鼠胚胎干细胞定向诱导为角膜上皮细胞的实验研究

目的建立能够稳定表达绿色荧光蛋白（green fluorescent protein,GFP）的鼠胚胎干细胞系,并研究体外诱导鼠胚胎干细胞（embryonic stem cell,Es细胞）向角膜上皮细胞分化的可能性及条件,为角膜损伤提供新型的上皮修复组织来源。方法质粒pEGFP-N1脂质体复合体转染鼠胚胎干细胞,对稳定表达GFP的ES细胞以Ⅳ型胶原作为诱导条件,定向诱导鼠ES细胞向角膜上皮细胞转化,观察培养后形态改变,免疫荧光及RT-PCR检测K12、K14及CD44的表达。结果转染后ES细胞稳定表达GFP,成功诱导出角膜上皮样细胞,呈典型上皮样细胞形态,免疫组化及RT-PCR检测显示,K12及CD44表达阳性,K14表达阴性。结论转染GFP的ES细胞,在Ⅳ型胶原的诱导下,成功向角膜上皮细胞分化。

Experimental study on differentiation of mouse embryonic stem cells into corneal epithelial cells

Objective To establish mouse embryonic stem cell lines which can stably express green fluorescent protein（green fluorescent protein,GFP）,and to study the possibility and conditions of differentiation of mouse embryonic stem（ES） cells into corneal epithelial cells in vitro,in order to serve new source of tissues for epithelial repair.Methods Plasmid pEGFP-N1 liposome complexes was used to transfect mouse ES cells and those cells stably expressing GFP were induced into corneal epithelial cells with type Ⅳ collagen as induction conditions.The resulting corneal epithelial cells were cultured and the morphological changes were observed.Immunofluorescence,RT-PCR were used to detect K12,K14 and CD44 expression.Results After transfection,the ES cells stably expressed GFP,and successfully induced to corneal epithelial cells with typical epithelioid cell morphology.Immunohistochemical and RT-PCR detection showed that the expression of K12 and CD44 were positive and the expression of K14 was negative.Conclusion In the induction of type Ⅳ collagen,ES cells transfect with GFP successfully differentiate to corneal epithelial cells.