非病毒载体负载增强型绿色荧光蛋白转染永生化神经前体细胞论文.docVIP

　　非病毒载体负载增强型绿色荧光蛋白转染永生化神经前体细胞论文 桂伶俐，刘志恒，张传汉，高峰 【关键词】 绿色荧光蛋白质类；,基因表达；,转染；,神经前体细胞 Transfection of enhanced green fluorescent protein into immortalized neural progenitor cells by nonviral vectors 【Abstract】 AIM: To investigate an effective transfection method of nonviral vectors and the expression of enhanced green fluorescent protein (EGFP) in the transfected immortalized neural progenitor cells (INPC). METHODS: The plasmid EGFPC1 ine 2000, TRANSfection and Sofast. The expression of EGFP and the transfection efficiency of the 3 vectors easured at 24 h after transfection. The transfection efficiency of the vector ost efficient, ine the expression peak of EGFP. The viability of transfected and nontransfected cells easured by trypan blue rejection test. After the plasmid EGFPC1 olecular marker of neural progenitor cells, nestin, munocytochemistry. After INPC/EGFP L/L fetal bovine serum, the cell morphology and the expression of EGFP ine 2000,.freeline 2000 ost efficient. Its transfection efficiency at 12, 24, 48 and 72 h after transfection as and processes. CONCLUSION: Extrinsic genes can be effectively transfected into INPC by Lipofectamine 2000. And EGFP is one of the valuable tracking tools for INPC. 【Keyine 2000，TRANSfection或阳离子聚合物Sofast分别负载质粒EGFPC1转染INPC，利用荧光显微镜检测转染后24 h的转染效率. 对转染效率最高的一种载体，观察其转染后12.freelL/L胎牛血清诱导INPC/EGFP分化，观察分化后细胞的形态及EGFP表达. 结果: Lipofectamine 2000，TRANSfection和Sofast转染INPC后24 h，转染效率分别为(25.5±2.9)%，(4.0±1.7)%，(7.9±1.4)%. Lipofectamine 2000的转染效率最高. 其转染后12，24，48和72 h的转染效率分别为(17.1±0.7)%，(25.5±2.9)%，(19.4±0.9)%，(15.6±1.4)%，EGFP在转染后24 h表达最高. INPC/EGFP中，EGFP阳性率约为95%. INPC/EGFP巢蛋白表达阳性，其分化后呈神经元或星形胶质细胞样，且胞体及突起中仍可见绿色荧光. 结论: Lipofectamine 2000可高效转染INPC. EGFP是INPC理想的示踪方法之一. 【关键词】 绿色荧光蛋白质类；基因表达；转染；神经前体细胞 0 引言 永生化神经前体细胞（immortalized neural progenitor cell, INPC）具有自我更新、无限增殖及多向分化的潜能，且较原代神经前体细胞易行基因操作，成为神经系统损伤细胞替代及基因治疗的理想靶细胞［1］. 而选择高效的外源基因导入系统对基因治疗的效果至关重要. 尽管病毒介导的基因转染具有更佳的转染效率［2］，但由于其安全性和技术方面的原因,探讨高效率的非病毒载体基因转染方法更具意义. 本实验拟以增强型绿色荧光蛋白(enhanced green fluorescent