慢病毒shRNA表达载体介导人肿瘤细胞基因稳定沉默的影响因素

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 以乏氧诱导因子-1α（Hypoxia-inducible factor-1α, HIF-1α）和乏氧诱导因子-1β（Hypoxia-inducible factor-1β, HIF-1β）基因为靶基因，采用Invitrogen公司的BLOCK-iT Lentiviral RNAi Expression System生产表达靶基因shRNA的慢病毒载体，转导Hela、SPCA1和A549，采用定量RT-PCR技术检测靶基因mRNA表达水平。结果 用此系统生产慢病毒，每一10cm培养皿可收获6.3×1010个病毒颗粒。浓度为2×1010copies/ml的Lenti6-HIF1α和Lenti6-HIF1β转导SPCA1、A549和Hela细胞的功能滴度分别为：1.8×106TU/ml、1.2×106TU/ml、1.75×106TU/ml和1.76×106TU/ml、1.21×106TU/ml和1.79×106TU/ml。延长病毒的吸附时间可以提高转导效率, 8小时以内转导效率与吸附时间呈正比，12小时开始进入平台期。1/4、1/2、1、2、4、8倍MOI的Lenti6-HIF1α病毒转导SPCA1和Hela细胞48小时后，RNAi效果与病毒量呈正相比。用筛选的转导细胞证实，RNAi长期效果与细胞类型无关，但与shRNA表达结构整合到靶细胞基因组的拷贝数呈正相关。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密切相关。

Factors concerned to gene stable silencing in human tumor cells by lentiviral vector based system

Objective To identify variables which affect the efficacy of gene silencing by lentiviral-mediated RNA interference. Methods Both hypoxia-inducible factor-1α (HIF-1α) and hypoxia-inducible factor-1β (HIF-1β) were chosen as the target genes. Lentiviral vectors harboring shRNA expressing constructs were packaged by BLOCK-iT Lentiviral RNAi Expression System (Invitrogen). Vectors were transduced to Hela, SPCA1 and A549 cells respectively, and mRNA abundance was determined by real time RT-PCR. Results The number of lentiviral particles produced by a 10cm tissue culture plate cells was 6.3×1010. Functional titers of Lenti6-HIF1α while transduced Hela, SPCA1 and A549 cells were 1.8×106TU/ml, 1.2×106TU/ml and 1.75×106TU/ml respectively, and they were 1.76×106TU/ml, 1.21×106TU/ml and 1.79×106TU/ml respectively for Lenti6-HIF1β. Longer period of the vector absorption enhanced virus transduction within 12 hours. And the transient RNAi efficacy was proportional related to the quantity of inoculums. Furthermore, the number of shRNA expression constructs integrated into genome of target cells was specifically associated with RNAi efficacy of stable transduced 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 efficacy was determined by viral gene copies integrated into genome of target cells.