【摘要】目的 探讨丝裂霉素C处理Swiss-3T3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Swiss-3T3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Swiss-3T3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。差异具有显著性。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Swiss-3T3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。
   【关键词】角膜缘干细胞；饲细胞；克隆化培养；增殖细胞核抗原；体外培养

【Abstract】 Objectives 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 trial.  Participants  The cultivated rabbit limbal stem cells and the cultivated rabbit limbal stem cells with Swiss-3T3 cells as feeder cells. Methods The cells were cultured in DMEM/F12 medium. Being treated with 4.0μg/ml 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 count/ml, CFE. Results The number of Swiss-3T3 cells treated with 4.0μg/ml 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.
【Key words】 Limbal stem cells; Feeder cells; Clone assay; PCNA; in vitro

   角膜缘干细胞移植是治疗角膜缘损伤导致干细胞功能障碍疾病的新手段，角膜缘干细胞的体外培养是进行细胞移植的基础，通过体外扩增将获得的少量角膜缘上皮细胞数量放大并保持干细胞特性，对于临床治疗角膜缘疾病具有重要意义。研究表明，角膜缘具有特殊的“Vogt”结构，通过RNA印迹杂交等技术证明角膜缘上皮细胞与周围成纤维细胞之间存在细胞因子调控网络。通过该网络，成纤维细胞可以间接地影响角膜缘上皮细胞的增殖和分化[1]。Swiss-3T3成纤维细胞作为饲细胞已成功地应用于包括兔[2]、人[3]等多种上皮细胞培养。本研究以Swiss-3T3成纤维细胞作为饲细胞，观察其对角膜缘干细胞克隆形成的影响，研究其增殖力的生物学特性，为角膜上皮干细胞体外扩增、保存和进一步的细胞移植奠定基础

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