Objective Enhanced green fluorescent protein was used as report gene to study the efficiency of gene transfection of Lipofectamine2000, CS ( N/P = 20 ) and PAMAM ( N/P = 20 ) , so as to lay the foundation of gene therapy for neurological diseases. Methods Complex of Iipofectamine2000, CS and PAMAM with plasmid DNA (pDNA) was prepared, and EGFP was transfected into Hek293 cells and SH-SY5Y cells cultured in vitro, MIT test was used to compare the toxicity of the three vectors in the cells, then the cell morphology was observed under confocal microscopy. EGFP expression intensity was observed by fluorescence microscope and transfection efficiency was evaluated by flow cytometry. Results ① The survival rate of Hek293 cells transfected by Lipofectamine2000, CS and PAMAM was 73. 09% , 73. 69% and 69.24% respectively, which showed no markedly differential in statistics; in SH-SY5Ycells, the survival rate respectively was 67.6% , 61.9% and 42.9% , the former two were higher than the third one( both P <0.05). ② The cell morphology of Hek293 cells transfected by Lipofectamine2000/pDNA complex and CS/pDNA complex showed no markedly change, and maintain difficile growth, however, the one transfected by PAMAM/pDNA complex shrinked and showed circular distribution growth. The cell morphology of SH-SY5Y cells transfected by three vectors all changed, but the cells transfected by PAMAM/pDNA complex had more morphological changes. ③The EGFP expression intensity, regardless of Hek293 cell or SH-SY5Y cells, which transfected by Lipofectamine2000/pDNA complex was the strongest, and followed was the one transfected by CS/pDNA complex and PAMAM/pDNA complex. ④The transfection efficiency of Lipo-fectamine2000, CS and PAMAM respectively was 60.9% , 45.2% and 42.7% in Hek293 cells, the former one was higher than the last two; in the SH-SY5Ycells, which respectively was 25. 3% , 19. 2% and 8. 9% , the former two was higher than the third one(both P <0.05). Conclusions At present, Lipofectamine2000 is the most suitable transfection medium for gene therapy for neurological diseases; the modified new type nanopaticle-CS has lower toxicity and highest transgene activity than PAMAM, hope to be used further in the study on neurological diseases.%目的 以增强型绿色荧光蛋白(EGFP)为报告基因,研究Lipofectamine2000、改良纳米材料CS(N/P=10)和纳米粒子PAMAM( N/P=20)介导的基因导入系统对神经母瘤细胞SH-SY5Y的毒性及基因转染效率,为神经系统疾病的基因治疗奠定基础.方法 制备Lipofectamine2000、CS和PAMAM与质粒DNA (pDNA)的复合物,分别转染在体外培养的人胚肾细胞Hek293和SH-SY5Y,利用噻唑蓝(MTT)比色实验比较三种载体的细胞毒性作用、用激光共聚焦显微镜观察转染后的细胞形态、倒置荧光显微镜检测转染细胞EGFP表达强度、流式细胞仪检测转染效率.结果 ①Lipofectamine2000、CS和PAMAM转染细胞存活率在Hek293细胞分别为73.09％、73.69％、69.24％,三者比较无显著差异；在SH-SY5Y细胞分别为67.69％、61.71％、42.92％,前两者均显著高于后者(P均＜0.05).②Hek293细胞经Lipofectamine2000和CS与pDNA的复合物分别转染后,细胞形态均未发生明显改变,保持梭状生长；而PAMAM/pDNA复合物转染后,细胞发生皱缩,呈圆形分布生长.经三种载体转染后的SH-SY5Y细胞均发生形态改变,其中PAMAM/pDNA复合物转染者形态改变程度相对较大.Hek293细胞和SH-SY5Y细胞中,均以Lipofectamine2000/pDNA复合物转染者EGFP表达强度最大,其次为CS/pDNA复合物、PAMAM/pDNA复合物转染者.④Lipofectamine2000、CS和PAMAM的转染效率在Hek293细胞分别为60.9％、45.2％、42.7％,前者显著高于后两者(P均＜0.05)；在SH-SY5Y细胞分别为25.3％、19.2％、8.9％,前两者均显著高于后者(P均＜0.05).结论 Lipofectamine2000为目前最适于神经系统疾病基因治疗研究的非病毒载体；改良后的新纳米材料CS毒性比PAMAM小、转染效率比PAMAM高,有望在神经系统疾病研究中得到进一步运用.
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