Isolation, culture, and characterization of endothelial cells from mouse glomeruli
Isolation, culture, and characterization of endothelial cells from mouse glomeruli
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DOI：
10.1111/j.1523-1755.2004.00634.x
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发表时间：
2004-06-01
影响因子：
19.6
通讯作者：
Madaio, MP
中科院分区：
文献类型：
--
作者：
Akis, N;Madaio, MP
背景。克隆肾小球内皮细胞（GENC）在免疫学和生理学研究中有许多潜在的用途和应用。GENC的繁殖一直很困难，而且可获得的同质GENC，特别是来自小鼠的基因有限。在此，我们报道了小鼠基因的分离、克隆、繁殖和鉴定。用tsA58永生小鼠分离肾小球细胞。通过差筛分离肾小球，并在内皮细胞允许和最佳条件下培养脱囊的外植体。利用对温度敏感的tsA58基因，扩增来自肾小球外生物的原代细胞，然后让细胞进行自发转化。然后利用抗cd31抗体及其摄取乙酰化低密度脂蛋白（LDL）的能力对细胞进行分类。通过斑块克隆分离得到的亚克隆个体用多个标记进行了特征鉴定。其中一个同质克隆在形态上呈内皮样，CD31、CD106、CD62E、CD54和乙酰化ldl摄取呈阳性，形成管状，上皮和系膜细胞标志物呈阴性。该GENC克隆的功能特性似乎是完整的，信号通路没有改变。其中两个克隆表现出内脏上皮细胞或系膜细胞的特征。确定的克隆应该在多个调查领域具有实用性。
Background. Cloned glomerular endothelial cells (GENC) have many potential uses and applications in immunologic and physiologic studies. Propagation of GENC has been difficult and available homogenous GENC, particularly from mice, are limited. Herein we report isolation, cloning, propagation, and characterization of GENC from mice.Methods. tsA58 immorto mice were used to isolate glomerular cells. Glomeruli were isolated by differential sieving, and decapsulated explants were cultured in permissive and optimal conditions for endothelial cells. The primary cells from glomerular outgrowths were expanded, taking advantage of the temperature-sensitive tsA58 gene, and then the cells were allowed to undergo spontaneous transformation. The cells were then sorted using anti-CD31 antibodies and their capacity to uptake acetylated-low-density lipoprotein (LDL). Individual subclones isolated by patch cloning were characterized using multiple markers.Results. One of the homogenous clones was morphologically endothelial-like, positive for CD31, CD106, CD62E, CD54, and acetylated-LDL uptake, formed tubes, and was negative for epithelial and mesangial cell markers. The functional properties of this GENC clone appeared to be intact, and signaling pathway was not altered. Two of the clones displayed the characteristics of either visceral epithelial or mesangial cells.Conclusion. The identified clones should have utility in multiple areas of investigation.