荧光激活细胞分离技术在角膜缘干细胞研究中的应用

目的探讨荧光激活细胞分离技术（FACS）在体外培养的兔角膜缘上皮细胞生物学特性研究中的应用。方法收集新西兰白兔的角膜缘组织进行组织块培养。采用FACS分离出体外培养的兔角膜缘上皮细胞中的侧群（SP）细胞和非SP细胞。用2%锥虫蓝染色法对分离出的SP细胞进行克隆形成率（CFE）检测以评估SP细胞的活性,用免疫组织化学染色检测K3/12和ABCG2蛋白在SP细胞和非SP细胞中的表达。结果SP细胞在体外培养的兔角膜缘上皮细胞中占（0.22±0.09）%,分离出的SP细胞的CFE为（5.52±0.45）%,而非SP细胞为（0.78±0.73）%,二者差异有统计学意义（t=2.17,P〈0.01）;大部分SP细胞呈K3/K12抗原阴性,ABCG2免疫标记呈阳性;而非SP细胞呈K3/K12抗原阳性,ABCG2免疫标记呈阴性。结论FACS可以应用于体外培养的兔角膜缘上皮细胞的SP细胞分离,分离出的SP细胞具有较强的增生能力。

Application of fluorescent-activated cell sorting (FACS) technique in the research of rabbit limbal stem cells

Objective The fluorescence-activated cell sorting (FACS) technique is a method for the identification and isolation of different cell populations.At present,the special surface marker for limbal stem cells has been not found yet.This study aimed to investigate the application of FACS technique in the research of rabbit limbal stem cells.MethodsCorneal limbal tissue was obtained from New Zealand white rabbits and cultured using the explant culture method in SHEM.Side population cells (SP cells) and non-SP cells were sorted from cultured rabbit limbal epithelium cells by FACS at a excitation wavelength 350 nm,and acquistion length 450 nm (blue light) and 675 nm (red light).The SP cells and non-SP cells were identified by detecting the expression of ABCG2 and K3/K12.The colony-forming efficiency of SP cells and non-SP cells were evaluated by the observation of cellular vitality with trypan blue staining.The percentage of colony formation was calculated as the colony number in various group/200×100%.ResultsIn 48-72 hours after primary culture,limbal epithelial cells migrated from the cultured tissue mass to form the mambrane-like structure and achieved 70%-80% confluence.The cells showed round,polygon and flattened shape.The proportion of SP cells in cultured limbal epithelial cells was 0.22%±0.09% with a colony-forming efficiency of 5.52±0.45% in SP cells and 0.78%±0.73% in non-SP cells,with a statistically significant difference between the two populations (t＝2.17,P＜0.01).After verapamil,an inhibitor of the expression of the ABCG2 protein,was added into the medium,the proportion of SP cells in the cultured limbal epithelial cells declined to 0.04%±0.006%.The SP cells presented a positive immunoresponse for ABCG2 and absence of immunoresponse for K3/K12,but a contradictory staining result was found in non-SP cells.ConclusionFACS can be applied in the research of limbal stem cells.