目的:比较小鼠角膜上皮细胞消化培养法和组织块培养法。方法:分别使用消化培养法和组织块培养法培养小鼠角膜上皮细胞。比较两种方法中小鼠角膜上皮细胞的克隆形成率(CFE)和群体倍增(PD)。通过Western blotting方法检测p63、角蛋白19以及角蛋白12的表达。结果:其中80%组织块培养法的原代培养可成功传代,而仅12%的消化培养法原代培养可成功传代;两者比较有显著性差异(P<0.05)。传代培养中,组织块培养法中55%的第一代(P1)细胞可以传代超过P10并继续稳定传代至少可传至P25。而消化培养法传代至P2即不能融合。在P1,组织块培养法细胞的CFE高于消化培养法(P=0·02);而组织块培养法P20细胞的CFE又显著高于其P1细胞(P=0.001)。免疫荧光染色显示消化培养法的P1细胞和组织块培养法的P1,P20细胞均表达p63和K19。K12仅在消化培养法的P1细胞和组织块培养法的P1中表达,而组织块培养法的P20细胞中,K12阴性表达。结论:小鼠角膜上皮细胞的培养,组织块培养法优于消化培养法。

AIM:To compare cell-suspension and explant culture of mouse corneal epithelial cells(MCEC).METHODS:MCEC were cultured by cell-suspension culture and explant culture,respectively.Colony forming efficiency(CFE) and cell proliferation were determined.The expression of corneal epithelial progenitor cell marker p63 and K19,as well as differentiation marker K12 was investigated by Western blotting.RESULTS:Twenty of 25(80%) cornea explant were successfully subcultured to passage1(P1),while only 12% cell-suspension culture were successfully subcultured to P1.There were statistical significance between explant culture and cell-suspension culture(P<0.01).Up to 55% of P1 cells in explant culture were passaged over P10 and were stably subcultured though at least 25 passages.However,cells cultured in suspension culture never achieved confluence in P2.CEF of P1 in explant culture was higher than P1 in cell-suspension culture(P=0.02) and CEF of P20 in explant culture was higher than P1 in explant(P=0.001).Immunostaining images showed expression of p63 and K19 in cell-suspension culture P1 and explant culture P1 and P20.K12 was expressed in P1 of both cell-suspension culture and explant culture,however,there was not K12 expressed in P20 of explant culture.CONCLUSION:In MECE culture,compared with cell-suspension culture,the explant culture is a preferable option.

R329.2

中国辽宁省自然科学基金资助项目 (No.20042081)