体外诱导人羊膜上皮细胞分化为角膜上皮样细胞的初步研究

Preliminary study of induced differentiation of human amniotic epithelial cells into corneal epithelial-like cells in vitro

目的探索以人永生化角膜上皮细胞( immortalized human corneal epithelial cells,ihCEC) 培养液体外模拟角膜上皮细胞微环境,诱导人羊膜上皮细胞( human corneal epithe-lial cells,hAEC) 分化为角膜上皮样细胞的可行性。方法取( 37 ± 1) 周剖宫产人羊膜组织,酶消化法提取 hAEC; 流式细胞仪测 CD29、CD90、CD105、CD34、HLA-DR 的表达。复苏培养 ihCEC,以 0 mg·L- 1、10 mg·L- 1、20 mg·L- 1、30 mg·L- 1、40 mg·L- 1丝裂霉素 37℃作用 2 h,吸去丝裂霉素,继续培养 72 h 后 CCK8 测吸光度并计算增殖抑制率。10 mg·L- 1、20 mg·L- 1丝裂霉素处理细胞后 12 h、24 h 收集细胞培养液培养 hAEC,CCK8 测吸光度绘制生长曲线; 收集 ihCEC 细胞培养液,制备条件培养基( CM) 培养 hAEC 10 d,倒置显微镜观察细胞形态,免疫荧光检测 CK12 的表达。结果 hAEC 可表达 CD29、CD90、D105,不表达 CD34、HLA-DR; 各浓度丝裂霉素组增殖抑制率分别为 10 mg·L- 1( 65. 48% ±1. 03) 、20 mg · L- 1( 77. 01% ± 0. 99) 、30 mg · L- 1( 75. 25% ± 0. 71) 、40 mg · L- 1( 76. 90% ±0. 97) ;20 mg·L- 1丝裂霉素培养 12 h 收集的细胞培养液对 hAEC 具有明显促增殖作用; 诱导分化后 hAEC 可表达 CK12。结论以 ihCEC 细胞培养液模拟的角膜上皮细胞微环境可诱导 hAEC 分化为角膜上皮样细胞。

Objective To explore the feasibility that induced differentiation of human amniotic epithelial cells（hAEC） into corneal epithelial-like cells by collecting culture media from immortalized human corneal epithelial cells （ihCEC）. Methods The human amniotic tissue was delivered from （37±1） weeks caesarean,and the hAEC were obtained from enzyme digestion method.The flow cytometry was used to detect the expression of CD29/90/105/34/HLA-DR.ihCEC was cultured and mitotically inactivated by adding 0 mg·L-1,10 mg·L-1,20 mg·L-1,30 mg·L-1,40 mg·L-1 mitomycin to the culture medium,and incubated these cells for 2 hours at 37°C,then removed mitomycin,cultured ihCEC for 72 hours,measured absorbance to calculate the proliferation inhibition rate by cell count kit-8（CCK8）;10 mg·L-1,20 mg·L-1 mitomycin was added to inactivate ihCEC,collected culture media after culturing for 12 hours or 24 hours,saved it at 4 ℃,then incubated hAEC,measured absorbance by CCK8 and drew the growth curve.After cultured hAEC for 10 days by conditioned media,the inverted phase contrast microscope was used to observe the change of morphology and the expression of CK12 were detected by immunofluorescence. Results hAEC expressed surface markers including CD29/90/105,the expression of CD34/HLA-DR were negative.The proliferative inhibition rates of 10 mg·L-1,20 mg·L-1,30 mg·L-1,40 mg·L-1 mitomycin were （65.48±1.03）%,（77.01±0.99）%,（75.25±0.71）% and （76.90±0.97）%.The collected culture media after 20 mg·L-1 mitomycin culturing for 12 hours could significantly promote the proliferation of hAEC.After cultured for 10 days by CM,the expression of CK12 was positive. Conclusion hAEC can differentiate into corneal epithelial-like cells by in vitro replication of the corneal epithelial microenvironment,using the culture media collected from ihCEC.