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.

该子项目是利用由NIH/NCRR资助的共享仪器赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是用于资助的，不一定是研究者的机构。描述（由申请人提供）：这是对带孵育室的立式落射荧光光学显微镜Leica工作站DM 6000 B的请求。显微镜设置是双面灌注上皮细胞单层的活细胞研究所需的。该显微镜是为一组研究细胞信号传导、基因治疗、运输和分化上皮细胞的细胞组织的动态和极性的研究人员而设计的。现在已经很好地认识到，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腔侧。类似地，管腔侧的流动影响细胞功能。研究这些调控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的、汇合的、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力学。在顶侧和基底侧用不同的溶液和/或速率灌注这样的上皮以模拟生理条件，即，在基底（血液）侧的营养物、氧气和某些调节剂以及在顶端（管腔）侧的其它调节剂和流动条件。特别是，我们将研究活上皮细胞：受体运输和信号传导的流量依赖性（肾近端小管细胞）;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）;野生型和多囊肾病小鼠β受体信号空间调控（肾集合管细胞），分化过程中SI 00蛋白的相互作用（表皮）、腔pH值调节（女性生殖道上皮）以及纤毛偏转所需的力（肾上皮细胞）。这些研究需要一个国家的最先进的直立epifluorescence光学显微镜工作站的温度和大气控制的活细胞和一个光学室，双灌注和水浸光学高数值孔径。