HORMONAL REGULATION OF CULTURED PANCREATIC ACINAR CELLS

我们最近开发了一种体外胰腺腺泡AR42J系统。 细胞，一种高度分化的大鼠细胞系。此外，我们还有 小鼠胰腺腺泡细胞原代单层培养体系的建立 细胞允许在一段时间内对正常细胞功能进行研究 几周。这项建议的目标是确定哪些激素具有 对胰腺腺泡细胞生长的直接调节作用 分化，并阐明所涉及的机制，通过利用 这两种文化体系。我们将测试：调节 细胞内钙离子，(例如，胆囊收缩素)；通过循环作用的激素 AMP(如分泌素)；通过酪氨酸激酶活性起作用的激素(如 胰岛素)和类固醇激素。以确定调节 生长，我们将测量DNA合成，蛋白质和DNA含量，以及 核标记指数。我们将检验CCK增加 胰腺腺泡细胞的生长机制与CCK的作用机制相同 通过评估其他钙离子介导的影响来诱导酶分泌 促分泌剂，以及直接增加 细胞内钙离子，或激活蛋白激酶C。AR42J变异株 具有独特生长特性的细胞也将被开发出来 阐明CCK诱导生长的机制。要确定 调节腺泡细胞分化的激素，我们将测试其效果 用生物合成标记法合成胰腺分泌酶， 免疫沉淀和双向凝胶电泳法。要确定 这些激素影响酶合成的机制，我们将 利用体外翻译和与cDNA的核酸杂交 用于定量检测腺泡细胞基因表达变化的探针。核径流 将进行检测以确定mRNA水平的变化 代表转录或信使核糖核酸稳定性的变化。对细胞的影响 形态将通过形态计量分析和 免疫细胞化学。CCK和CCK对CCK受体的长期调节作用 其他因素将通过激素结合分析来确定， 内化和合成。

We have recently developed an in vitro system of pancreatic acinar AR42J cells, a highly differentiated rat cell line. In addition, we have developed a primary monolayer culture system of mouse pancreatic acinar cells which allows studies of normal cell function over a period of several weeks. The objective of this proposal is to determine which hormones have direct regulatory effects on pancreatic acinar cell growth and differentiation, and to elucidate the mechanisms involved, by utilizing these two culture systems. We will test: hormones that regulate intracellular Ca2+, (e.g. cholecystokinin); hormones that act via cyclic AMP (e.g. secretin); hormones that act via tyrosine kinase activity (e.g. insulin); and steroid hormones. To identify hormones which regulate growth, we will measure DNA synthesis, protein and DNA content, and the nuclear labeling index. We will test the hypothesis that CCK increases the growth of pancreatic acinar cells by the same mechanisms that mediate CCK induced enzyme secretion, by evaluating the effects of other Ca2+-mediated secretagogues, and of pharmacological agents which directly increase intracellular Ca2+, or activate protein kinase C. Variant strains of AR42J cells with unique growth properties will also be developed to help elucidate the mechanism involved in CCK induced growth. To identify hormones which regulate acinar cell differentiation we will test effects on pancreatic secretory enzyme synthesis using biosynthetic labeling, immunoprecipitation, and two-dimensional gel electrophoresis. To determine the mechanisms by which these hormones affect enzyme synthesis, we will utilize in vitro translation and nucleic acid hybridization with cDNA probes to quantitate changes in the acinar cell mRNA. Nuclear run-off assays will be conducted to determine whether changes in mRNA levels represent changes in transcription, or mRNA stability. Effects on cellular morphology will be evaluated by morphometric analysis and immunocytochemistry. The longterm regulation of CCK receptors by CCK and other factors will be determined by analysis of hormone binding, internalization and synthesis.