Isolation and characterization of conditionally immortalized mouse glomerular endothelial cell lines
Isolation and characterization of conditionally immortalized mouse glomerular endothelial cell lines
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DOI：
10.1111/j.1523-1755.2004.66009.x
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发表时间：
2004-12-01
影响因子：
19.6
通讯作者：
Berden, JH
中科院分区：
文献类型：
--
作者：
Rops, AL;van der Vlag, J;Berden, JH
背景肾小球细胞系的培养和建立已被证明是了解肾小球细胞在肾小球生理和病理中功能的重要工具。特别是最近建立的条件永生化内脏上皮细胞系，极大地推动了足细胞生物学的研究。从H-2K（B）-tsA 58转基因小鼠中分离肾小球，所述转基因小鼠含有编码SV 40大肿瘤抗原的温度敏感变体的基因，其促进在33 ℃下的增殖生长和在37 ℃下的分化。通过负载有CD 31、CD 105、GSL I-B4和ULEX的磁珠从肾小球生长物中分离肾小球内皮细胞。克隆细胞系的特征在于免疫荧光染色的抗体/凝集素特异性标记的内皮细胞，足细胞，系膜细胞。对假定的肾小球内皮细胞系进行分析，以确定（1）苦参碱诱导的粘附分子表达;（2）基质胶涂层上的管形成;（3）窗孔的存在。通过对肾母细胞瘤-1、平滑肌肌动蛋白（SMA）、足细胞标记蛋白和血管性血友病因子（vWF）的免疫染色判断，我们获得了假定的内皮细胞、足细胞和系膜细胞系。小鼠肾小球内皮细胞克隆#1（mGEnC-1）对vWF、足糖萼蛋白、CD 31、CD 105、VE-钙粘蛋白、GSL I-B4和ULEX、内化乙酰化低密度脂蛋白（LDL）呈阳性，并在用促炎细胞因子活化后显示粘附分子表达增加。此外，mGEnC-1形成管状结构，并含有非封闭的窗孔。mGEnC-1代表一种条件永生化细胞系，在37 ℃培养时具有分化的肾小球内皮细胞的各种特征。最重要的是，mGEnC-1含有非血管性窗孔，这是肾小球内皮细胞的独特特征。
Background. The culture and establishment of glomerular cell lines has proven to be an important tool for the understanding of glomerular cell functions in glomerular physiology and pathology. Especially, the recent establishment of a conditionally immortalized visceral epithelial cell line has greatly boosted the research on podocyte biology.Methods. Glomeruli were isolated from H-2K(b)-tsA58 transgenic mice that contain a gene encoding a temperature-sensitive variant of the SV40 large tumor antigen, facilitating proliferative growth at 33 C and differentiation at 37 C. Glomerular endothelial cells were isolated from glomerular outgrowth by magnetic beads loaded with CD31, CD105, GSL I-B4, and ULEX. Clonal cell lines were characterized by immunofluorescence staining with antibodies/lectins specific for markers of endothelial cells, podocytes, and mesangial cells. Putative glomerular endothelial cell lines were analyzed for (1) cytokine-induced expression of adhesion molecules; (2) tube formation on Matrigel coating; and (3) the presence of fenestrae.Results. As judged by immunostaining for Wilms tumor-1, smooth muscle actin (SMA), podocalyxin, and von Willebrand factor (vWF), we obtained putative endothelial, podocyte and mesangial cell lines. The mouse glomerular endothelial cell clone #1 (mGEnC-1) was positive for vWF, podocalyxin, CD31, CD105, VE-cadherin, GSL I-B4, and ULEX, internalized acetylated-low-density lipoprotein (LDL), and showed increased expression of adhesion molecules after activation with proinflammatory cytokines. Furthermore, mGEnC-1 formed tubes and contained nondiaphragmed fenestrae.Conclusion. The mGEnC-1 represents a conditionally immortalized cell line with various characteristics of differentiated glomerular endothelial cells when cultured at 37 C. Most important, mGEnC-1 contains nondiaphragmed fenestrae, which is a unique feature of glomerular endothelial cells.