UPRIGHT LIGHT MICROSCOPE: CYSTIC FIBROSIS GENE THERAPY

正置光学显微镜：囊性纤维化基因治疗

• 批准号: 7335075
• 负责人: Ulrich Hopfer
• 金额: $ 4.68万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-04-01 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7335075
• 关键词: epithelium; gene therapy; lighting

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.

该子项目是利用 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) 可能已从另一个 NIH 来源获得主要资金，因此可以在其他 CRISP 条目中出现。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）：这是对带孵化室的直立式落射荧光光学显微镜 Leica 工作站 DM 6000B 的请求。双面灌注上皮细胞单层的活细胞研究需要显微镜装置。一组研究人员需要使用该显微镜，这些研究人员关注细胞信号传导的动态和极性、基因治疗、运输和分化上皮细胞的细胞组织。现在人们已经充分认识到，激素或生长因子的调节不仅可以通过血液进行，还可以通过极化细胞的管腔侧进行。同样，管腔侧的流量也会影响细胞功能。对这些调节过程的动力学和空间组织的研究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建立许多不同类型上皮细胞的体外模型。为了获得高度分化的细胞并模拟体内情况，细胞在可渗透的支持物上生长以形成极化、汇合、分化的上皮细胞层，其中可以通过荧光显微镜研究不同细胞过程的动态。这种上皮细胞在顶端和基底侧用不同的溶液和/或速率灌注以模拟生理条件，即基底（血液）侧的营养物、氧气和某些调节剂以及顶端（管腔）侧的其他调节剂和流动条件边。特别是，我们将研究活上皮细胞：受体运输和信号传导（肾近曲小管细胞）的流量依赖性；用于基因治疗（肺上皮）的浓缩聚赖氨酸/DNA复合物的细胞内命运；野生型和多囊肾病小鼠（肾集合管细胞）的 EOF 受体信号传导的空间调节、分化过程中 SI00 蛋白的相互作用（表皮）、管腔 pH 值（雌性生殖道上皮细胞）的调节以及纤毛所需的力偏转（肾上皮细胞）。这些研究需要最先进的直立式落射荧光光学显微镜工作站，对活细胞进行温度和大气控制，以及具有双灌注和高数值孔径水浸光学器件的光学室。