IDENTIFICATION AND CHARACTERIZATION OF MATERIALS SECRETED BY CLARA CELLS

克拉拉细胞分泌物质的鉴定和表征

• 批准号: 4693207
• 负责人: G R HOOK
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/4693207
• 关键词: antibiotics; biological models; bronchus; cortisol; density gradient ultracentrifugation; endoplasmic reticulum; epidermal growth factor; gel electrophoresis; granule; growth media; insulin; methionine; model design /development; radiotracer; respiratory epithelium; secretion; tissue /cell culture; transferrin

The functions of the bronchiolar Clara cell are not known although numerous morphological investigations in many species have led to the hypothesis that the cell is secretory. The objectives of this research are to elucidate the secretory nature of the Clara cell, identify and characterize those secretions and determine their extracellular functions. We have developed a model system for the study of Clara cell functions and metabolism using cells isolated from the lungs of rabbits. Clara cells are dispersed from the lungs with pancreatic proteases and purified to over 90% by using, sequentially, a variety of techniques involving centrifugal elutriation, density gradient centrifugation, differential adherence and in vitro cultivation. Incubation of the isolated Clara cells with 35S-methionine for 4 hrs results in the radiolabeling of many intracellular proteins some of which were released into the incubation medium. Biosynthesis and secretion of proteins by Clara cell have been compared with those synthesized and secreted by other isolated and purified pulmonary cells including alveolar macrophages, Type II cells, and basal cells. The labeled cellular proteins and proteins released from the cells were identified by using sodium dodecylsulfate-polyacrylamide gel electrophoresis under reducing conditions followed by fluorography and quantified by microdensitometry. Four major newly synthesized proteins with molecular weights of 9 kd, 30 kd, 90 kd and 180 kd were released from the Clara cells. The 9 kd protein accounted for 42% of the released and 10% of the cellular 35S-protein after 4 hrs of incubation. The 9 kd and 180 kd proteins were not synthesized by any of the other cell types. In addition, antisera prepared against Clara cell antigens were used to identify the presence of the 9 kd protein in lavage effluents from the lungs of rabbits. These data demonstrate that Clara cells make and secrete proteins different from those made and secreted by several other major pulmonary cells and that some of those proteins are present in the pulmonary extracellular lining.

细支气管Clara细胞的功能尚不清楚， 对许多物种的形态学研究导致了这样的假设 细胞是分泌性的 本研究的目的是 阐明Clara细胞的分泌性质，鉴定和表征 这些分泌物并决定其细胞外功能。 我们有 开发了一个用于研究Clara细胞功能的模型系统， 使用从兔子肺中分离的细胞进行代谢。 克拉拉细胞是 用胰蛋白酶从肺中分散出来，并纯化到90%以上， 通过依次使用各种技术， 淘洗，密度梯度离心，差速贴壁和离心， 离体培养 将分离的Clara细胞与 35 S-甲硫氨酸4小时导致许多细胞内的放射性标记。 其中一些蛋白质被释放到孵育介质中。 比较了Clara细胞蛋白质的合成和分泌 与其他分离和纯化的合成和分泌的那些 肺细胞，包括肺泡巨噬细胞、II型细胞和基底细胞 细胞 标记的细胞蛋白质和从细胞中释放的蛋白质 采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳进行鉴定 在还原条件下进行电泳，然后进行荧光照相， 通过显微密度测定法定量。 四种新合成的蛋白质 释放分子量为9 kd、30 kd、90 kd和180 kd的 克拉拉细胞 9 kd蛋白占释放量的42%， 孵育4小时后10%的细胞35 S-蛋白。 9 kd和 180 kd蛋白质不被任何其它细胞类型合成。 在 此外，针对Clara细胞抗原制备的抗血清用于 鉴定来自胃肠道的灌洗流出物中9 kd蛋白的存在， 兔子的肺 这些数据表明，克拉拉细胞制造和分泌 蛋白质不同于其他几种主要的蛋白质 肺细胞，其中一些蛋白质存在于 肺细胞外膜