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。 CFTR依赖性C1通道功能将 在用CFTR基因治疗之前和之后的细胞中测量 替代药品 它将用于临床项目， 以及所有涉及矢量的基础科学子项目 生产或使用。 该核心的功能包括（1）测量 利用短路技术研究了生物电特性，（2）净C1~ 在培养的单层，单细胞， 以及成像技术，在混合细胞群中，（3）膜片钳， （4）双层重构。 将汇合的细胞培养物 在Ussing室中进行短路电流分析 专为培养的融合细胞而设计。 网的测量 ~C1通量将使用荧光染料6-甲氧基-N-（3-甲氧基苯基）-N-（2 磺丙基）喹啉鎓（SPQ）。 细胞上的荧光测量 单层或单细胞水平将被采用时，培养物是 不汇合。 或者，将使用荧光比率成像 为了测量C1，其中仅稀疏或混合的细胞群，例如可能 从患者那里获得，可以获得。 拟采用的方案 是那些专门设计用来区分CFTR- - 介导的跨上皮C1-通量或CFTR介导的来自其他细胞的C1-通量 上皮细胞离子转运功能 这些协议包括离子 取代、离子梯度和使用特异性抑制剂或活化剂 CFTR相对于分泌型细胞的其他离子转运蛋白 上皮细胞 放射性同位素程序将用于评价 细胞膜和细胞的净离子通量和屏障性质 cultures. 技术人员将进行短路电流 测量和支持核心，和一个全职博士后助理 将可用于图像分析和荧光研究。 的 核心将在一个完整的电生理环境中， 膜运输设施，从而产生巨大的节省， 成本和努力。 因此，膜片钳，双层，正面双发射- 双激发荧光分光光度计，基于双激发的滤光片 荧光分光光度计，显微荧光分光光度计的能力，和一个尤辛 将根据需要向核心小组提供试验室设置。 只 部分资金（20%）用于购买图像分析系统， 所要求的额外费用将在其他来源之间分配。 由于在该CORE中工作的人员将处于 一个最先进的生理学实验室，他们将与支持 人员，博士后，学生，访问科学家和高级 教师谁提供专业知识的方法，技术和 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.