| 摘要: |
| 【目的】对小鼠胚胎干细胞（mESCs）转染条件进行优化。 【方法】分别将0.5×105,1×105,2×105,4×105个细胞接种24孔板（各细胞量均接种2孔），培养48 h在其细胞融合度分别为30%，50%，70%，90%左右时，分别设置相同的（0.8 μg DNA/2 μL脂质体）和不同的DNA/脂质体用量（DNA量（μg）/Lipo量（μL）分别为0.4/1，0.8/2，1.6/4，3.2/8），采用脂质体（LipofectamineTM 2000）转染法，将pEGFP N1报告基因载体转染到mESCs中，48 h后通过荧光观察和流式分析检测各组EGFP荧光强度，比较转染效率。 【结果】在质粒及脂质体用量相同（0.8 μg DNA/2.0 μL 脂质体）的条件下，随着细胞融合度的增加，EGFP荧光强度逐渐减弱，在细胞融合度为30%，50%，70%，90%左右时转染，各组EGFP阳性细胞率分别为56.4%，51.8%，40.8%和23.3%。提高转染质粒及脂质体量可在一定程度上提高转染效率，但脂质体的细胞毒性也随之增加，不利于细胞生长。 【结论】低融合度（30%～50%）条件下，使用0.8 μg DNA/2.0 μL 脂质体对mESCs进行转染，可获得较高的转染效率（＞50%），同时可维持良好的细胞状态。 |
| 关键词: 小鼠胚胎干细胞 细胞融合度 脂质体法 pEGFP-N1 转染效率 |
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| 基金项目:国家自然科学基金项目(30570937)；国家高技术研究发展计划项目(2006AA02A102)；中南大学研究生创新基金项目(2340-74334000003) |
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| Optimization of the transfection efficiency of mouse embryonic stem cells |
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| Abstract: |
| 【Objective】The study was done to optimize the transfection efficiency of mouse embryonic stem cells (mESCs).【Method】mESCs were seeded into 24 well plates at a density of 0.5×105,1×105,2×105,4×105 per well (each for two wells).48 h later,cells were transfected when cell confluence was 30%,50%,70%,90% respectively.The reporter plasmid pEGFP-N1 was transfected into cells using LipofectamineTM 2000 by using the same amount (0.8 μg DNA/2 μL liposome) or different amounts of DNA and liposome (DNA(μg)/liposome (μL):0.4/1,0.8/2,1.6/4,3.2/8) respectively.48 hr later,the fluorescence intensities in each group were observed and the transfection efficiencies were compared.【Result】The result of fluorescence microscopy and FACS analysis showed that the percentage of EGFP positive cells gradually decreased (56.4%,51.8%,40.8% and 23.3% respectively) as the cell confluence increased (30%,50%,70% and 90% respectively) when using the same amount of DNA and liposome ((0.8 μg DNA/2.0 μL liposome/well of 24 well plate).When using more DNA and liposome,transfection efficiency further increased.However,obvious cytotoxicity could be observed.【Conclusion】 High transfection efficiency (＞50%) on mESCs could be achieved by using 0.8 μg DNA/2.0 μL liposome/well of 24 well plate under condition of low cell confluences (30%-50%). |
| Key words: mouse embryonic stem cells (mESCs) cell confluence Liposome mediated pEGFP-N1 transfection efficiency |
