CF TS TRANSFORMANTS--BIOCHEMICAL AND GENETIC ANALYSIS

CF TS 转化体--生化和遗传分析

• 批准号: 3359775
• 负责人: Dieter C Gruenert
• 金额: $ 13.47万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-09-30 至 1993-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3359775
• 关键词: G protein; bronchus; cell differentiation; chlorine; clone cells; complementary DNA; cyclic AMP; cystic fibrosis; dimethylsulfoxide; electron microscopy; fluorescent dye /probe; gene expression; genetic manipulation; human tissue; keratin; membrane channels; molecular genetics; mucosa; nose neoplasm; oncogenes; phorbols; plasmids; protein kinase; pulsed field gel electrophoresis; respiratory epithelium; retinoate; secretion; simian virus 40; temperature sensitive mutant; trachea; transfection; viral carcinogenesis; virus antigen; voltage /patch clamp

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.

该项目的目标是建立和表征 囊性纤维化（CF）气道上皮细胞的永久培养物。 将增殖的CF上皮细胞的原代培养物进行培养。 用ptsA 209 ori质粒转染。 质粒含有一个 SV 40基因组在复制起点有缺陷， 大T抗原的温度敏感性突变 质粒 将通过磷酸钙沉淀引入。 无性系 转化的CF上皮细胞来源于气管， 支气管、粘膜下层和鼻息肉。 变形人将 首先测定它们的上皮来源、转化- 特异性抗原和有缺陷的Cl离子转运。 个人 将在允许温度（33 ℃）下分析菌落， 非允许温度（40摄氏度），与正常温度相比 上皮细胞 细胞将被全面监控 分析离子转运、分泌和癌基因表达。 SV 40 T抗原表达、角蛋白产生和分泌 在浆液细胞、粘液细胞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 将被用来表征的形态特征， 细胞，例如，连接形成、微绒毛和分泌 颗粒。 将通过膜片钳测量氯离子转运， 通过摄取36 Cl，使用Ussing室和荧光Cl- 敏感喹啉染料。 还将分析细胞的 cAMP的β-肾上腺素能刺激和癌基因产物的作用 在这条通路中以及在离子运输中起作用。 变化 用佛波醇酯、维甲酸处理后的癌基因表达 酸，二甲亚砜将比较离子的变化 运输和分泌。 将使用永生CF细胞 构建cDNA文库，用脉冲- 现场凝胶碎片 通过筛选分离的候选克隆， 脉冲场凝胶片段将用于互补研究。