慢病毒载体介导RNA干扰质粒转染小鼠血管瘤内皮细胞的条件优化

Optimization of transfection conditions for transfecting shRNA to endothelium of mouse angioma cells mediated by lentiviral vector.

目的优化在慢病毒介导下将含有针对小鼠α1，3GT的RNA干扰质粒导入小鼠血管瘤内皮细胞（EOMA）时的转染条件。方法按是否加入促转染剂Polybrene（8μ/ml），感染复数（MOI）分别为1、5、10、15和20，感染时间分别为4、6、12和24h，将EOMA细胞分为40个组，分别加入相应的慢病毒转染混合液，于转染后140h在荧光显微镜下计数阳性细胞率，台盼蓝染色法检测各转染条件下的EOMA细胞活性。结果不同病毒感染复数、不同感染时间及有、无促转染剂Polybrene的各组间病毒对EOMA细胞的转染效率差异有统计学意义，其中当加入Polybrene（8μg／ml），MOI=5，感染时间为6h时，转染效率达到最高（83％），细胞活性良好，存活率为96％，此后再增加MOI值和感染时间，转染效率未继续增加，而对细胞的毒性作用明显增强。结论加入Polybrene（8μg／ml），MOI=5，感染时间为6h时可以实现慢病毒载体对EO-MA细胞的高效转染并保持较高的细胞活性。

Objective To optimize the transfection parameters for transfecting small interfering RNA （siRNA） to endothelium of mouse angioma （EOMA） cells mediated by lentiviral vector. Methods According to Polybrene existing or not （8 μg/ml）, series of multiplicity of infection （MOI） （ 1,5, 10, 15, 20）and the time of infection （4, 6, 12,24 h）, EOMA cells were divided into 40 groups. 140 hours after transfection, cells were counted under fluorescent microscope to determine the percentage of the cells transfected under each condition. The cell viability was calculated by using Typan Blue. Results The percentage of positive cells was as high as 83% in the group with Polybrene （8 μg/ml）, MOI（5） and the time of tranfection （ 6 h）, and the livability of EOMA in this group was 96%. After that, the transfection efficiency was not increased with the increasing of MOI, the time of transfection, but the mortality of EOMA was increased markedly. Conclusion The transfection efficiency and cell viability are dependent on the Polybrene（8 μg/ml） , correct MOI （5）and the time of transfection（6 h）.