电穿孔介导外源基因转染兔成体成纤维细胞的初步研究

目的采用电穿孔方法，介导质粒载体pEGFP-cl转入兔成体成纤维细胞，探讨建立电穿孔介导外源基因转染兔成体成纤维细胞的基本条件。方法电穿孔介导质粒载体pEGFP—cl转入7．10代兔耳成体成纤维细胞系GG，研究不同电压、电容、DNA剂量，孵育温度的条件下，观察细胞存活数，流式细胞仪测试GFP的含量。结果以PBS作为电击基础液、细胞浓度2×10^6个／ml，电压250V、电容700μF，DNA用量30μg时，兔耳成纤维细胞的转染率和存活率最佳。且电击前冰浴10min，电转染后37℃孵育10min有利于提高细胞的转染率和存活率。结论在合适电压、电容、DNA剂量条件下，电转染提供了外源基因转染兔耳成体成纤维细胞适用性；电转染前的一定时间冰浴和电转染后一定温度孵育可使电转染效率得到改善。

Preliminary Study on Conditions of Transfection of Rabbit Adult Fibroblasts by Electroporation

Objective To investigate and explore conditions oftansfection on the rabbit adult fibroblasts by means of electroporation, the plasmid vector of pEGFP-c 1 was successfully transfected into the rabbit adult fibroblasts by electroporation. Methods The rabbit fibroblasts at 7-10 passages were transferred with pEGFP-cl by means ofelectroporation with different electric voltages, capacitors, the doses of DNA, different incubation time before electroporation, incubation temperature after electroporation. The cell survival rate was counted under visible light microscope, and the transfection efficiency and production of protein were identified through FACS analysis. Results transfection efficiency and cell survival rate were of the higher value, when The rabbit fibroblast cell with concentration of 2 × 10^6 ells/ml （electroporation medium： PBS） and mixed with 30 Mg vector DNA, the electric voltage of electropration apparatus was set at 250 V, and the capacitors was 700 μF. Moreover, the electransfected efficiency and cell survival rate was increased with 10 rain of ice incubation before electroporation and 10 min of 37℃ incubation after electroporation. Conclusion The electrotransfection is very critical for the tansfection on the rabbit adult fibroblasts, and incubation temperature could improve transfection efficiency