活体染料羧基荧光素乙酰乙酸对兔眼虹膜色素上皮细胞体外染色的研究  

Iris pigment epithelial cells of rabbits stained by carboxyfluorescein diacetate succinimidyl ester in vitro

作　　者：彭绍民[1] 张中宇[2] 黎晓新[1] 丛丽丹[2] 

机构地区：[1]北京大学人民医院眼科中心,100005 [2]哈尔滨医科大学附属二院眼科

出　　处：《中华眼底病杂志》2006年第4期261-264,共4页Chinese Journal of Ocular Fundus Diseases

基　　金：国家自然科学基金资助项目(30271395);黑龙江省海外学人科研资助项目(1053HQ014);黑龙江省自然科学基金资助项目(D0241)

摘　　要：目的探讨活体染料羧基荧光素乙酰乙酸琥珀酰亚胺酯（CFSE）标记兔眼虹膜色素上皮（IPE）细胞的可行性。方法使用酶消化加机械分离的方法分离培养兔眼IPE细胞，将传3～4代的IPE细胞用2．5、5、10、20、40μmol／L浓度的CFSE分别作用1、5、10min进行染色，通过观察染色后的细胞荧光强度和细胞贴壁率筛选出最理想的标记条件。采用流式细胞术追踪检测标记的IPE细胞的荧光强度变化。用免疫荧光抗体标记法检测此染色剂有无渗漏以及是否对周围细胞着染。结果浓度为20μmol／L的CFSE 37℃下水浴作用1min为IPE细胞染色、观测、研究的最适条件。流式细胞仪和荧光显微镜观察结果显示，CFSE对IPE细胞染色可持续4周以上，未发生染料渗漏或转染其它细胞，且此浓度的CFSE不影响细胞的性状。结论活体染料CFSE是一种染色率高，追踪时间长，应用简便安全可靠的标记兔IPE细胞的新方法。Objective To investigate the feasibility of labeling iris pigment epithelial（IPE）cells of rabbits with 5- （and 6-） carboxyfluorescein diacetate succinimidyl ester （CFSE）. Methods Enzymeassisted microdissection was used to isolate the cultured rabbit＇s IPE cells. The third or forth subcultured IPE cells were incubated with 2.5,5,10,20,and 40 μmol/L of CFSE for 1,5,and 10 min respectively. The fluorescence intensity was detected by flow cytometry, and the leakage of CFSE and its dyeing were observed by fluorescence antibody labeling. Results Incubation with 20 μmol/L CFSE under 37℃ forlminute was the most optimal condition for IPE cells labeling. The coloration of IPE cells stained by CFSE lasted 4 weeks. There was no leakage of dye from labeled rabbit IPE cells to non-labeled human IPE cells in mixed culture process. Conclusion With the advantages of high rate of dyeing, long time of tracing,safety and convenience ,CFSE can be used as a new method to label the rabbit＇s IPE cells.

关 键 词：荧光染料 虹膜 色素上皮 眼 染色与标记 

分 类 号：R773.1[医药卫生—眼科] R446.39[医药卫生—临床医学]