Conditionally immortalized retinal capillary endothelial cell lines (TR-iBRB) expressing differentiated endothelial cell functions derived from a transgenic rat
Conditionally immortalized retinal capillary endothelial cell lines (TR-iBRB) expressing differentiated endothelial cell functions derived from a transgenic rat
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DOI：
10.1006/exer.2000.0941
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发表时间：
2001-02-01
影响因子：
3.4
通讯作者：
Terasaki, T
中科院分区：
文献类型：
--
作者：
Hosoya, K;Tomi, M;Terasaki, T
本研究的目的是建立和表征视网膜毛细血管内皮细胞系（TR-iBRB）从一个新开发的转基因大鼠携带的温度敏感的猿猴病毒40（SV 40）大T抗原基因（Tg大鼠）。从Tg大鼠分离视网膜毛细血管内皮细胞，并在胶原包被的培养皿中于37 ℃培养48小时。随后在33 ℃培养细胞以激活大T抗原。在第三代时，通过集落形成克隆细胞并与其它细胞分离。从Tg大鼠获得9个视网膜毛细血管内皮细胞的永生化细胞系（TR-iBRB 1类似于9）。这些细胞系具有梭形纤维形态，表达典型的内皮标志物血管性血友病因子和内化的乙酰化低密度脂蛋白。此外，血管内皮生长因子（VEGF）受体-2在TR-iBRB中表达。TR-iBRB表达大T抗原，在33 ℃下生长良好，倍增时间为19-21小时。相反，由于大T抗原表达减少，细胞在37和39 ℃下不生长，支持温度依赖性细胞生长。TR-iBRB表达GLUT 1并显示出3-O-甲基-D-葡萄糖（3-OMG）摄取活性。该3-OMG摄取可饱和，米氏常数为5.56 +/- 0.51 mM，最大摄取速率为45.3 +/- 2.6 nmol min（-1）mg蛋白（-1）。在TR-iBRB中表达了分子量约为180 KDa的P-糖蛋白。RT-PCR检测TR-iBRB 2中mdr 1a、mdr 1b和mdr 2基因的表达。总之，条件永生化视网膜毛细血管内皮细胞系建立从一个转基因大鼠窝藏的温度敏感SV 40大T抗原基因，这些线显示出视网膜毛细血管内皮细胞的特性。(C)北京：科学出版社.
The objective of this study was to establish and characterize a retinal capillary endothelial cell line (TR-iBRB) from a newly developed transgenic rat harboring the temperature-sensitive simian virus 40 (SV 40) large T-antigen gene (Tg rat). Retinal capillary endothelial cells were isolated from a Tg rat and cultured in collagen-coated dishes at 37 degreesC for a period of 48 hr. Cells were subsequently cultured at 33 degreesC to activate the large T-antigen, At the third passage, cells were cloned by colony formation and isolated from other cells. Nine immortalized cell lines of retinal capillary endothelial cells (TR-iBRB1 similar to 9) were obtained from a Tg rat. These cell lines had a spindle-fiber shape morphology, expressed the typical endothelial marker, von Willebrand factor, and internalized acetylated-low density lipoprotein. Moreover, vascular endothelial growth factor (VEGF) receptor-2 was expressed in TR-iBRBs. TR-iBRBs expressed a large T-antigen and grew well at 33 degreesC with a doubling time of 19-21 hr. In contrast, cells did not grow at 37 and 39 degreesC due to the reduced expression of large T-antigen, supporting temperature-dependent cell growth. TR-iBRBs expressed GLUT1 and exhibited 3-O-methyl-D-glucose (3-OMG) uptake activity This 3-OMG uptake was saturable with a Michaelis-Menten constant of 5.56 +/- 0.51 mM and a maximum uptake rate of 45.3 +/- 2.6 nmol min(-1) mg protein(-1). P-Glycoprotein, with a molecular weight of similar to 180 KDa, was expressed in TR-iBRBs. In addition, mdr 1a, mdr 1b and mdr 2 were detected in TR-iBRB2 using RT-PCR. In conclusion, conditionally immortalized retinal capillary endothelial cell lines were established from a transgenic rat harboring the temperature-sensitive SV 40 large T-antigen gene and these lines were shown to exhibit the properties of retinal capillary endothelial cells. (C) 2001 Academic Press.