Satchell S C, Tasman C H, Singh A, Ni L, Geelen J, von Ruhland C J, O'Hare M J, Saleem M A, van den Heuvel L P, Mathieson P W
Academic Renal Unit, University of Bristol, Southmead Hospital, Bristol, UK.
Kidney Int. 2006 May;69(9):1633-40. doi: 10.1038/sj.ki.5000277.
Glomerular endothelial cells (GEnC) are specialized cells with important roles in physiological filtration and glomerular disease. Despite their unique features, GEnC have been little studied because of difficulty in maintaining them in cell culture. We have addressed this problem by generation of conditionally immortalized (ci) human GEnC using technology with which we have previously produced ci podocytes. Primary culture GEnC were transduced with temperature-sensitive simian virus 40 large tumour antigen and telomerase using retroviral vectors. Cells were selected, cloned, and then characterized by light and electron microscopy (EM), response to vascular endothelial growth factor (VEGF), and tumour necrosis factor (TNF)alpha, expression of endothelial markers by focused gene array, immunofluorescence and Western blotting, and formation and behaviour of monolayers. CiGEnC proliferated at the permissive temperature (33 degrees C) and became growth arrested at the non-permissive temperature (37 degrees C). CiGEnC retained morphological features of early-passage primary culture GEnC up to at least p41, confirming successful immortalization. EM demonstrated fenestrations, increased in number by VEGF. mRNA analysis confirmed expression of the endothelial markers platelet endothelial cell adhesion molecule 1, intercellular adhesion molecule 2, VEGF receptor 2, and von Willebrand factor, validated by immunofluorescence and Western blotting. CiGEnC also expressed Tie2, and TNFalpha upregulated E-selectin. CiGEnC formed monolayers with barrier properties responsive to cyclic adenosine 3',5' monophosphate (cAMP) and thrombin. CiGEnC retain the markers and behaviour of primary culture GEnC. They express fenestrations which are upregulated in response to VEGF. These cells are a unique resource for further study of GEnC and their roles in glomerular filtration, glomerular disease, and response to glomerular injury.
肾小球内皮细胞（GEnC）是在生理滤过和肾小球疾病中起重要作用的特殊细胞。尽管它们具有独特的特征，但由于在细胞培养中难以维持，GEnC的研究很少。我们通过使用先前用于产生条件永生化（ci）足细胞的技术来生成条件永生化（ci）人GEnC，解决了这个问题。使用逆转录病毒载体将温度敏感的猿猴病毒40大肿瘤抗原和端粒酶转导至原代培养的GEnC。对细胞进行筛选、克隆，然后通过光镜和电镜（EM）、对血管内皮生长因子（VEGF）和肿瘤坏死因子（TNF）α的反应、聚焦基因阵列、免疫荧光和蛋白质印迹法检测内皮标志物的表达以及单层的形成和行为进行表征。CiGEnC在允许温度（33℃）下增殖，在非允许温度（37℃）下生长停滞。CiGEnC在至少传代41次时仍保留早期传代原代培养GEnC的形态特征，证实永生化成功。EM显示有窗孔，VEGF可使其数量增加。mRNA分析证实了内皮标志物血小板内皮细胞黏附分子1、细胞间黏附分子2、VEGF受体2和血管性血友病因子的表达，免疫荧光和蛋白质印迹法验证了这一点。CiGEnC还表达Tie2，TNFα上调E-选择素。CiGEnC形成具有对环磷酸腺苷（cAMP）和凝血酶有反应的屏障特性的单层。CiGEnC保留了原代培养GEnC的标志物和行为。它们表达可被VEGF上调的窗孔。这些细胞是进一步研究GEnC及其在肾小球滤过、肾小球疾病和对肾小球损伤反应中作用的独特资源。
