Iredale J P, Benyon R C, Arthur M J, Ferris W F, Alcolado R, Winwood P J, Clark N, Murphy G
University of Medicine, Southampton General Hospital, Southampton, England.
Hepatology. 1996 Jul;24(1):176-84. doi: 10.1002/hep.510240129.
Liver fibrosis results from a relative imbalance between synthesis and degradation of matrix proteins. We have previously described release of the protein collagenase inhibitor, tissue inhibitor of metalloproteinase-1 (TIMP-1), by culture-activated human hepatic stellate cells (HSCs). In this study, we have investigated the relative expression of TIMP-1 and interstitial collagenase in culture-activated rat HSCs and rat models of liver injury and fibrosis. The complementary DNA (cDNA) for rat TIMP-1 was obtained by homology polymerase chain reaction (PCR) and sequenced. By Northern analysis using this probe, TIMP-1 messenger RNA (mRNA) expression was up-regulated with HSC activation by culture on plastic as defined by cellular expression of procollagen-1. Interstitial collagenase mRNA was expressed in early 1. Interstitial collagenase mRNA was expressed in early culture (<4 days) but became undetectable in more activated cells (7-21 days). By activity assay of serum-free cell-conditioned media, TIMP-1 was found to be released in increasingly concentrations with duration of culture on plastic. Expression of TIMP-1 interstitial collagenase, and procollagen-1 mRNAs were studied in rat models of biliary and parenchymal injury (bile duct ligation and CC14 administration) by ribonuclease protein assay. TIMP-1 mRNA expression was increased at 6, 24 hours, and 3 days after bile duct ligation and was also shown to rise in acute CC14 liver injury and remain elevated as the liver became fibrotic. TIMP-1 expression preceded procollagen-1 expression in both models. In contrasts, interstitial collagenase mRNA levels remained similar to control values throughout both models of liver injury. Total cellular RNA from hepatocytes, HSCs, and kupffer cells freshly isolated from livers after acute CC14 injury was subjected to Northern analysis. TIMP-1 transcripts were observed in nonparenchymal cells only. We suggest that increased expression of TIMP-1 relative to interstitial collagenase by HSCs may promote progression of liver fibrosis in these rat models by preventing degradation of secreted collagens.
肝纤维化是由基质蛋白合成与降解之间的相对失衡所致。我们之前曾描述过培养激活的人肝星状细胞（HSC）可释放蛋白胶原酶抑制剂——金属蛋白酶组织抑制剂1（TIMP-1）。在本研究中，我们调查了培养激活的大鼠HSC以及大鼠肝损伤和纤维化模型中TIMP-1和间质胶原酶的相对表达情况。通过同源聚合酶链反应（PCR）获得大鼠TIMP-1的互补DNA（cDNA）并进行测序。使用该探针通过Northern印迹分析发现，如根据前胶原-1的细胞表达所定义，随着在塑料上培养导致HSC激活，TIMP-1信使核糖核酸（mRNA）表达上调。间质胶原酶mRNA在培养早期（<4天）表达，但在激活程度更高的细胞（7 - 21天）中无法检测到。通过无血清细胞条件培养基的活性测定发现，随着在塑料上培养时间的延长，TIMP-1以逐渐增加的浓度释放。通过核糖核酸酶蛋白测定法研究了胆管和实质损伤（胆管结扎和给予CCl4）大鼠模型中TIMP-1、间质胶原酶和前胶原-1 mRNA的表达。胆管结扎后6、24小时和3天TIMP-1 mRNA表达增加，在急性CCl4肝损伤中也显示升高，并在肝脏纤维化时持续升高。在两种模型中TIMP-1表达均先于前胶原-1表达。相比之下，在两种肝损伤模型中，间质胶原酶mRNA水平始终与对照值相似。对急性CCl4损伤后从肝脏新鲜分离的肝细胞、HSC和库普弗细胞的总细胞RNA进行Northern印迹分析。仅在非实质细胞中观察到TIMP-1转录本。我们认为，在这些大鼠模型中，相对于间质胶原酶，HSC中TIMP-1表达增加可能通过阻止分泌型胶原蛋白的降解而促进肝纤维化进展。
