稳定表达RFP-GFP-LC3的RAW264.7细胞株的建立  

Establishment of RAW264.7 cell strain stably expressing RFP-GFP-LC3

作　　者：王婉[1] 张庆[1] 赵润鹏[1] 徐雪维 邢应如[2] 张荣波[1] 吴静[1] 胡东[1] 

机构地区：[1]安徽理工大学医学院医学免疫学与检验教研室,安徽淮南232001 [2]安徽理工大学医学院附属肿瘤医院检验科,安徽淮南232001

出　　处：《细胞与分子免疫学杂志》2015年第9期1175-1178,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(81302524;81202294;81172778);安徽省自然科学基金(1208085QH162;KJ2013A105);大学生创新创业训练计划项目(201310361096;AH201310361188;AH201310361189);安徽理工大学大学生科研项目(2013-2014年)

摘　　要：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 murine macrophage RAW264. 7 cell strain with stable expression of red fluorescent protein-green fluorescent protein-microtubule associated protein light chain 3（ RFP-GFP-LC3）. Methods A lentiviral vector containing RFP-GFP-LC3 gene was constructed and then packaged in HEK293 T cells with the packaging plasmids. The viral supernatant was collected to infect RAW264. 7 cells. The RAW264. 7 cell strain with stable expression of RFP-GFP-LC3 was screened with puromycin and analyzed with flow cytometry and fluorescent microscopy for infection efficiency. The number of RFP-GFP-LC3 puncta was observed using florescence microscopy following starvation treatment. Results The recombinant lentivirus p LV-CMV-RFP-GFP-LC3 was successfully constructed. The RAW264. 7 cells with stable expression of RFP-GFPLC3 were obtained by viral infection and puromycin screening. Fluorescent microscopy and flow cytometry demonstrated the expression rates of RFP and GFP reached to 100%. The number of autophagic puncta significantly increased after starvation treatment. Conclusion The RAW264. 7 cell strain with stable expression of RFP-GFP-LC3 has been successfully constructed,which provides a reliable cellular platform for autophagy research.

关 键 词：自噬 慢病毒 RAW264.7细胞 稳定表达 

分 类 号：R392.12[医药卫生—免疫学] Q344.13[医药卫生—基础医学]