DIFFERENTIATION OF RENAL EPITHELIAL CELLS IN CULTURE

发展肾近端小管特性的LLC-PK1细胞系 在培养的MDCK细胞系中，表现为肾远端小管 肾上皮细胞系长期极化的特征 起源。一系列不同功能的表达在 细胞融合，并可被环氧亚甲基等化学物质加速 双乙酰胺，称为分化诱导剂。我们将描述 微绒毛水解酶作为分化标志物的调控 LLC-PK1细胞对细胞融合和化学诱导剂的反应 特别强调两种最有效的诱导剂，环己烷 双乙酰胺和5-氮胞苷与海藻糖酶和麦芽糖酶的调节 活性，我们已经证明了六亚甲基可以诱导这些活性 双乙酰胺。针对纯化的海藻糖酶的抗血清将用于 生物合成标记和免疫沉淀研究，免疫电印迹 分析和荧光组织化学研究，以探讨 海藻糖酶活性诱导表达的基础。生理学 细胞融合、培养底物、激素、周期等变量 将测试核苷酸水平和营养物质对表达的影响 差别化标记。我们的变异克隆亚系在 对诱导剂的反应将被表征和新的变异细胞系 与世隔绝。其他诱导剂反应的分化标记将是 在MDCK和LLC-PK1细胞中鉴定，以确定 差异化物业受到协调或单独监管。 这项工作的根本目标是定义和复制正常的， 程序性上皮细胞发育和形态发生的顺序 细胞培养。这些研究与我们对 肾脏的细胞和发育生物学以及这一现象 衰老，可以被视为正常的程序化和内在的一部分 发展，受随机环境影响的影响。

The LLC-PK1 cell line, which develops properties of renal proximal tubule in culture and the MDCK cell line, which exhibits renal distal tubule characteristics are longterm polarized epithelial cell lines of renal origin. Expression of an array of differentiated functions develops at cell confluence and can be accelerated by chemicals such as hexamethylene bisacetamide, known as differentiation inducers. We will characterize the regulation of microvillar hydrolases as markers of differentiation in LLC-PK1 cells in response to cell confluence and chemical inducers, with particular emphasis on the two most potent inducers, hexamethylene bisacetamide and 5-azacytidine, and the regulation of trehalase and maltase activities, which we have shown to be inducible by hexamethylene bisacetamide. Antisera directed against purified trehalase will be used in biosynthetic labelling and immunoprecipitation studies, immunoelectroblot analysis and fluorescence histochemistry studies in order to probe the basis for induced expression of trehalase activity. Physiological variables such as cell confluence, culture substratum, hormones, cyclic nucleotide levels and nutrients will be tested for effects on expression of differentiated markers. Our variant clonal sublines which are altered in response to inducers will be characterized and new variant cell lines isolated. Additional inducer -responsive differentiation markers will be identified in MDCK and LLC-PK1 cells in order to determine if differentiated properties are co-ordinately or individually regulated. The fundamental goal of this work is to define and reproduce normal, programmed epithelial developmental and morphogenic sequences of events in cell culture. These studies are relevant to our understanding of the cellular and developmental biology of the kidney, as well as the phenomenon of aging, which can be viewed as a programmed and intrinsic part of normal development, modulated by stochastic environmental influences.