人类膀胱移行上皮细胞的体外培养与鉴定

Culture and identification of human bladder transitional cells in vitro

背景：移行上皮细胞的分离方法有酶消化法、组织块法及刮削法3种。目的：探讨培养人类膀胱移行上皮细胞和其传代的最佳方法。方法：培养人类膀胱移行上皮细胞，观察细胞在MEM-F12和KSFM培养基中的生长增殖以及形态变化。免疫荧光染色观察上皮细胞特异性蛋白标记AEl及仅在尿路上皮组织中特异表达的尿空斑蛋白UroplakinⅢ，以鉴定膀胱移行上皮细胞。分别利用4步胰蛋白酶消化法和组织块再植法对细胞进行传代培养。结果与结论：①细胞在MEM-F12培养基中较KSFM培养基中爬出及融合均快，生长状态良好。②细胞免疫荧光鉴定证实为移行上皮细胞。⑨胰蛋白酶消化传代细胞，传代第18-24h贴壁，贴壁后无法继续生长，60-72h后细胞开始凋亡：组织块再种传代细胞生长稳定迅速，24-48h细胞开始爬出，第10-16天达到80％融合，传至第4代后开始出现衰退。说明以DMEM-F12培养基进行组织块培养法和组织块再种法是人类膀胱移行上皮细胞原代培养和传代培养经济有效的方法。

BACKGROUND： Methods to separate the transitional epithelial cells include enzyme digestion method, tissue block method and spinning scraping method. OBJECTIVE： To investigate the best approach to culture and passage the human bladder transitional cells in vitro. METHODS： Human bladder transitional epithelial cells were cultured in MEM-F12 and KSFM media. Then the cell morphological changes, growth and proliferation were observed. The bladder transitional epithelial cells were identified by immunofluorescent staining based on the presence of uothelium specific cell markers AE1 and Uroplakin Ill, the latter one specifically expressed in urothelial tissue. The cells were subcultured by 4-step trypsin digestion method and tissue block replantation method, respectively. RESULTS AND CONCLUSION： The cells in EVE-F12 media grew well, and the cell climbing-out and cell fusion occurred earlier than cells in KSFM media. The cells were identified as transitional epithelial cells by immunofluorescence. Passage cells subcultured by trypsin digestion began to adhere to the wall at 18-24 hours after passage. The cells stopped growing after adherence. Cellapoptosis appeared after 60-72 hours. The passage cells subcultured by tissue block replantation displayed a stable and fast growth. The cell began to climb out at 24 48 hours. Cell fusion rate achieved 80% on 10- 16 days. Cells began to degenerate at the fourth passage. These results demonstrate that tissue block cultivation and replantation based on DMEM-F12 is an economic and effective way to culture and subculture the human bladder transitional cells.