Core--Tissue /biophysical

该计划项目中的组织培养/生物物理核心在该PPG中扮演着三个关键角色，是项目之间整合协作的关键机制。首先，核心中心将为五个项目中每一个项目使用的八种肺内皮细胞培养提供一个集中的资源设施。具体而言，该细胞培养核心将继续提供新鲜分离和培养的小鼠肺内皮细胞、人和牛肺动脉内皮细胞系、人和牛肺微血管内皮细胞系、人真皮微血管内皮细胞系、高表达野生型或酪氨酸缺失蛋白酪氨酸磷酸酶Mu突变体的细胞系和高表达蛋白激酶C-Delta的大鼠脂肪垫微血管细胞系。其次，这个核心将利用尖端技术仔细暴露肺部 与体内生理和病理生理条件相关的机械活动环境中的内皮。这项工作的重要工具包括在受控循环拉伸和受控层流剪应力条件下培养内皮细胞。最后，核心将量化计划项目中提出的每项研究的生理意义：(1)测量融合内皮细胞的白蛋白清除；(2)使用内皮细胞-底物阻抗传感系统(ECIS)测量静态培养和循环拉伸预适应内皮细胞单层的跨单分子层电阻；(3)实时同时测量剪切应力下的内皮细胞跨内皮细胞电阻；以及(4)内皮细胞 使用ECIS电穿孔/创伤模块实时监测EC伤口愈合的伤口分析。生物物理和组织培养核心人员的丰富经验继续发挥组织培养核心的作用(之前的项目GRANT HL-58064)，确保高质量细胞的持续供应，在精确表征的剪应力或循环拉伸条件下培养细胞，并在EC单层屏障功能的量化方面提供专家协助。组织培养和生物物理设施的接近将提供在计划项目中获得的结果的重复性和一致性，并确保最经济地使用核心以充分供应计划项目的所有研究人员。

The tissue culture/biophysical Core in this program project serves three key roles in this PPG and is a key mechanism for integrated collaboration between the projects. First, the Core will provide a centralized resource facility for eight lung endothelial cell cultures used in each of five projects. Specifically, the cell culture core will continue provide freshly isolated and cultured murine lung endothelial EC, cell lines of human and bovine pulmonary artery EC, human and bovine pulmonary microvascular EC, human dermal microvascular EC, native cell lines and cell lines over-expressing wild type or tyrosine-deficient mutant of protein tyrosine phosphatase mu, and rat fat pad microvascular cells over-expressing protein kinase C-delta. Second, this Core will utilize sophisticated state of the art technologies to carefully expose lung pulmonary endothelium in mechanically active settings relevant to physiological and pathophysiological in vivo conditions. Important tools for this work include culturing EC under conditions of controlled cyclic stretch and controlled laminar shear stress. Finally, the Core will quantitate the physiological significance of each of the studies proposed in the Program Project: (1) measurements of albumin clearance across confluent EC, (2) measurements of trans-monolayer electrical resistance of static cultures and cyclic stretch-preconditioned EC monolayers using endothelial cell-substrate impedance sensing system (ECIS); (3) real time simultaneous measurements of EC trans-endothelial electrical resistance under shear stress; and (4) EC wound assay with real time monitoring of EC wound healing using ECIS lectroporation/wounding module. The extensive experience of the biophysical and tissue culture core personnel continue to function as a tissue culture core (previous Program Project Grant HL-58064) assuring the continuous supply of high quality cells, culturing cells under precisely characterized conditions of shear stress or cyclic stretch and expert assistance in quantitation of EC monolayer barrier function. The close proximity of the tissue culture and biophysical facilities will provide reproducibility and consistency of results obtained in the Program Projects, and ensure the most economical use of the Core to adequately supply all the investigators of the Program Project.