Retinoid Metabolism in Hepatitic Stellate Cells

描述(由申请人提供)：这项建议的总体目标是了解特定的大鼠视黄酸代谢酶(尤其是视黄酸酯水解酶)在调节肝星状细胞中的视黄醇代谢中的作用。肝星状细胞是肝脏中维生素A的主要储存库，维生素A主要以维生素A棕榈酸酯的形式储存在细胞内高度可见的脂滴中。当动物暴露在肝毒素中或人类长期酗酒时，星状细胞被激活并转化为肌成纤维细胞样细胞。这些转化的细胞是酒精性肝纤维化中胶原和细胞外基质蛋白形成的部位。星状细胞激活的最早事件之一是视黄酸酯的水解和细胞内脂滴的耗尽。最有可能的视黄酸酯水解酶是广泛底物专一性脂肪酶和羧酸酯酶家族的成员。肝星状细胞表达的特定视黄酸酯水解酶及其在星状细胞激活过程中调节活性的机制尚不清楚。在初步数据中，我们发现最常见的五种具有视黄酸棕榈酸酯活性的大鼠肝脏羧酸酯酶在星状细胞中不高表达。然而，激素敏感型脂肪酶基因在大鼠肝星状细胞中表达，并在体外具有视黄酸棕榈酸酯水解酶活性。该基金的目标是通过实时、定量的RNA聚合酶链式反应和带有酶特异性抗体的星状细胞免疫荧光显微镜，鉴定在分离的大鼠肝细胞(星状细胞、枯否细胞、肝细胞)中表达的特定视黄酯水解酶(脂肪酶和羧酸酯酶)。视黄酸酯水解酶和视黄醇脱氢酶的表达将与脂滴中的视黄醇自身荧光和培养的大鼠星状细胞在激活成肌成纤维细胞样细胞时细胞特异性标志物的表达有关。在可行的情况下，我们将测量视黄酸棕榈酸酯水解酶的活性，并对细胞提取物进行蛋白质凝胶电泳酯酶活性染色。我们将研究纯化的大鼠星状细胞视黄酸酯水解酶的动力学，并检测酶激活剂和抑制剂的影响。抑制星状细胞中视黄酸酯的水解可能是阻止肝纤维化早期星状细胞激活的有效治疗策略。

DESCRIPTION (provided by applicant): The overall goal of this proposal is to understand the role of specific rat retinoid metabolizing enzymes (especially retinyl ester hydrolases) in the regulation of retinoid metabolism in hepatic stellate cells. Hepatic stellate cells are the main reservoir of Vitamin A in the liver where it is stored mainly as retinyl palmitate in highly visible intracellular lipid droplets. When animals are exposed to hepatotoxins or humans chronically abuse alcohol, the stellate cells become activated and transform into myofibroblast-like cells. These transformed cells are the sites of collagen and extracellular matrix protein formation in alcohol-induced hepatic fibrosis. One of the earliest events in stellate cell activation is the hydrolysis of retinyl esters and the depletion of the intracellular lipid droplets. The most likely retinyl ester hydrolase candidates are the members of the families of broad substrate specificity lipases and carboxylesterases. The specific retinyl ester hydrolases expressed in hepatic stellate cells and the mechanisms for regulation of their activity during stellate cell activation is not known. In preliminary data, we show that the five most common rat liver carboxylesterases with retinyl palmitate activity are not highly expressed in stellate cells. However, the hormone-sensitive lipase gene is expressed in rat hepatic stellate cells and it has retinyl palmitate hydrolase activity in vitro. The goals of the grant are to identify the specific retinyl ester hydrolases (lipases and carboxylesterases) that are expressed in isolated rat liver cells (stellate cells, Kupffer cells, hepatocytes) by real-time, quantitative PCR of RNA and immunofluorescence microscopy of stellate cells with enzyme-specific antibodies. The expression of retinyl ester hydrolases and retinol dehydrogenases will be correlated with retinoid autofluorescence in lipid droplets and expression of cell-specific markers in cultured rat stellate cells as they undergo activation to myofibroblast-like cells. When feasible, we will measure retinyl palmitate hydrolase activity and perform protein gel electrophoresis of cellular extracts with esterase activity staining. We will examine the kinetics of purified rat stellate cell retinyl ester hydrolases and examine the effects of enzyme activators and inhibitors. Inhibitors of retinyl ester hydrolysis in stellate cells may be an effective therapeutic strategy for arresting stellate cell activation early in hepatic fibrosis.