CF TS TRANSFORMANTS--BIOCHEMICAL AND GENETIC ANALYSIS

该项目的目标是建立和表征 囊性纤维化(CF)呼吸道上皮细胞的永久培养。 增殖的CF上皮细胞的原代培养将是 将ptsA209ori-载体导入ptsA209ori-pTX。该质粒含有一种 SV40基因组在复制起始处有缺陷，并具有 大T抗原的温度敏感型突变。该质粒 将采用磷酸钙沉淀法。的克隆 转化的CF上皮细胞将来自气管， 支气管、粘膜下层和鼻息肉。变形金刚将 最初要检测它们的上皮来源，转化- 特异性抗原和氯离子转运缺陷。个体 菌落将在允许的温度(33摄氏度)和 不允许(40摄氏度)温度，与正常温度相比 上皮细胞。将对牢房进行全面监测和 分析离子转运、分泌和癌基因表达。 SV40T抗原的表达、角蛋白的产生和分泌 在浆液性、粘液性或杯状细胞中表达的抗原 通过免疫细胞化学染色确定。电子显微镜 将被用来表征 细胞，例如，连接形成、微绒毛和分泌 颗粒。氯离子传输将通过膜片钳进行测量， 用Ussing小室吸收~(36)Cl和荧光氯- 敏感的喹啉染料。这些细胞也将被分析 β-肾上腺素能刺激cAMP及其癌基因产物的作用 在这一途径中发挥作用，也在离子运输中发挥作用。中的更改 维甲酸佛波酯治疗后癌基因的表达 酸，和二甲基亚砜会比较离子的变化 运输和分泌。将使用永生的CF细胞 目的：构建脉冲法筛选的cDNA文库。 现场凝胶碎片。通过筛选和筛选分离的候选克隆 脉冲场凝胶碎片将用于互补研究。

The goal of this project is establishment and characterization of permanent cultures of cystic fibrosis (CF) airway epithelial cells. Primary cultures of proliferating CF epithelial cells will be transfected with the ptsA209ori- plasmid. The plasmid contains an SV40 genome defective in the origin of replication and has a temperature-sensitive mutation in the large T-antigen. The plasmid will be introduced by calcium phosphate precipitation. Clones of transformed CF epithelial cells will be derived from trachea, bronchi, submucosa, and nasal polyps. Transformants will initially be assayed for their epithelial origin, transformation- specific antigens, and defective Cl ion transport. Individual colonies will be analyzed at the permissive (33 degrees C) and nonpermissive (40 degrees C) temperatures and compared to normal epithelial cells. Cells will be monitored and comprehensively analyzed for ion transport, secretion, and oncogene expression. SV40 T-antigen expression, keratin production, and secretory antigens expressed in serous, mucous, or goblet cells will be determined by immunocytochemical staining. Electron microscopy will be used to characterize the morphological features of the cells, e.g., junction formation, microvilli, and secretory granules. Chloride ion transport will be measured by patch clamp, by the uptake of 36Cl, with Ussing chambers, and fluorescent Cl- sensitive quinolinium dyes. The cells will also be analyzed for beta-adrenergic stimulation of cAMP and the role oncogene products play in this pathway as well as in ion transport. Changes in oncogene expression after treatment with phorbol esters, retinoic acid, and dimethyl sulfoxide will be compared to changes in ion transport and secretion. Immortal CF cells will be used to construct cDNA libraries which will be screened with pulsed- field gel fragments. Candidate clones isolated by screening and pulsed-field gel fragments will be used in complementation studies.