Vyas S K, Leyland H, Gentry J, Arthur M J
University of Southampton, Hampshire, England.
Gastroenterology. 1995 Sep;109(3):889-98. doi: 10.1016/0016-5085(95)90399-2.
BACKGROUND & AIMS: Hepatic lipocyte proliferation and activation are pivotal in liver fibrosis. Disruption of normal lipocyte-matrix interactions may contribute to this process. The synthesis of transin, which degrades normal liver matrix, by culture-activated hepatic lipocytes was investigated.
Lipocytes were isolated by pronase/collagenase perfusion, density gradient centrifugation, and centrifugal elutriation. Transin messenger RNA in lipocytes was analyzed by Northern blotting. Transin activity was analyzed by zymography, Western blotting, immunocytochemistry, and quantitative [14C]beta-casein degradation assay.
Transin messenger RNA was detected in early primary culture (3-5 days) but not in freshly isolated lipocytes or late primary culture. Zymography of lipocyte medium showed caseinolytic activity (relative molecular weight, 57 kilodaltons and 60 kilodaltons) inhibited by ethyl-enediaminetetraacetic acid but not thiol or serine protease inhibitors. Immunoblotting and immunocytochemistry confirmed the presence of transin in media and cells. Quantitative transin activity decreased progressively with increasing duration of primary lipocyte culture and myofibroblastic transformation.
Rat hepatic lipocytes express the transin gene and secrete its product during the early phase of lipocyte activation in primary culture. Because this enzyme degrades a wide spectrum of normal basement membrane proteins and activates progelatinase B and interstitial collagenase, it may have an important role in liver injury and fibrosis.
肝脂肪细胞的增殖和激活在肝纤维化过程中起关键作用。正常脂肪细胞与基质相互作用的破坏可能促使这一过程发生。本研究调查了培养激活的肝脂肪细胞中降解正常肝基质的转胶酶的合成情况。
通过链霉蛋白酶/胶原酶灌注、密度梯度离心和离心淘析法分离脂肪细胞。采用Northern印迹法分析脂肪细胞中转胶酶信使核糖核酸。通过酶谱分析、蛋白质印迹法、免疫细胞化学和定量[14C]β-酪蛋白降解试验分析转胶酶活性。
在原代培养早期（3 - 5天）检测到转胶酶信使核糖核酸，但在新鲜分离的脂肪细胞或原代培养后期未检测到。脂肪细胞培养基的酶谱分析显示酪蛋白溶解活性（相对分子质量为57千道尔顿和60千道尔顿）受乙二胺四乙酸抑制，但不受硫醇或丝氨酸蛋白酶抑制剂抑制。蛋白质印迹法和免疫细胞化学证实培养基和细胞中存在转胶酶。随着原代脂肪细胞培养时间的延长和肌成纤维细胞转化，转胶酶活性逐渐降低。
大鼠肝脂肪细胞在原代培养脂肪细胞激活的早期阶段表达转胶酶基因并分泌其产物。由于该酶可降解多种正常基底膜蛋白并激活前明胶酶B和间质胶原酶，它可能在肝损伤和纤维化中起重要作用。
