REGULATION OF EXTRACELLULAR MATRIX BIOSYNTHESIS

细胞外基质生物合成的调控

• 批准号: 3342890
• 负责人: MICHAEL A HARALSON
• 金额: $ 6.81万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-09-30 至 1986-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3342890
• 关键词: Escherichia coli; bacterial DNA; bacterial RNA; cell growth regulation; cell type; connective tissue development; connective tissue metabolism; electron microscopy; extracellular matrix; gel electrophoresis; genetic strain; lung; messenger RNA; molecular cloning; nucleic acid sequence; plasmids; respiratory epithelium; retinoate

Basement membranes are the specialized extracellular matrices which interface with both alveolar epithelial and capillary endothelial cells in the lung. Current data indicate these structures contain three types of macromolecules - type IV collagen, laminin and proteoglycans. This type of extracellular maxtrix (ECM) functions not only as a cellular supporting element and a molecular filter, but recent evidence suggests the components of the matrix may influence cell growth and differentiation. As these structures are critical to the normal maintenance of lung function, knowledge about the regulation of these ECM components is essential for understanding lung development and response of the tissue during disease. It is the long-term objective of the proposed investigations to delineate the mechanisms and factors which control the expression of the basement membrane components and the mechanisms through which the components influence cellular function. It is felt that these objectives can be achieved by combining in vitro studies with recombinant DNA techniques to dissect at the molecular level the regulation of ECM biosynthesis in the lung. Recent work in this laboratory has established that cultured fetal rat lung cells (clone 2G3) offer a unique opportunity to investigate in vitro the biosynthesis of ECM components. Thus the first major objective of this proposal has two specific aims: 1) to determine if genetic types of collagen other than type IV are synthesized by the cells; and 2) to assess the effects of EGF and retinoic acid on this biosynthetic parameter. The second major approach to be used for acquiring insight into the regulation of ECM biosynthesis in pulmonary systems will be to construct cloned DNA sequences complimentary to the Alphal(IV) procollagen mRNA isolated from cultured ED-PYS cells for the purpose of creating probes for this rate-limiting element in the biosynthesis of type IV collagen, and this major objective has four specific aims: 1) to construct cloned cDNA sequences in bacteria against ED-PYS mRNA; 2) to identify those recombinant molecules containing sequences complimentary to the Alphal(IV) procollagen mRNA; 3) to use these clones to identify and isolate the gene coding for this ECM component; and 4) to develop the experimental methodologies using the cloned cDNA sequences to quantitate and measure changes in Alphal(IV) procollagen mRNA levels in vivo. It is felt that this combination of approaches will establish a legitimate experimental milieu for future investigations aimed at elucidating the mechanisms that regulate extracellular matric biosynthesis in the lung.

基底膜是特化的细胞外基质， 与肺泡上皮细胞和毛细血管内皮细胞的界面 肺 目前的数据表明，这些结构包含三种类型的 大分子-IV型胶原蛋白、层粘连蛋白和蛋白聚糖。 这种类型的 细胞外基质（ECM）不仅作为细胞支持， 元素和分子过滤器，但最近的证据表明， 可能影响细胞的生长和分化。 因为这些 结构对于肺功能的正常维持是至关重要的， 了解这些ECM组件的调节方法，对于 了解肺部发育和疾病期间组织的反应。 拟议调查的长远目标是， 基底膜表达的调控机制及影响因素 膜组件和通过其组件的机制 影响细胞功能。 人们认为，这些目标可以 通过将体外研究与重组DNA技术相结合， 在分子水平上剖析ECM生物合成的调节， 肺。 本实验室最近的工作已经证实， 大鼠肺细胞（克隆2G 3）提供了研究 ECM组分的体外生物合成。 第一个主要目标 这项建议有两个具体目标：1）确定是否遗传类型 IV型以外的胶原蛋白由细胞合成;和2） 评估EGF和维甲酸对这种生物合成的影响， 参数. 第二个主要的方法是用来获得洞察力， 肺系统中ECM生物合成调节将 构建与Alphal（IV）前胶原互补的克隆DNA序列 从培养的ED-PYS细胞中分离的mRNA用于产生探针 对于IV型胶原生物合成中的这种限速元件，以及 该主要目标有四个具体目标：1）构建克隆的cDNA 针对ED-PYS mRNA的细菌中的序列; 2）鉴定那些重组的 含有与Alphal（IV）前胶原互补序列的分子 3）使用这些克隆鉴定和分离编码 该ECM组件;以及4）开发实验方法， 克隆的cDNA序列，以定量和测量Alphal（IV） 体内前胶原mRNA水平。 据认为，这种结合 这些方法将为未来建立一个合法的实验环境， 调查旨在阐明调节 细胞外基质的生物合成。