CORE--CELL BIOLOGY LABORATORY

核心--细胞生物学实验室

• 批准号: 6105620
• 负责人: Jonathan H Widdicombe
• 金额: $ 5.2万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-09-01 至 1999-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6105620
• 关键词: biomedical facility; calcium flux; cyclic AMP; cystic fibrosis; cytology; fluorescence microscopy; hematopoietic stem cells; in situ hybridization; laboratory mouse; membrane permeability; phosphorylation; protein kinase; radiotracer; respiratory epithelium; tissue /cell culture; transfection; voltage /patch clamp; western blottings

The Cell Biology CORE will provide cultured cells, assays for determining the viability and function of cells following transfection, and the technology needed for studies of transfected hemopoietic stem cells. Highly differentiated primary cultures of surface and gland epithelium from human trachea (both CF and non-CF) will be initiated in Dr. Finkbeiner's laboratory, and maintained in Dr. Widdicombe's laboratory. Airway gland cell cultures were initially developed at UCSF, and are not generally available at other institutions. Increasing evidence that abnormal gland secretion contributes to the airway pathology of CF greatly enhances the value of these cells. Transformed airway epithelial cell lines (gland and surface epithelium, CF and non-CF) will be made available by Dr. Cruenert. Other cell lines will be initiated and maintained in culture as required. A wide variety of techniques are currently established in Dr. Widdicombe's laboratory for assessing the Cl secretory function of cell -cultures. These include Ussing chambers, radiotracer fluxes, measurements of SPQ fluorescence, and patch-clamping. In addition to transport assays, other cell biological techniques will be available, including tests of viability, cell fractionation, detection of proteins by Western blotting, measurement of [Ca2+]i and cAMP, determination of protein kinase activities and protein phosphorylation. The effectiveness of in vivo CFTR transfection in experimental animals and humans will be assessed from measurements of transepithelial airway potential difference (p.d.) (under the appropriate conditions, this p.d. is a measure of cAMP- dependent Cl secretion). In Dr. Pallavicini's laboratory, expertise is available to a) enrich either murine or human hemopoietic stem cells from marrow specimens (after transfection), b) transplant murine stem cells and/or whole marrow into murine recipients, and c) assess stem and progenitor frequencies in both human and mouse hemopoietic tissues. The frequency of hemopoietic cells carrying the transfected gene will be assessed in hemopoietic progenitors using colony assays and in whole tissues. Expression of transfected genes will be determined using molecular/biochemical assays (to be provided by each investigator). Engraftment of donor cells will be monitored using molecular analysis of genetically tagged donor cells. Integration of the transfected DNA sequences will be measured using fluorescence in situ hybridization.

细胞生物学核心将提供培养的细胞，测定 转染后细胞的活力和功能，以及 研究转染造血干细胞所需的技术。 表面和腺上皮的高分化原代培养物 将在Dr. Finkbeiner的实验室，并在Widdicombe博士的实验室维护。 气道腺细胞培养最初是在加州大学旧金山分校开发的，现在还没有。 一般在其他机构。越来越多的证据表明 CF的气道病理改变与腺体分泌异常有关 提高了这些细胞的价值。转化气道上皮细胞 将提供线（腺体和表面上皮，CF和非CF） Cruenert博士。其他细胞系将在 文化是必需的。目前有各种各样的技术 在Widdicombe博士的实验室建立，用于评估Cl分泌 细胞培养的功能。其中包括尤辛室，放射性示踪剂 通量、SPQ荧光测量和膜片钳。此外 对于转运测定，其它细胞生物学技术也是可用的， 包括生存力、细胞分级分离、蛋白质检测， 蛋白质印迹法，[Ca 2 +]i和cAMP的测量， 蛋白激酶活性和蛋白磷酸化。有效性 在实验动物和人类中的体内CFTR转染将是 通过测量跨上皮气道电位差进行评估 （p.d.）（在适当的条件下，这一点。是一种cAMP的测量方法 依赖性Cl分泌）。在帕拉维奇尼博士的实验室里， 可用于a）富集鼠或人造血干细胞， 骨髓样本（转染后），B）移植鼠干细胞 和/或全骨髓移植到鼠受体中，和c）评估干细胞和 人类和小鼠造血组织中的祖细胞频率。的 携带转染基因的造血细胞的频率将是 使用集落测定法在造血祖细胞中评估， 组织中转染基因的表达将使用 分子/生化分析（由每位研究者提供）。 供体细胞的植入将使用以下分子分析来监测： 基因标记的供体细胞转染DNA的整合 使用荧光原位杂交测量序列。