Immortalized liver endothelial cells: a cell culture model for studies of motility and angiogenesis.
Immortalized liver endothelial cells: a cell culture model for studies of motility and angiogenesis.
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DOI：
10.1038/labinvest.2010.132
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发表时间：
2010-12
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肝窦内皮细胞（HSEC）是一种独特的有孔内皮细胞亚群，排列在肝窦内，构成肝脏内大部分内皮细胞。HSEC细胞在血液清除、血管张力和免疫中起重要作用，但也经历促成纤维化、血管生成和门静脉高压的病理变化。由于原代细胞分离耗时，数量有限，并且通常缺乏病理性脉管系统的特征，因此用于运动性和血管生成的体外研究的细胞培养模型很少。本研究的目的是产生一个永生化的细胞系来源于HSEC，模仿病理性血管系统，并允许详细的分子干预进行追求。采用基于CD31的免疫磁性分离法从小鼠肝脏中分离HSEC，用SV40大T抗原永生化，并基于其内吞乙酰化低密度脂蛋白（AcLDL）的能力进行亚克隆。所得到的细胞系，转化的窦状内皮细胞（TSEC），保持内皮表型以及一些HSEC特异性的功能。这通过内皮的典型显微特征得到证明，包括板状伪足和丝状伪足的形成以及细胞单层的鹅卵石形态。电子显微镜检查表明，在筛板中组织的窗孔数量有限。TSEC表达多种内皮特异性标志物，包括通过PCR阵列、免疫印迹和免疫荧光检测的CD31和von Willebrand因子。在功能上，TSEC保持许多关键的内皮特征，包括响应血管生成因子的迁移、血管管的形成、AcLDL的内吞作用和细胞外基质的重塑。它们的表型最接近与慢性肝病相关的病理性新生血管，其中细胞变得增殖、脱膜和血管生成。重要的是，可以用病毒载体有效地转导细胞。TSEC应该提供一个可重复的细胞培养模型，用于高通量的体外研究，涉及广泛的肝内皮细胞功能，但也可能涉及更广泛的内皮细胞生物学。
Hepatic sinusoidal endothelial cells (HSEC) are a unique subpopulation of fenestrated endothelial cells lining the hepatic sinusoids and comprising the majority of endothelial cells within the liver. HSEC cells play important roles in blood clearance, vascular tone, and immunity, but also undergo pathologic changes contributing to fibrosis, angiogenesis, and portal hypertension. There are few cell culture models for in vitro studies of motility and angiogenesis since primary cells are time-consuming to isolate, limited in number, and often lack features of pathologic vasculature. The aim of this study was to generate an immortalized cell line derived from HSEC that mimics pathologic vasculature and allows detailed molecular interventions to be pursued. HSEC were isolated from mouse liver using CD31-based immunomagnetic separation, immortalized with SV40 large T antigen, and sub-cloned based on their ability to endocytose acetylated low density lipoprotein (AcLDL). The resulting cell line, transformed sinusoidal endothelial cells (TSEC), maintains an endothelial phenotype as well as some HSEC-specific features. This is evidenced by typical microscopic features of endothelia, including formation of lamellipodia and filopodia and a cobblestone morphology of cell monolayers. Electron microscopy demonstrated maintenance of a limited number of fenestrae organized in sieve plates. TSEC express numerous endothelia-specific markers including CD31 and von Willebrand's factor as detected by PCR array, immunoblotting, and immunofluorescence. Functionally, TSEC maintain a number of key endothelial features including migration in response to angiogenic factors, formation of vascular tubes, endocytosis of AcLDL, and remodelling of extracellular matrix. Their phenotype most closely resembles the pathologic neovasculature associated with chronic liver disease in which cells become proliferative, defenestrated, and angiogenic. Importantly, the cells can be transduced efficiently with viral vectors. TSEC should provide a reproducible cell culture model for high-throughput in vitro studies pertaining to a broad range of liver endothelial cell functions, but likely broader endothelial cell biology as well.