Establishment and characterization of conditionally immortalized endothelial cell lines from the thoracic duct and inferior vena cava of tsA58/EGFP double-transgenic rats
Establishment and characterization of conditionally immortalized endothelial cell lines from the thoracic duct and inferior vena cava of tsA58/EGFP double-transgenic rats
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DOI：
10.1007/s00441-006-0229-x
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发表时间：
2006-12-01
影响因子：
3.6
通讯作者：
Saiki, Ikuo
中科院分区：
文献类型：
--
作者：
Matsuo, Mitsuhiro;Koizumi, Keiichi;Saiki, Ikuo
血液血管内皮细胞的基本生物学已被很好地记录。然而，淋巴管内皮细胞，尽管其在正常和病理条件下的重要性，知之甚少。淋巴管内皮细胞系的缺乏阻碍了这一领域的进展。本研究的目的是建立和表征淋巴管和静脉内皮细胞系来自新开发的tsA 58/EGFP转基因大鼠窝藏的温度敏感的猿猴病毒40（SV 40）大T-抗原和增强的绿色荧光蛋白（EGFP）。通过管腔内酶消化从转基因大鼠中分离内皮细胞。将克隆的细胞系分别命名为TR-LE（来自胸导管的温度敏感的大鼠淋巴管内皮细胞）和TR-BE（来自下腔静脉的温度敏感的大鼠血管内皮细胞），并在添加有20%胎牛血清和内皮有丝分裂原的HuMedia-EG 2中在允许温度33 ℃下在纤连蛋白包被的培养皿上培养。温度变化到37 ℃导致增殖减少，大T抗原降解和聚（ADP-核糖）聚合酶裂解。TR-LE细胞表达淋巴管内皮标记物VEGFR-3（血管内皮生长因子受体）、LYVE-1（淋巴管内皮受体）、Prox-1（同源框基因产物）和podoplanin（肾小球足细胞膜粘蛋白），以及内皮标记物CD 31、Tie-2和VEGFR-2，而TR-BE细胞表达CD 31、Tie-2和VEGFR-2，但不表达淋巴管内皮标记物。因此，这些条件永生化和EGFP表达的淋巴管和血管内皮细胞系可能代表了一个重要的工具，在体外内皮细胞功能的研究。
The basic biology of blood vascular endothelial cells has been well documented. However, little is known about that of lymphatic endothelial cells, despite their importance under normal and pathological conditions. The lack of a lymphatic endothelial cell line has hampered progress in this field. The objective of this study has been to establish and characterize lymphatic and venous endothelial cell lines derived from newly developed tsA58/EGFP transgenic rats harboring the temperature-sensitive simian virus 40 (SV40) large T-antigen and enhanced green fluorescent protein (EGFP). Endothelial cells were isolated from the transgenic rats by intraluminal enzymatic digestion. The cloned cell lines were named TR-LE (temperature-sensitive rat lymphatic endothelial cells from thoracic duct) and TR-BE (temperature-sensitive rat blood-vessel endothelial cells from inferior vena cava), respectively, and cultured on fibronectin-coated dishes in HuMedia-EG2 supplemented with 20% fetal bovine serum and Endothelial Mitogen at a permissive temperature, 33 degrees C. A temperature shift to 37 degrees C resulted in a decrease in proliferation with degradation of the large T-antigen and cleavage of poly (ADP-ribose) polymerase. TR-LE cells expressed lymphatic endothelial markers VEGFR-3 (vascular endothelial growth factor receptor), LYVE-1 (a lymphatic endothelial receptor), Prox-1 (a homeobox gene product), and podoplanin (a glomerular podocyte membrane mucoprotein), together with endothelial markers CD31, Tie-2, and VEGFR-2, whereas TR-BE cells expressed CD31, Tie-2, and VEGFR-2, but no lymphatic endothelial markers. Thus, these conditionally immortalized and EGFP-expressing lymphatic and vascular endothelial cell lines might represent an important tool for the study of endothelial cell functions in vitro.