CAJ | 학술논문

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(8μg/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24 h,将EOMA细胞分为40个纽,分别加入相应的慢病毒转染混合液,于转染后140 h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(8 μg/ml),MOI=5,感染时间为6 h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(8 μg/ml),MOI=5,感染时间为6 h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.
목적 우화재만병독개도하장함유침대소서α1,3GT적RNA간우질립도입소서혈관류내피세포(EOMA)시적전염조건.방법 안시부가입촉전염제Polybrene(8μg/ml),감염복수(MOI)분별위1、5、10、15화20,감염시간분별위4、6、12화24 h,장EOMA세포분위40개뉴,분별가입상응적만병독전염혼합액,우전염후140 h재형광현미경하계수양성세포솔,태반람염색법검측각전염조건하적EOMA세포활성.결과 불동병독감염복수、불동감염시간급유、무촉전염제Polybrene적각조간병독대EOMA세포적전염효솔차이유통계학의의,기중당가입Polybrene(8 μg/ml),MOI=5,감염시간위6 h시,전염효솔체도최고(83%),세포활성량호,존활솔위96%,차후재증가MOI치화감염시간,전염효솔미계속증가,이대세포적독성작용명현증강.결론가입Polybrene(8 μg/ml),MOI=5,감염시간위6 h시가이실현만병독재체대EO-MA세포적고효전염병보지교고적세포활성.
Objective To optimize the transfecfion parameters for transfecting small interfering RNA (siR-NA) to endothelium of mouse angioma (EOMA) cells mediated by lentiviral vector. Methods According to Poly-brene existing or not(8 μg/ml), series of multiplicity of infection (MOI) (1, 5, 10, 15, 20)and the time of infec-tion(4, 6, 12,24 h), EOMA cells were divided into 40 groups. 140 hours after transfection, cells were counted un-der 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 tranfectian (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).