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目的观察人角膜上皮细胞(corneal limbal epithelial cells,CLEC)的生物学特性,探讨CLEC在普通培养皿和聚乙二醇水凝胶膜(poly(ethylene glycol)-based hydrogel films,PHF)上的增殖率差异。设计实验研究。研究对象人角膜上皮细胞。方法采用组织块培养法体外扩增CLEC,待细胞生长至指数生长期,分别种植于普通培养皿和PHF上,倒置显微镜下观察培养细胞的生长特点,利用免疫荧光对其鉴定,利用CCK-8比色法观察两组细胞相对增殖率(relative growth rate,RGR)的差异。主要指标光镜下CLEC的生长,AE1染色,CCK-8比色法的吸光度(optical density,OD)值。结果光镜下可见CLEC在普通培养皿和PHF上均能移行扩增、融合成片,在PHF上的生长速度与普通培养皿组相近。两组细胞AE1荧光染色均呈阳性,PHF组阳性率(73.26%±8.84%)与普通培养皿组(70.84%±3.51%)基本相同。PHF组在12 h(0.89±0.06)、24 h(1.13±0.10)、48 h(1.24±0.03)的OD值与普通培养皿组(0.89±0.03、1.08±0.04、1.28±0.09)差异无统计学意义(P=0.79,0.36,0.76)。PHF组细胞在12 h(99.12%±4.81%)、24 h(103.74%±5.55%)、48 h(97.83%±13.37%)的RGR均大于75%,各时间点间差异无统计学意义(P=0.37,0.90)。结论PHF可以作为体外扩增角膜上皮细胞的良好载体。
Objective To observe the biological characteristics of human corneal limbal epithelial cells (CLECs) and to investigate the proliferation of CLECs on common petri dishes and poly (ethylene glycol) -based hydrogel films (PHFs) Rate difference. Design experiment research. Study target human corneal epithelial cells. Methods CLECs were expanded in vitro by tissue culture method. The cells were grown in exponential growth phase. The cells were seeded on ordinary Petri dishes and PHF. The growth of cultured cells was observed under inverted microscope. The cells were identified by immunofluorescence and identified by CCK-8 Colorimetric method was used to observe the difference of relative growth rate (RGR) between the two groups. The main indexes were the growth of CLEC under light microscope, AE1 staining and the optical density (OD) value of CCK-8 colorimetric method. Results Under light microscope, CLEC could migrate and proliferate on ordinary petri dishes and PHFs, and then fuse into tablets. The growth rate on PHF was similar to that of ordinary petri dishes. AE1 fluorescence staining of both groups of cells was positive, the positive rate of PHF group (73.26% ± 8.84%) and ordinary culture dish group (70.84% ± 3.51%) were basically the same. There was no statistical difference between the ODF value of PHF group at 12 h (0.89 ± 0.06), 24 h (1.13 ± 0.10), 48 h (1.24 ± 0.03) and normal culture dish group (0.89 ± 0.03,1.08 ± 0.04,1.28 ± 0.09) Significance (P = 0.79,0.36,0.76). The RGR of PHF group was more than 75% at 12 h (99.12% ± 4.81%), 24 h (103.74% ± 5.55%) and 48 h (97.83% ± 13.37%) respectively. There was no significant difference between the time points P = 0.37, 0.90). Conclusion PHF can be used as a good carrier of corneal epithelial cells in vitro.