角膜缘干细胞克隆化培养影响因素的研究  

Study on the effect factors of cloning culture rabbit limbal stem cells

作　　者：王颖[1] 潘志强[1] 张文华[1] 武宇影[1] 接英[1] 

机构地区：[1]首都医科大学附属北京同仁医院眼科中心北京市眼科研究所

出　　处：《眼科》2005年第3期191-194,共4页Ophthalmology in China

摘　　要：目的探讨丝裂霉素C处理Swiss3T3成纤维细胞作为饲细胞的条件,了解兔角膜缘干细胞克隆化培养的增殖状况。设计对照实验研究。研究对象兔角膜缘干细胞单纯培养组,兔角膜缘干细胞与Swiss3T3成纤维细胞作为饲细胞的混合培养组。方法采用DMEM/F12培养基进行细胞培养,观察4.0μg/ml丝裂霉素C作用不同时间后3T3细胞数量的变化。DispaseⅡ酶消化兔角膜缘上皮细胞,与饲细胞混合接种,观察干细胞克隆形成情况,比较各代、各组的细胞克隆形成率(CFE)。用EDTA去除饲细胞,间接免疫荧光染色检测角膜缘干细胞克隆团表达增殖细胞核抗原(PCNA)情况。主要指标细胞数/毫升,细胞克隆形成率。结果4.0μg/ml丝裂霉素C处理2.5小时的Swiss3T3细胞,培养期间(约12日)细胞数量无明显变化,可作为饲细胞。与饲细胞共同培养的兔角膜缘干细胞形成细胞克隆团,细胞的平均CFE比较:有饲细胞组角膜缘干细胞明显高于无饲细胞组(t=2.982,P<0.01)。兔角膜缘干细胞冻存复苏后平均CFE比较,有饲细胞组明显高于无饲细胞组(t=3.007,P<0.01)。兔角膜缘干细胞克隆团中细胞PCNA染色为阳性。结论角膜缘干细胞与作为饲细胞的Swiss3T3成纤维细胞共同培养形成细胞克隆,其中含有干细胞,且具有高增殖力。Objective To research the preparation of Swiss-3T3 cells with mitomycin C as feeder cells,examine the cloning and proliferation power of rabbit limbal stem cells.Design Comparative experimental study.Participants The cultivated rabbit limbal stem cells and the cultivated rabbit limbal stem cells with Swiss-3T3 cells as feeder cells.Methods Cells were cultured in DMEMF12 medium.Being treated with 4.0μgml mitomycin C at different time,the proliferation power of Swiss-3T3 cells were evaluated with counting cells.Rabbit limbal stem cells were digested by DispaseⅡand cultivated with the feeder cells.The cloning growth of cultivated cells was measured with colony-forming efficiency (CFE) in different groups.After feeder cells were discarded by 0.04% EDTA, the cultivated limbal stem cells were examined with immunofluorescence staining of PCNA.Main Outcome Measures Cell countml,CFE.Results The number of Swiss-3T3 cells treated with 4.0μgml mitomycin C for 2.5 hours had no significant changed during the culture period (about 12 days).Limbal stem cells with feeder cells grew as clones.The mean CFE of limbal stem cells in the group with feeder cells was more than that in the group without feeder cells (t=2.982,P<0.01).Recovering from frozen,the cells with feeder cells had higher proliferating power than cells without feeder cells had (t=3.007,P<0.01).The clones of cultivated limbal stem cells were positively stained by anti-PCNA in nucleolus with green fluorescence.Conclusion Limbal stem cells co-cultivated with feeder cells grow as clones,which had higher proliferating power and hinted stem cells existing.

关 键 词：角膜缘干细胞 饲细胞 体外培养 

分 类 号：R779.65[医药卫生—眼科]