脂质体法和电穿孔法体外转染SD大鼠视网膜Mller细胞的比较

Comparison of electroporation and lipofection efficiency in retinal Müller cells

目的:探讨脂质体法及电穿孔法介导增强型绿色荧光蛋白(EGFP)基因转染体外培养的视网膜Mller细胞的可行性和区别。方法:体外培养出生后7～10d的SD大鼠视网膜Mller细胞,免疫荧光染色法鉴定95%以上为视网膜Mller细胞。分别用阳离子脂质体Lipofectamine2000介导的脂质体转染法和电穿孔法将质粒PEGFP-N1转染视网膜Mller细胞,荧光显微镜下检测转染后1,2,3,4d的转染效率,并持续观察至转染后14d,比较两者基因表达持续时间。结果:荧光显微镜下检测转染后1d,两组均可见少量细胞表达EGFP绿色荧光蛋白。转染后2d,两者转染效率均达到最大,并且电穿孔法介导质粒PEGFP-N1转染Mller细胞效率约为31.0%±2.8%,较脂质体法转染效率(10.5%±2.4%)更高。两者比较有统计学意义(P<0.01)。随后,两者转染效率均逐渐降低。电穿孔转染后的PEGFP-N1可在视网膜Mller细胞内持续表达近14d,而脂质体转染后仅能表达约7d。结论:脂质体法和电穿孔法均适用于视网膜Mller细胞的基因转染,但电穿孔法效率更高,表达时间更长。

AIM：To evaluate the possibility of transferring enhanced green fluorescent protein （EGFP） to cultured retinal Mueller cells （RMCs） via electroporation and lipofection and to compare the transfection efficiency of electroporation with lipofection in cultured RMCs of rats. METHODS： First of all, Mueller cells were isolated from rat retina,and proliferating cells were expanded in serumcontaining medium. Secondly,the third or fourth passage of cells were identified by glutamatespartate transporters （GLAST） and glutamine synthetase （GS）. Thirdly, cultured RMCs were transfected either by electroporation or by lipofection using a PEGFP-N1 plasmid. At last,the cells were analyzed 24,48 hours, 3,4 days,1 week and 2 weeks after transfection by fluorescence microscopy. RESULTS： Ninety-five percent cultured RMCs were positively reacted for GLAST and GS. Twenty-four hours after transfection there are only few cells transfected in these two groups. However, the percentage of transfected cells was significantly higher when electroporation was used as compared with lipofection in forty-eight hours after transfection. At this time, the transfection efficiency was superior with electroporation （31. 0% ± 2.8%） as compared to lipofection （10.5% ±2.4%）. And there were significant differences between them （P 〈 0.01）. The expression of EGFP could be detected for at most 1 week after lipofection and more than 2 weeks after electroporation. CONCLUSION： Our results show the feasibility of a gene tranfer into RMCs via electroporation and lipofection. Electroporation is superior to lipofection in RMCs.This study may provide a novel tool for our future targeted gene therapy on RMCs.