绿色荧光蛋白基因转染小鼠胚胎干细胞及向神经细胞的分化

Transfection of embryonic stem cells with green fluorescent protein gene and their differentiation into neural cells

目的建立能够稳定表达绿色荧光蛋白（GFP）的小鼠胚胎干细胞（ESC），并诱导其向神经细胞分化，为临床移植治疗神经系统疾病提供方便追踪观察的神经细胞。方法分别用脂质体、电穿孔转染法转染小鼠胚胎干细胞系R1，检测48h转染效率；经G418筛选后，得到稳定高效表达GFP的ESC克隆，通过碱性磷酸酶（AKP）染色检测ESC的生物学特性；利用单层分化法诱导转染GFP的胚胎干细胞向神经细胞分化，通过免疫荧光检测神经细胞特异标记Tuj1。结果转染48h的GFP阳性细胞转染率脂质体组为65％，电穿孔组为79％，两组比较差异无统计学意义（Х^2=3.14，P〉0．05）。转染GFP的胚胎干细胞AKP染色阳性，并可诱导分化为Tuj1阳性的神经细胞。结论建立稳定表达GFP基因的胚胎干细胞克隆，获得的转染GFP的胚胎干细胞能够分化为神经细胞。

Objective To establish embryonic stem cells （ESC） that can express green fluorescent protein （GFP） and differentiate them into neurons. It would provide tagging neurons for clinical transplantation to cure neural system diseases. Methods ESC （R1） was transfected with a plasmid containing the GFP by electroporation. A transgenic cell line was obtained after selection with G418. The ESCs were characterized by AKP staining. Monolayer differentiation method was used to induce neural differentiation derived from GFP-ESC and immunofluorescence method was used to identify Tuj 1 positive cells. Results There was no significant difference （Х^2 = 3.14,P 〉 0.05 ） in transfect rates between liposome and electroporation （65% vs 79% ）. The AKP staining of GFP-ESC was positive. GFP-ESC could be differentiated into neural cells. Conclusions These results show that ESC expressing GFP has been established, which can be differentiated into neurons.