| Objectives; Hepatic stellate cells ( HSCs, Ito cells, fat - storing cells) are located in the perisinusoidal space of Disse in hepatic lobule. They contain 80%- 90% of hepatic vitamin A and are multifunctional. Presently, HSCs are considered to be main cells of producing extracellular matrix and play a pivotal role in the cellular and molecular events that lead to hepatic fibrosis. The process of HSC activation is complicated with changes including morphology and function. It has been demonstrated that HSC can stay quiescent in culture with normal extracellular matrixs(ECM) for a longer period of time than that with collagen I in vitro. These results indicated that the changing of ECM component around cells could activate HSC and induce the changing of function including retinoid metabolism, morphology and proliferation.Matrix Metalloproteinases ( MMPs) form a family of proteases, which are capable of degrading most of ECM components. Now, it has been paid attention to the relationship between hepatic fibrosis and MMPs. Matrix Metalloproteinase- 2 ( MMP - 2 ) as a major enzyme involved in extracellular matrix remodeling, is one of the members of this super family of MMPs. Developed studies indicated that the expression and activation of MMP - 2 produced by HSC is associated with development of hepatic fibrosis and an important pathological factor during the process of hepatic fibrosis. At the present time, MMP - 2 is secreted as an inactive proenzyme , which is activated by a membrane - linked process that involves MT1 - MMP and tissue inhibitor metalloproteiase - 2 ( TIMP - 2). The viewpoint about activation of MMP - 2 as the follow: MT1 - MMP complexed with TIMP -2 has been found to serve as a cell receptor for MMP -2. The activation of bound MMP - 2 has been proposed to occur through an initial cleavageby other (free) MT, - MMP molecules to an intermediate form followed by auto-proteolytic activation of MMP -2 to fully active enzyme. But detailed activation mechanism is not clear until now. It has been little known about the mechanism of activation of MMP - 2 in HSC. In the present paper we for the first time established the experimental culture system for studying the interaction of ECM and HSC by using extracellular matrix components such as collagen I and IV as substratum mimicking the in vivo structure. The conditional media from HSC culture were analyzed for MMP - 2, MMP - 9 by gelatin zymography. Meanwhile , we collected cells from different cultures and observed the expression of MT, - MMP by Western blot, the expression of MMP - 2mRNA,MT, - MMP mRNA ,TIMP - 2 mRNA by RT - PCR. The aim of this paper is to study whether ECM components including collagen I and IV modulate the activation of MMP - 2, MMP - 9 in rat HSC cultures. We also discuss the biological functions of ECM, the activation of MMP -2, the effect of MT, - MMP^TIMP -2 on proM-MP - 2 activation and biological mechanism of ECM remodling by using cultured HSC in different substratum. This study is to provide the theory bases for hepatic fibrosis study through trying to find the method to changing interaction between activated HSC and the activation of MMP - 2 in order to prevent or hold up the development of hepatic fibrosis clinically.Materials and Methods1. Isolation of rat HSC.Rat HSCs were isolated by the collagenase shaking method and then purified by different centrifugation over the density gradient, as previously reference described. HSCs were cultured in Dulbecco 's modified Eagle 's medium (DMEM) supplemented with 10% (v/v) fetal bovine serum(FBS) and antibodies at 371 in a humidified atmosphere of 95% air and 5% C02. HSCs were subcultured at 1 ;3 split and used during 5-10 passages in this study.2. Preparation of culture dishes with several types of substrata. Polystyrene dishes (Po. ) ,type I collagen - coated dishes (C. C. ) ,type I collagen gel (C. G. ) , Matrigel dishes ( M. G. )3. The culture of rat HSCs in serum -free culture medium.HSCs were seeded on every substratum in DMEM...
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