HORMONAL REGULATION OF CULTURED PANCREATIC ACINAR CELLS

培养的胰腺腺泡细胞的激素调节

• 批准号: 3234188
• 负责人: Craig D Logsdon
• 金额: $ 3.47万
• 依托单位: MOUNT ZION HOSPITAL AND MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-08-01 至 1987-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3234188
• 关键词: biotechnology; cell differentiation; cell growth regulation; cholecystokinin; cyclic AMP; electron microscopy; endocrine pharmacology; gel electrophoresis; genetic regulation; growth factor; histochemistry /cytochemistry; hormone regulation /control mechanism; immunofluorescence technique; insulin; messenger RNA; mutant; pancreas; pancreas enzyme; pancreas hormone; protein kinase; radiotracer; secretin; secretion; steroid hormone; tissue /cell culture

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.

我们最近开发了胰腺腺泡AR 42 J的体外系统， 细胞，高度分化的大鼠细胞系。 另外我们有 建立了小鼠胰腺腺泡原代单层培养体系 细胞，允许研究正常细胞功能超过几个月的时间， 周 这项提案的目的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，利用 这两个文化体系。 我们将测试：调节 细胞内Ca 2+（如胆囊收缩素）;通过循环作用的激素 AMP（如促胰液素）;通过酪氨酸激酶活性起作用的激素（如促胰液素）; 胰岛素）;和类固醇激素。 为了确定调节 生长时，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通过相同的机制介导CCK 诱导的酶分泌，通过评估其他Ca 2+介导的 促分泌素和直接增加 细胞内Ca 2+或活化蛋白激酶C。 AR 42 J变异株 还将开发具有独特生长特性的细胞， 阐明CCK诱导生长的机制。 以识别 我们将测试调节腺泡细胞分化的激素对 使用生物合成标记的胰腺分泌酶合成， 免疫沉淀和二维凝胶电泳。 以确定 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 探针定量腺泡细胞mRNA的变化。 核径流 将进行测定以确定mRNA水平的变化是否 代表转录或mRNA稳定性的变化。 对细胞的影响 将通过形态测定分析评价形态， 免疫细胞化学 CCK对CCK受体的长时程调节作用及其机制 其它因素将通过分析激素结合来确定， 内化和合成。