脂质体介导转染N2a细胞的优化方法

Optimization of N2a cell transfection mediated by liposome

背景:阳离子脂质体介导的细胞转染具有结果可靠、可重复性强的特点,但面临一个共同的缺点,就是转染效率常较低。目的:探讨阳离子脂质体转染N2a细胞(小鼠神经胶质瘤细胞)的优化方法。方法:采用24孔培养板,1.5μL阳离子脂质体Lipofectamine?LTX介导,通过贴壁法和悬浮法,将500 ng带有绿色荧光蛋白(GFP)基因的重组质粒pcDNA3-GFP转入N2a细胞,倒置荧光显微镜观察细胞内绿色荧光蛋白表达情况,并比较二者转染效率。采用悬浮转染法,将500 ng质粒DNA分别用1.0,1.5,2.0,2.5μL Lipofectamine?LTX进行转染,探讨最适脂质体和DNA比例。结果与结论:1.5μL脂质体/500 ng DNA,悬浮法转染效率显著高于贴壁法转染效率(P<0.01);1.0,1.5,2.0,2.5μL Lipofectamine?LTX对500 ng质粒DNA的转染效率分别为(76.60±3.85)%,(80.00±4.17)%,(88.00±5.89)%,(54.96±4.23)%,提示脂质体2.0μL与质粒DNA 500 ng的转染效率最高。

BACKGROUND： Cationic iiposome-mediated cell transfection is reliable and repeatable. However the transfection efficiency is often low. OBJECTIVE： To study the optimized methods for gene transfection mediated by iiposome into N2a cells （mouse neuroblastma cells）. METHODS： Using traditional adherent method and improved Suspension method, 500 ng recombinant plasmid pcDNA3-GFP carrying green fluorescence protein was transfected into N2a cells in 24-well culture plate, which was mediated by 1.5 μL LipofectamineTM LTX Reagent. The expression of green fluorescent protein was observed by inverted fluorescence microscope, and the transfection efficiencies at different transfection ways were calculated. By using improved suspension transfection method, 500 ng plasmid DNA was transfected with different doses of LipofectamineTM LTX Reagent （1.0, 1.5, 2.0, 2.5 μL）. The optimal ratio of liposome and DNA was explored. RESULTS AND CONCLUSION： The transfection efficiency of suspension transfection method was significantly higher than that of the tranditional adherent method （P 〈 0.01） when using 1.5μL liposome/500 ng DNA. The transfection efficiency of the 1.0, 1.5, 2.0, 2.5 μL LipofectamineTM LTX on 500 ng plasmid DNA was respectively （76.60±3.85）%, （80.00±4.17）%, （88.00±5.89）%, （54.96±4.23）%. It showed the 500 ng DNA and 2.0 μL liposome achieve the highest transfection efficiency.