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目的建立能够稳定表达红色荧光蛋白-绿色荧光蛋白-微管相关蛋白轻链3(RFP-GFP-LC3)的小鼠RAW264.7巨噬细胞。方法构建含RFP-GFP-LC3基因的慢病毒重组质粒载体,将重组质粒与慢病毒包装质粒共转染HEK293T细胞,收集病毒上清后感染RAW264.7细胞。经嘌呤霉素筛选获得稳定表达RFP-GFP-LC3的RAW264.7细胞株,用荧光显微镜和流式细胞术分析感染效率。饥饿处理稳定感染细胞,荧光显微镜下观察RFP-GFP-LC3斑点。结果成功构建了慢病毒p LV-CMV-RFPGFP-LC3,经感染和嘌呤霉素筛选获得稳定表达RFP-GFP-LC3的RFP-GFP-LC3-RAW264.7细胞;荧光显微镜和流式细胞术显示RFP和GFP荧光率均达到100%,饥饿处理后自噬斑点显著增多。结论成功建立稳定表达RFP-GFP-LC3的RAW264.7细胞株,为自噬研究建立了可靠细胞平台。
Objective To establish mouse RAW264.7 macrophages that stably express the red fluorescent protein (GFP) - microtubule-associated protein light chain 3 (RFP-GFP-LC3). Methods The recombinant lentiviral vector containing RFP-GFP-LC3 gene was constructed and transfected into HEK293T cells with lentivirus packaging plasmid. RAW264.7 cells were infected with virus supernatant. The RAW264.7 cell line stably expressing RFP-GFP-LC3 was obtained by puromycin screening, and the infection efficiency was analyzed by fluorescence microscopy and flow cytometry. Stably infected cells were starved, and RFP-GFP-LC3 spots were observed under a fluorescence microscope. Results The lentivirus p LV-CMV-RFPGFP-LC3 was successfully constructed and RFP-GFP-LC3-RAW264.7 cells stably expressing RFP-GFP-LC3 were obtained by infection and puromycin screening. Fluorescence microscopy and flow cytometry The fluorescence of RFP and GFP reached 100%, and the number of autophagy spots increased significantly after starvation treatment. Conclusion The RAW264.7 cell line stably expressing RFP-GFP-LC3 was successfully established and established a reliable cell platform for autophagy research.