CORE--PHYSIOLOGY

生理学核心致力于满足以下项目的需求 CFTR功能的测量。依赖于cftr的c1~通道函数将 在CFTR基因治疗前后的细胞中进行测量 替代药品。它将被临床项目使用， 以及所有与矢量有关的基础科学子项目 生产或使用。该核心的能力包括(1)测量 使用短路技术的生物电学特性，(2)净c1~ 使用荧光染料在培养的单层、单细胞、 利用成像技术，在混合细胞群体中，(3)膜片钳， (4)双层重组。融合的细胞培养将是 Ussing电离室短路电流分析 专门为培养的融合细胞而设计。网的测量 ~使用荧光染料6-甲氧基-N-(3-甲氧基-N-(3- 磺丙基喹啉(SPQ)。细胞的荧光测量 当培养时，将采用单层或单细胞水平 不是融合的。或者，将使用荧光比率成像 为了测量只有稀疏或混合的细胞群体的c1~ 从患者那里获得的，都是可用的。将采用的协议 是专门为区分CFTR而设计的- 介导的跨上皮细胞C1~流量或CFTR介导的来自其他来源的C1~流量 上皮细胞离子转运功能。这些协议包括ION 取代、离子梯度和特定抑制剂或激活剂的使用 与其他分泌物离子转运蛋白的比较 上皮细胞。将使用放射性同位素程序来评估 细胞膜和细胞的净离子通量和屏障特性 文化。一名技术人员将实施短路电流 测量和支持核心，以及一名全职博士后助理 将可用于图像分析和荧光研究。这个 核心将在一个完整的电生理学和 膜运输设施，从而节省了大量的 成本和努力。因此，膜片钳、双层、正面双发射- 双激发光谱仪--基于滤光片的双激发 荧光分光光度计、微量荧光分光光度计性能和一次使用 如有需要，将向堆芯提供密封室设置。仅限 用于购买图像分析系统的部分资金(20%)是 如有请求，额外费用将在其他来源之间分配。 因为在这个核心工作的人员将处于 一个最先进的生理学实验室，他们将与支持人员互动 人员、博士后、学生、访问科学家和高级 提供方法、技术和技术方面的专业知识的教师 将需要的分析。

The Physiology Core is dedicated to the needs of the program project for measurement of CFTR function. CFTR-dependent C1~ channel function will be measured in cells before and after treatment with CFTR genetic replacement pharmaceuticals. It will be used by the clinical project, and all of the basic science subprojects which are involved in vector production or use. The capabilities of this Core include (1) measurement of bioelectric properties using short circuit techniques, (2) net C1~ transport using fluorescent dyes in cultured monolayers, single cells, and with imaging techniques, in mixed cell populations, (3) patch clamp, and (4) bilayer reconstitution. Confluent cell cultures will be subjected to short circuit current analysis in Ussing chambers specifically designed for cultured confluent cells. Measurements of net ~C1 flux will be accomplished using the fluorescent dye, 6-methoxy-N-(3- sulfopropyl) quinolinium (SPQ). Fluorescence measurements on cell monolayers or at the single cell level will be employed when cultures are not confluent. Alternatively, fluorescence ratio imaging will be used to measure C1~ where only sparse or mixed cell populations such as might be obtained from patients, are available. The protocols to be employed are those which have been specifically designed to distinguish CFTR- mediated transepithelial C1~ flux or CFTR-mediated C1~ flux from other epithelial ion transport function. These protocols include ion substitution, ion gradients, and use of specific inhibitors or activators of the CFTR versus other ion transport proteins of the secretory epithelia. Radioisotopic procedures will be employed for evaluation of net ionic flux and barrier properties of cell membranes and cell cultures. A technician will carry out the short circuit current measurements and support the core, and a full time postdoctoral associate will be available for the image analysis and fluorescence studies. The Core will be in the environment of a complete electrophysiology and membrane transport facility, thereby producing an enormous saving in costs and efforts. Thus, patch clamp, bilayer, front face dual emission- dual excitation spectrofluorimeter, a filter based dual excitation spectrofluorimeter, microspectrofluorimeter capabilities, and one Ussing chamber set-up will be made available to the CORE as needed. Only partial funding (20%) for the purchase of an image analysis system is requested, the additional costs to be distributed amongst other sources. Since the personnel working in this CORE will be in the environment of a state-of-the-art physiology laboratory, they will interact with support personnel, postdoctorals, students, visiting scientists and senior faculty who provide expertise in the approaches, technologies and analyses which will be needed.