人结膜上皮细胞的培养鉴定及液氮冻存  

The Culture and Cryopreservation of Human Conjunctival Epithelium in Vitro

作　　者：郑健樑[1] 卢蓉[1] 张洁[1] 林明楷[1] 林建贤[1] 

机构地区：[1]中山医科大学中山眼科中心,广州510060

出　　处：《眼科学报》2000年第2期135-138,共4页Eye Science

摘　　要：目的:探索人眼结膜上皮细胞体外培养和细胞保存的最佳方法,建立人结膜上皮细胞系,进一步研究人结膜的病理、生理特点及为毒理试验提供可靠的细胞模型。方法:分别用组织块培养法、机械分离法及混合消化液培养法体外培养正常成年人结膜上皮细胞,通过观察细胞形态、生长特性并用原位免疫组化方法鉴定培养细胞;收集第3代和第4代融合的细胞液氮冻存,保存30天后复苏,观察复苏成功率。结果:3种取材方法中,混合消化液培养法细胞生长繁殖较快,培养细胞形态多样,贴壁生长。免疫组化Keratin染色阳性,细胞冻存复苏的成功率均达到90％。组织块培养法细胞生长比较缓慢,而机械分离法细胞未见贴壁生长。结论:混合消化液培养法是人结膜上皮细胞培养的最佳方法,通过液氮冻存可保存培养的人结膜上皮细胞。眼科学报2000;16:135-138。Purposes: To detect the best method of culture and preservation of human conjunctival epithelia. Methods: Human conjunctival epithelia were cultured by tissue inoculation, mechanical separation or enzyme digestion with 0. 25% trypsin. The cultured cells were identified through their morphology, growing features and immunohistochemical staining. The third and fourth passage confluent cells were frozen in liquid nitrogen, and resuscitated 30 days later. Results: Cells from tissue digested with 0.25% Trypsin grew much better than those from tissue inoculation. No cell outgrew in mechanical separating group. The cultured cells spread along the flask in polygonal shape and positive in pan-Keratin staining. Ninty percent of the cryopreserved cells were successfully resuscitated . Conclusion: Tissue digestion with trypsin is the best approach to culture human conjunctival epithelium, and the cultured cells can be cryopreserved in liquid nitrogen. Eye Science 2000; 16: 135 - 138,

关 键 词：结膜上皮细胞 免疫组化 冻存复苏 

分 类 号：R77[医药卫生—眼科]