PC12细胞中增强型绿色荧光蛋白基因的表达

Expression of enhanced green fluorescent protein gene in PC12 cells

目的:研究PC12细胞中增强型绿色荧光蛋白(GFP)基因的表达。方法:用脂质体转染法将增强型GFP基因转入PC12细胞,在荧光显微镜下观察转染细胞GFP表达及荧光强度。用碘化丙啶(PI)标记缺氧血清剥夺再灌流后的PC12/GFP细胞,在流式细胞仪上分选GFP阳性(活)细胞并测定其比例。结果:克隆转移后几乎所有细胞表达GFP,可连续稳定传40代以上。流式细胞仪可精确分选出GFP(+)/PI(-)、GFP(+)/PI(+)、GFP(-)/PI(-)、GFP(-)/PI(+)4组细胞。结论:增强型GFP基因可在PC12细胞中稳定高表达,PC12/GFP细胞株的建立为短暂脑缺血再灌流后干预治疗的研究提供了理想的对照实验细胞模型。

Aim: To study expression of enhanced GFP gene in PC12 cells and its application. Methods: PC12 cells were transfected by enhanced GFP gene in liposome-mediated gene transfer, GFP-expression and fluorescent intensity were detected under fluorescent microscopy. The PC12/GFP cells after anoxia-serium deprivation/reperfusion were stained by PI and we tried to sort the GFP positive survival cells and determine the percentage of them. Results: Almost all of the cells expressed high-level GFP after being selected by G418, and there was no significant difference between fluorescent intensity of the 5th generation PC12/GFP cells and that of the 40th. Four kinds of cells including GFP( + ) / PI( - ) ,GFP( + ) / PI( + ) ,GFP( - ) / PI( - ) ,GFP( - ) / PI( + ) were accurately sorted by flow cytometry. Conclusions: The PC12/ GFP cell line is a stable, high-level GFP-expression cell line, and it can be served as an ideal control group for the study of neuroprotection after anoxia-serium deprivation/reperfusion.