大鼠角膜缘上皮细胞的体外培养以及碱烧伤后β-连环蛋白表达的实验研究

Expression of β-catenin in rat's limbal epithelial cells cultured in vitro after alkali burn injury

目的探讨角膜缘上皮细胞的体外培养以及碱烧伤后β-连环蛋白(β-catenin)的表达规律。方法消化法培养大鼠角膜缘上皮细胞,并对其生物学特征[细胞形态、泛角质蛋白抗体(AE1/AE3)和细胞核增殖抗原(PCNA)]做初步鉴定;构建体外碱烧伤模型,利用实时定量聚合酶链反应和免疫荧光技术检测伤后各时相点β-catenin的表达。结果体外培养的角膜缘上皮细胞具典型的上皮细胞特点,大部分细胞分化程度较低且生活状态良好;核酸和蛋白水平均显示,伤后24小时β-catenin的表达有显著升高(P<0.05),至伤后48小时达到峰值(P<0.01)。结论在一定程度上可以利用角膜缘上皮细胞的研究结果来近似反映角膜干细胞的特点;Wnt经典信号途径参与了碱烧伤后的细胞修复过程。

Objective To investigate the expression of β-catenin on limbal epithelial cells cultured in vitro after alkali burn injury.Methods Limbal epithelial cells in rat were separated and cultured by trypsin digestion,then its biological characterization（such as cellular morphology and expressions of AE1/AE3 and PCNA） was primarily identified.After establishing the model of alkali burn in vitro,real-time quantitive PCR and immunofluorescent technique were used to detect the expression of β-catenin in different postburn time points.Results Limbal epithelial cells in vitro had typical features of other epithelial cells,most of them with lower differentiation and normal animation.Both levels of the nucleic acid and protein showed that the expressions of β-catenin were significantly increased at postburn 24hours（P0.05）,reached the peak at postburn 48hours（P0.01）.Conclusion The feature of limbal epithelial cells is similar to that of the corneal stem cells in some degree.Wnt signal pathway plays a role in the course of cellular repair after alkali burn injury.