EPITHELIAL CELL LINES FROM CF AND NORMAL TRACHEAS

囊性纤维化（CF）是最常见的致死性遗传疾病， 高加索人。 其主要临床症状均与 外分泌腺的上皮，包括粘膜下腺和粘膜 主要气道的分泌细胞。 最近，重大进展 发生在定义CF的遗传学和表征的缺陷， 各种上皮细胞关于电解质和液体运输和粘液 分泌物 尽管取得了这些进展，CF研究现在正陷入僵局。 主要的障碍包括组织和细胞的稀缺 以及体外繁殖的人类上皮细胞的“有限”寿命。 能够维持免疫原性的永生化细胞系的开发 分化的CF表型将极大地促进生物化学和 对这些细胞的生理学研究。 本项目的主要目标是 建议采用无血清培养基繁殖，低传代 上皮细胞来源于粘膜下腺体和 从CF患者获得的气管;将细胞表征为上皮细胞， 组织化学、超微结构、免疫细胞化学和 生化特性;使CF和正常上皮细胞永生化 （已经建立）使用致癌病毒，用病毒转染 DNA，并通过改变培养条件。 具体来说，我们计划使用 涉及用含有以下的亚基因组区段进行克隆的方法 Ad 12-SV 40的完整转化早期区域和质粒载体 含有致癌基因如v-Ha-ras 病毒DNA整合 病毒基因的表达将 分别用Southern印迹和免疫细胞化学方法进行检测。 如果这些程序不成功，我们将测试涉及 感染细胞以激活氯离子通道。 为此， 将使用单通道膜片钳技术。 在正常细胞中， 在细胞贴附模式下，C1通道应被8-溴-cAMP激活 记录，但通道不应在CF细胞中激活。 后 分离膜补片，内外模式Cl-通道应 随后在正常和CF细胞中使用180 nM Ca++激活， 水浴;测定生长动力学（倍增时间、细胞周期参数） 来源于正常和CF组织的低传代和永生化细胞; 比较正常和CF细胞中粘蛋白和细胞角蛋白的性质 在低传代和永生化细胞中;建立程序， 冷冻细胞并将其分发给对CF感兴趣的研究人员 research. 正常和CF气管上皮细胞在研究中的可用性 社区将促进对CF研究的不同方面的研究， 可能有助于发现这种致命疾病的新疗法。

Cystic fibrosis (CF) is the most common lethal genetic disease among the Caucasian population. Its major clinical symptoms are all related to epithelial of exocrine glands including the submucosal glands and mucosal secretory cells of the major airways. Recently, major advances have occurred in defining the genetics of CF and characterizing the defects in various epithelia with regards to electrolyte and fluid transport and mucus secretion. Despite these advances, CF research is now reaching an impasse. Major impediments includes the scarcity of tissues and cells to work with and the 'finite' life span of human epithelial cells propagated in vitro. The development of immortalized cell lines capable of maintaining the differentiated CF phenotypes would greatly facilitate biochemical and physiological studies with these cells. The major objectives of this proposal are the following: propagate in serum-free medium, low passage epithelial cells derived from submucosal glands and surface mucosa of trachea obtained from CF patients; characterize the cells as epithelial on the basis of histochemical, ultrastructural, immunocytochemical, and biochemical properties; Immortalize the CF and normal epithelial cells (already established) using oncogenic viruses, transfection with viral DNAs, and by modifying culture conditions. Specifically, we plan to use procedures involving transfecting with subgenomic segments containing the intact transforming early regions of Ad12-SV40, and plasmid vectors containing oncogenes such as the v-Ha-ras. Integration of viral DNA sequences into host cell chromosomes and the expression of viral genes will be examined by Southern blot and immunocytochemical analyses, respectively. If these procedures are not successful, we will test procedures involving infection of the cell for the activation of chloride channels. For this, single channel patch clamp techniques will be used. In normal cells, the C1 channels should be activated by 8-bromo-cAMP in the cell-attached mode of recording, but channels should not be activated in CF cells. After detaching the membrane patch, inside-out mode Cl- channels should be activated in normal and CF cells subsequently by using 180 nM Ca++ in the bath; determine the growth kinetics (doubling times, cell cycle parameters) of low passage and immortalized cells derived from normal and CF tissues; compare the nature of mucoproteins and cytokeratins in normal and CF cells at a low passage and in immortalized cells; establish procedures for freezing the cells and distributing them to investigators interested in CF research. The availability of normal and CF tracheal epithelial cells to the research community would facilitate studies on different aspects of CF research and may be instrumental in discovering new therapies for the lethal disease.