EPITHELIAL CELL LINES FROM CF AND NORMAL TRACHEAS

囊性纤维化（CF）是最常见的致死性遗传病 在高加索人群中。 其主要临床症状为 都与外分泌腺的上皮细胞有关， 粘膜下腺体和主要气道的粘膜分泌细胞。 最近，在定义遗传学方面取得了重大进展， CF和表征各种上皮中的缺陷， 电解质和液体运输以及粘液分泌。 尽管 这些进展，CF研究现在陷入僵局。 主要 这些障碍包括缺乏组织和细胞 以及在体外繁殖的人类上皮细胞的“有限”寿命， 体外 具有以下能力的永生化细胞系的开发： 保持分化的CF表型将大大 促进这些细胞的生物化学和生理学研究。 该提案的主要目标如下： 在无血清培养基中，低传代上皮细胞来源于 从CF获得的气管粘膜下腺体和表面粘膜 患者;根据以下特征将细胞表征为上皮细胞： 组织化学、超微结构、免疫细胞化学和生物化学 特性;使CF和正常上皮细胞永生化（已经 建立），用病毒DNA转染， 以及通过改变培养条件。 具体来说，我们计划使用 涉及用亚基因组片段进行克隆的方法 含有Ad 12-SV 40的完整转化早期区域，和 含有致癌基因如v-Ha-ras的质粒载体。 病毒DNA序列整合到宿主细胞染色体中， 通过Southern印迹检测病毒基因的表达， 免疫细胞化学分析。 如果这些程序 如果不成功，我们将测试涉及感染的程序。 用于激活氯离子通道。为此，单身 将使用通道膜片钳技术。 在正常细胞中， C1通道应该被细胞中的8-溴-cAMP激活- 附加录制模式，但不应激活通道 在CF细胞中。 在分离膜贴片后，由内而外模式 Cl-通道在正常和CF细胞中应被激活 随后通过在浴中使用180 nM Ca++;测定生长 低传代动力学（倍增时间、细胞周期参数）和 来源于正常和CF组织的永生化细胞;比较 正常和CF细胞中粘蛋白和细胞角蛋白的性质 低传代和永生化细胞;建立程序， 冷冻细胞并分发给研究人员 对CF研究感兴趣。 正常和CF气管上皮细胞的可用性， 研究界将促进不同方面的研究 CF的研究，并可能有助于发现新的治疗方法 这种致命的疾病

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.