评价基于 HIV-1 的慢病毒载体在巨噬细胞集落刺激因子分化的人巨噬细胞中的转导效率。
Evaluation of transduction efficiency in macrophage colony-stimulating factor differentiated human macrophages using HIV-1 based lentiviral vectors.
机构信息
Experimental Atherosclerosis Section, National Heart, Lung, and Blood Institute, NIH, Bethesda, MD, USA.
出版信息
BMC Biotechnol. 2011 Jan 31;11:13. doi: 10.1186/1472-6750-11-13.
BACKGROUND
Monocyte-derived macrophages contribute to atherosclerotic plaque formation. Therefore, manipulating macrophage function could have significant therapeutic value. The objective of this study was to determine transduction efficiency of two HIV-based lentiviral vector configurations as delivery systems for the transduction of primary human blood monocyte-derived macrophages.
RESULTS
Human blood monocytes were transduced using two VSV-G pseudotyped HIV-1 based lentiviral vectors containing EGFP expression driven by either native HIV-LTR (VRX494) or EF1α promoters (VRX1090). Lentiviral vectors were added to cultured macrophages at different times and multiplicities of infection (MOI). Transduction efficiency was assessed using fluorescence microscopy and flow cytometry. Macrophages transduced between 2 and 120 hours after culturing showed the highest transduction efficiency at 2-hours transduction time. Subsequently, cells were transduced 2 hours after culturing at various vector concentrations (MOIs of 5, 10, 25 and 50) to determine the amount of lentiviral vector particles required to maximally transduce human monocyte-derived macrophages. On day 7, all transduced cultures showed EGFP-positive cells by microscopy. Flow cytometric analysis showed with all MOIs a peak shift corresponding to the presence of EGFP-positive cells. For VRX494, transduction efficiency was maximal at an MOI of 25 to 50 and ranged between 58 and 67%. For VRX1090, transduction efficiency was maximal at an MOI of 10 and ranged between 80 and 90%. Thus, transductions performed with VRX1090 showed a higher number of EGFP-positive cells than VRX494.
CONCLUSIONS
This report shows that VSV-G pseudotyped HIV-based lentiviral vectors can efficiently transduce human blood monocyte-derived macrophages early during differentiation using low particle numbers that do not interfere with differentiation of monocytes into macrophages.
背景
单核细胞衍生的巨噬细胞有助于动脉粥样硬化斑块的形成。因此，调节巨噬细胞的功能可能具有重要的治疗价值。本研究的目的是确定两种基于 HIV 的慢病毒载体构型作为转导主要人血单核细胞衍生的巨噬细胞的转导系统的转导效率。
结果
使用两种含有 EGFP 表达的 VSV-G 假型 HIV-1 基于慢病毒载体，通过天然 HIV-LTR（VRX494）或 EF1α 启动子（VRX1090）驱动，转导人血单核细胞。将慢病毒载体添加到培养的巨噬细胞中不同的时间和感染复数（MOI）。使用荧光显微镜和流式细胞术评估转导效率。在培养后 2 至 120 小时之间转导的巨噬细胞在 2 小时转导时间时显示出最高的转导效率。随后，在各种载体浓度（MOI 为 5、10、25 和 50）下在培养后 2 小时转导细胞，以确定最大程度转导人单核细胞衍生的巨噬细胞所需的慢病毒载体颗粒量。第 7 天，所有转导的培养物在显微镜下均显示出 EGFP 阳性细胞。流式细胞术分析显示，所有 MOI 均存在 EGFP 阳性细胞的峰移。对于 VRX494，在 MOI 为 25 至 50 时转导效率最大，范围在 58%至 67%之间。对于 VRX1090，在 MOI 为 10 时转导效率最大，范围在 80%至 90%之间。因此，与 VRX494 相比，VRX1090 转导显示出更多的 EGFP 阳性细胞。
结论
本报告表明，VSV-G 假型 HIV 基于慢病毒载体可以在单核细胞分化的早期通过使用不会干扰单核细胞向巨噬细胞分化的低颗粒数有效地转导人血单核细胞衍生的巨噬细胞。
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