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目的:观察层粘连蛋白和纤维连接蛋白对原代和传代培养的人晶状体上皮细胞(hLECs)形态学和波状蛋白表达的影响,探讨更适合的hLECs体外培养条件。方法:以层粘连蛋白(Ln)和纤维连接蛋白(Fn)处理培养皿表面,以倒置显微镜观察原代和传代培养的hLECs生长的时间和形态,MTT法记录传代后第3代细胞的生长曲线,免疫荧光法显示第3代细胞的波形蛋白表达形态的变化。结果:在层粘连蛋白和纤维连接蛋白处理组,原代hLECs长出的时间明显早于对照组;两处理组的原代细胞可以正常传代,对照组细胞传代后不能存活。传代晚期层粘连蛋白处理组细胞发生凋亡,而纤维连接蛋白处理组出现多数晶状体小结。MTT实验显示,传代后4～7d纤维连接蛋白处理组细胞数多于层粘连蛋白处理组。免疫荧光反应中,两组波状蛋白形态有明显差别。结论:层粘连蛋白可以提供适宜hLECs保持上皮细胞状态的体外培养微环境,而纤维连接蛋白可以促进hLECs的增殖和分化。
OBJECTIVE: To observe the effects of laminin and fibronectin on the morphology and wavy protein expression of primary and subcultured human lens epithelial cells (hLECs) and to explore the more suitable hLECs culture conditions in vitro. Methods: The surface of culture dishes was treated with laminin (Ln) and fibronectin (Fn). The growth time and morphology of primary and subcultured hLECs were observed with inverted microscope. The growth curve of the third passage cells was recorded by MTT assay Immunofluorescence showed the changes of vimentin expression patterns in the third generation cells. Results: In the laminin and fibronectin groups, the primary hLECs grew significantly earlier than the control group. The primary cells of the two treatment groups could be passaged normally, while the control group could not survive after passage. Apoptosis occurred in the late passage laminin group, while most of the lens tubules appeared in the fibronectin group. MTT experiments showed that the number of fibronectin-treated cells in 4 ~ 7 days after passage was more than that in laminin-treated group. Immunofluorescence reaction, the two groups of wavy protein morphology were significantly different. CONCLUSION: Laminin can provide an in vitro culture microenvironment suitable for hLECs to maintain the epithelial state, whereas fibronectin promotes the proliferation and differentiation of hLECs.