UPRIGHT LIGHT MICROSCOPE: CYSTIC FIBROSIS GENE THERAPY

该子项目是利用由NIH/NCRR资助的共享仪器补助金提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是资助机构，不一定是研究者的机构。描述（由申请人提供）：这是一份带有孵育室的徕卡工作站DM 6000B立式荧光显微镜的申请。双面灌注的上皮细胞单层活细胞研究需要显微镜设置。该显微镜适用于研究细胞信号传导、基因治疗、运输和分化上皮细胞的细胞组织的动力学和极性的研究小组。目前已充分认识到，激素或生长因子的调节不仅可以发生在血液中，也可以发生在极化细胞的管腔侧。同样，管腔侧的血流也会影响细胞功能。研究这些调节过程的动力学和空间组织对于理解上皮细胞的正常和病理行为至关重要。细胞培养方法的进步使我们能够为许多不同类型的上皮建立体外模型。为了获得高度分化的细胞并模拟体内的情况，细胞在可渗透的载体上生长，形成极化、融合、分化的上皮细胞层，荧光显微镜可以研究不同细胞过程的动力学。这种上皮在根尖和基侧灌注不同的溶液和/或速率，以模拟生理条件，即基础侧（血液）的营养物质、氧气和某些调节剂，以及根尖侧（管腔）的其他调节剂和流动条件。特别是，我们将研究活上皮：受体运输和信号（肾近端小管细胞）的流量依赖性；用于基因治疗的浓缩聚赖氨酸/DNA复合物的细胞内命运（肺上皮）；野生型和多囊肾病变小鼠（肾收集管细胞）EOF受体信号的空间调节，SI00蛋白在分化过程中的相互作用（表皮），腔内pH值的调节（雌性生殖道上皮），以及纤毛偏转所需的力（肾上皮细胞）。这些研究需要一个最先进的立式荧光显微镜工作站，具有活细胞的温度和大气控制，以及具有双灌注和高数值孔径的水浸光学的光学室。

This subproject is one of many research subprojects utilizing the resources provided by a Shared Instrumentation Grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the grant, which is not necessarily the institution for the investigator. DESCRIPTION (provided by applicant): This is a request for the upright epifluorescence light microscope Leica workstation DM 6000B with incubation chamber. The microscope set-up is needed for live cell studies of epithelial cell monolayers with dual-sided perfusion. The microscope is requested for a group of investigators concerned with investigating the dynamics and polarity of cell signaling, gene therapy, transport, and cellular organization of differentiated epithelial cells. It is well recognized by now that hormonal or growth factor regulation can occur not only from the blood, but also the luminal side in polarized cells. Similarly, flow on the luminal side affects cellular functions. Investigations of the dynamics and spatial organization of these regulatory processes is crucial for progress in understanding normal and pathological behavior of epithelial cells. Advances in cell culture methods have allowed us to establish in-vitro models for many different types of epithelia. To obtain highly differentiated cells and mimic the in-vivo situation, cells are grown on permeable support to form polarized, confluent, differentiated epithelial cell layers in which the dynamics of different cellular processes can be studied by fluorescence microscopy. Such epithelia are perfused with different solutions and/or rates on apical and basal side to mimic physiological conditions, i.e., nutrients, oxygen and certain regulatory agents on the basal (blood) side and other regulatory agents and flow conditions on the apical (luminal) side. In particular, we will study in live epithelia: flow dependence of receptor trafficking and signaling (renal proximal tubule cells); the intracellular fate of condensed polylysine/DNA complexes used for gene therapy (pulmonary epithelia); spatial regulation of EOF receptor signaling from wild type and polycystic kidney diseased mice (renal collecting duct cells), interactions of SI00 proteins during differentiation (epidermis), regulation of luminal pH (epithelia of the female reproductive tract), and the force required for cilia deflection (renal epithelial cells). These studies require a state-of-the-art upright epifluorescence light microscope workstation with temperature and atmospheric control for live cells and an optical chamber with dual perfusion and water immersion optics with high numerical aperture.