摘要 目的 用增强绿色荧光蛋白基因(EGFP)的表达载体pIRES2EGFP转染神经干细胞(neural stem cells，NSCs)，检测该真核表达载体对NSCs的转染效率及EGFP的表达状态，为用NSCs为载体基因治疗中枢神经系统疾病提供实验依据。 方法 体外扩增、酶切鉴定pIRES2-EGFP质粒；悬浮培养NSCs；阳离子脂质体lipofectamineTM2000介导增强绿色荧光蛋白质粒转染NSCs；庆大霉素的氨基糖苷 (G418) 筛选重组子；荧光显微镜观察转染效率及表达情况；免疫细胞化学鉴定重组子。 结果 转染后6 h，荧光蛋白偶见表达，24 h表达量明显增加，48 h达到最高峰，1个月后抗性细胞有克隆球形成。 结论 脂质体介导报告基因—pIRES2-EGFP转染NSCs的方法简单、效率高、易成功，是一较为理想的基因转染方法。
Abstract Objective To transfect neural stem cells (NSCs) with enhanced green fluorescent protein expression vector pIRES2-EGFP, to detect transfection efficiency and expression of EGFP and to obtain experimental data concerning NSCs used as a delivery vehicle for gene therapy in gene diseases of central nervous system. Methods LipofectamineTM 2000 liposome mediated pIRES2-EGFP plasmid transfected NSCs. Transfected NSCs were screened with G418. Fluorescence microscope observed transfection efficiency and expression of EGFP. And the resistant NSCs were evaluated by immunocytochemical method. Results Fluorescence proteins were expressed by chance 6 h after transfection . And their expression increased 24 h after transfection with a summit at 48 h. After one month, resistant cells developed great neurospheres. Conclusion Lipofectamine TM2000 liposomes mediated pIRES2-EGFP plasmids transfecting NSCs is simple and efficient, and is an ideal method for gene transfection. |
