摘要
目的通过实验观察无外源基因的慢病毒颗粒对HEK-293细胞产生的生物学影响,为选择以何种方式获得重组慢病毒感染的目的细胞提供基础依据。方法以pLentiLox3.7慢病毒核心空载体和包装质粒VSVG、RSV-REV和pMDLg/pRRE按一定比例混合,采用脂质体共转染,将慢病毒载体系统的4质粒共转染HEK-293T细胞,收获病毒原液,再以浓缩的病毒原液或等体积的培养基加入培养中的HEK-293细胞,观察细胞的形态变化,分析其染色体并采用流式细胞术分析细胞周期。结果按照本实验中慢病毒包装的质粒比例及包装过程获得的浓缩病毒液使得HEK-293细胞具有融合趋势,染色体分析后表明细胞尚未真正融合,但流式分析显示一定量的慢病毒可使得S期的细胞比例显著增多。结论一定量的慢病毒对细胞具有融合趋势,建议取低浓度含有荧光筛选标签的重组慢病毒感染目的细胞,培养之后再进行流式无菌分选以获得较纯的目的细胞。
Objective To observe the effect of intact lentiviral particles without exogenous gene on HEK - 293 cells and provide the basis for recombinant lentivirus infecting target cells. Methods According to a certain proportion, four kinds of plasmids, including pLentiLox3.7, VSVG, RSV - REV and pMDLg/pRRE, were mixed and co - transfected with liposome into HEK - 293T cells for packa- ging the lentivirus. Then concentrated virus supernatant was dropped into HEK -293 cells. Twenty -four hours later, cellular morpholo- gy, chromosome and cell cycle were analyzed. Results The HEK - 293 ceils, which grew in the culture including concentrated virus, had a trend of fusion. Chromosomal analysis showed that the cells had not been true integrated with each other. Flow analysis, however, showed that a certain amount of lentiviruses could leal the proportion of ceils in S phase to increase. Conclusion Highly concentrated lentiviruses can cause cell fusion. It is suggested that lowly concentrated recombinant lentivirus with fluorescent screening marker was a- dopted and infected target cells. After cell culture, pure objective cells were obtained by flow sorting.
出处
《医学研究杂志》
2013年第7期58-61,共4页
Journal of Medical Research
基金
国家自然科学基金青年科学基金资助项目(31200811)
关键词
慢病毒
转染
细胞融合
染色体
细胞周期
Lentivirus
Transfection
Cell fusion
Chromosome
Cell cycle
