摘要
目的:探讨慢病毒载体介导人肿瘤细胞RNA干扰的影响因素。方法:用介导RNAi的慢病毒载体Lenti6-HIF1α和Lenti6-HIF1β分别转导人肿瘤细胞系Hela、SPCA1和A549,筛选靶基因稳定表达沉默的细胞株。病毒RNA分子拷贝数和靶基因mRNA水平用实时定量RT-PCR确定。结果:针对不同靶基因的慢病毒转导肿瘤细胞的效率与靶基因无关、存在细胞类型特异性。8h内病毒转导效率与吸附时间呈正关,12h后继续延长吸附时间不能提高转导效率。对靶基因表达的特异性抑制效果与病毒量呈正相关。靶基因稳定沉默细胞株的RNAi效果与细胞类型无关,与靶细胞基因组整合的shRNA表达结构的拷贝数呈正相关。结论:慢病毒载体介导人肿瘤细胞RNA干扰,短期基因抑制效果取决于细胞类型、病毒量和病毒的吸附时间,稳定基因沉默效果与病毒整合到靶细胞基因组的拷贝数相关,筛选稳定基因沉默细胞株时,应尽可能提高病毒量以提高病毒整合的拷贝数。
Objective： To explore factors concerned for stable gene silencing in human cancer cells by lentiviral vector based system. Methods： Lentiviral vectors Lenti6-HIF1 α and Lenti6-HIF1β which mediated gene silencing of HIF-1α and HIF-1β were used to transduce Hela, SPCA1 and A549 cells respectively. Cell sub- clones in which target mRNA silenced were screened. The RNA copies of viral vectors and mRNA levels of target genes were determined by quantitative real-time RT-PCR. Results： The transduction efficiency of lentiviral vectors is related to host cell type but not genes being targeted. Longer period of the vector absorption enhanced virus transduction within 12 hours. The transient RNAi efficiency was proportional related to the quantity of inoculums. Furthermore, the RNAi efficiencies in stable gene silenced sub-clones were non-cell-type specific, which were specifically associated with number of shRNA expression constructs integrated into the genome of target cells. Conclusion： In lentiviral-mediated human tumor RNA interference, the transient gene expression inhibition was determined by cell type, quantity of vectors and absorption time, but the stable gene silencing efficiency was determined by viral gene copies integrated into the genome of target cells. To construct a stable gene silenced cell line, it is important to improve integration of virus into host cell genome by increasing viral vector copies.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2007年第2期19-23,共5页
China Biotechnology
基金
国家自然科学基金资助项目(30570547)
