase digestion and purified by centrifugal elutriation, cell vitality were analyzed by Trypan blue staining, and were cultured on plastic dishes until they were activated to a myofibroblastic phenotype after 7-10 days, then we detected the expression of Desmin and α-SMA in HSC by immnohistochemistry. The antisense recombinant plasmid and the pcDNA3 empty plasmid were transfected into HSC by Effectene (QIAGEN) separately, HSC were selected by growing in DMEM containing 400μ g/ml G418.We evaluated the transfecting rate by use of pEGFP-C1 plasmid. Expression of exogenous plasmid was assessed by Northern blot, and expression of TIMP-1 in HSC was determined by Northern blot and Western blot. We tested the interstitial collagenase activity with FITC-labled type I collagen as substrate. Ultimately, we quantified the type I,III collagen by Western blot. All values were expressed as mean±SD, ANOVA was used to determined the significance of differences among the three groups. Results: HSC were isolated from normal male Sprague-Dawley rat (200 ?...
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