悬浮细胞电转染条件的优化及影响因素

Optimization and influencing factor of electroporafion parameters for cells cultured in suspension

目的以绿色荧光蛋白（GFP）为报告基因，优化悬浮培养的白血病细胞的电穿孔转染条件。方法通过控制电压（200～400V）、电容（450～1200μF）、细胞状态及缓冲液血清浓度（0％、10％、15％）等转染条件，采用不同条件组合后用电穿孔法将质粒转入悬浮培养的人白血病细胞株K562，通过流式细胞仪和荧光显微镜分析转染率。结果K562细胞在310V、1050μF条件下转染率最高，pEGFP-C2／K562为67．04％，pEGFP-C2／BRD7／K562为59．29％。对数生长期细胞电转染率高于生长过老期细胞；而缓冲液中的血清浓度与电转染率无关。结论电穿孔是一种高效的基因转染法，通过优化转染条件、控制影响因素，可提高转染率。

Objective To optimize the electroporation parameters in leukemia cell lines cultured in suspension using green fluorescent protein （GFP） as a reporter gene. Methods The GFP plasmid was transferred into leukemia cell lines K562 by electroporation using differently experimental conditions such as the voltage （200-400 V） ,the electric capacity （450-1200 μF） ,the state of cell vitality and the serum concentration with buffer solution （0,10%, 15% ）. The electroporation efficiency was evaluated by flow cytometry and fluorescent microscopy. Results The highest electropration efficiency with leukemia cell lines k562 cultured in suspension was obtained under the condition of voltage 310 V, electric capacity of 1050 μF （for pEGFP-C2/K562,67.04% ; for pEGFP-C2/BRD7/K562,59.29% ）. The electroporation efficiency for the cells in logarithmic growth phase was increased highly. The serum concentration in buffer solution was not related with the electropration efficiency. Conclusion Electroporation is a method with high efficiency to gene transfection, and optimizing electroporation parameters and controlling the related factors can increase the electroporation efficiency.