DIFFERENTIATION OF RENAL EPITHELIAL CELLS IN CULTURE

LLC-PK 1细胞系，其具有肾近端小管的特性 在培养物和MDCK细胞系，其显示肾远端小管 特征是长期极化的肾上皮细胞系， 起源 一系列分化功能的表达在 细胞融合，并可以加速化学品， 双乙酰胺，被称为分化诱导剂。 我们将描述 微绒毛水解酶作为分化标志物的调节 LLC-PK 1细胞对细胞融合和化学诱导剂的反应，以及 特别强调两个最有效的诱导剂， 双乙酰胺和5-氮杂胞苷，以及海藻糖酶和麦芽糖酶的调节 活动，我们已经证明，这是诱导的， 二乙酰胺 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，以探测 海藻糖酶活性诱导表达的基础。 生理 变量，如细胞融合，培养基质，激素，周期性 将测试核苷酸水平和营养物对表达的影响， 差异化标记 我们的变异克隆亚系， 对诱导剂的反应将被表征，新的变异细胞系 与世隔绝 另外的诱导物应答分化标志物将被 在MDCK和LLC-PK 1细胞中鉴定，以确定 对不同的属性进行协调或单独调节。 这项工作的基本目标是定义和复制正常， 程序化上皮发育和形态发生序列 细胞培养 这些研究有助于我们理解 肾脏的细胞和发育生物学，以及 老化，这可以被视为正常的程序和内在的一部分， 发展，受随机环境影响的调制。

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.