Division of Nephrology, Department of Internal Medicine, Juntendo University Faculty of Medicine, Bunkyo-ku, Tokyo 113-8421, Japan.
Nephrol Dial Transplant. 2010 Jun;25(6):1825-32. doi: 10.1093/ndt/gfp742. Epub 2010 Jan 18.
Establishing a peritoneal mesothelial cell (MC) line in which the native characteristics of primary MCs are constantly maintained in vivo is of great significance for investigating their morphological and functional changes in peritoneal dialysis. We established transgenic (Tg) rats that expressed the temperature-sensitive tsA58 mutant of the simian virus 40 large T-antigen (tsSV40T), which served as a source of immortalized rat cell lines. The cells were immortalized at a permissive temperature of 33 degrees C, although they were differentiated at a non-permissive temperature of 38 degrees C. In this study, we established a novel MC line from tsSV40T Tg rats and evaluated its characteristics.
MCs were isolated from 8-week-old tsSV40T Tg rats and cloned. MCs from 8-week-old Wistar rats were used as controls. These cells were immunohistochemically and phenotypically evaluated by immunofluorescence, phase contrast and electron microscopy. The production of plasminogen activator inhibitor 1 (PAI-1) from MCs stimulated by tumour necrosis factor-alpha (TNF-alpha) was measured.
The tsSV40T MCs showed a cobblestone-like appearance at 33 and 38 degrees C, which was similar to normal primary cultured MCs. Microvilli-like structures were observed on the cell surface by a scanning electron microscope at 33 and 38 degrees C. Wilms tumour-1 and pancytokeratin, as MC markers, were expressed at 33 and 38 degrees C. Following TNF-alpha stimulation, PAI-1 production of tsSV40T MCs was similar to that of normal primary cultured MCs.
We established a novel, conditionally immortalized MC line that continuously retained the characteristics of primary cultured peritoneal MCs. This cell line might be a useful tool for various types of in vitro biological research on peritoneal dialysis.
建立一个腹膜间皮细胞（MC）系，使其在体内持续保持原代 MC 的固有特性，对于研究腹膜透析中 MC 的形态和功能变化具有重要意义。我们建立了表达猿猴病毒 40 大 T 抗原（tsSV40T）温度敏感 tsA58 突变体的转基因（Tg）大鼠，该突变体作为永生化大鼠细胞系的来源。这些细胞在允许温度 33℃下被永生化，尽管在非允许温度 38℃下它们会分化。在这项研究中，我们从 tsSV40T Tg 大鼠中建立了一种新型 MC 系，并对其特性进行了评估。
从 8 周龄 tsSV40T Tg 大鼠中分离并克隆 MC。将 8 周龄 Wistar 大鼠的 MC 作为对照。通过免疫荧光、相差和电子显微镜对这些细胞进行免疫组织化学和表型评估。测量由肿瘤坏死因子-α（TNF-α）刺激的 MC 产生纤溶酶原激活物抑制剂 1（PAI-1）的情况。
tsSV40T MC 在 33℃和 38℃下呈鹅卵石样外观，与正常原代培养 MC 相似。在 33℃和 38℃下，扫描电子显微镜观察到细胞表面有微绒毛样结构。Wilms 肿瘤-1 和广谱细胞角蛋白作为 MC 标志物，在 33℃和 38℃下均有表达。在 TNF-α刺激后，tsSV40T MC 的 PAI-1 产生与正常原代培养 MC 相似。
我们建立了一种新型的、条件永生化的 MC 系，其连续保留了原代培养腹膜 MC 的特征。该细胞系可能是各种腹膜透析体外生物学研究的有用工具。
