摘要
目的构建稳定表达GFP-LC3的人急性单核细胞白血病细胞(THP-1)。方法采用p CDH-CMV-GFP-LC3-EF1α-puro转染293T细胞构建慢病毒质粒系统,获取的慢病毒感染THP-1细胞,用嘌呤霉素(Puromycin)筛选稳定表达GFP-LC3蛋白的细胞系。通过Western blot和流式细胞术检测THP-1细胞中GFP-LC3蛋白的表达,并利用该稳定表达细胞系观察饥饿和雷帕霉素诱导时细胞发生的自噬变化。结果筛选得到稳定表达GFP-LC3蛋白的THP-1细胞系,在倒置荧光显微镜下观察可见绿色荧光。Western blot和流式细胞术检测证实了GFP-LC3融合蛋白的表达。激光共聚焦法和Western blot均证实饥饿和雷帕霉素可以诱导自噬的发生,且GFP-LC3融合蛋白可以反映内源LC3蛋白的变化,包括蛋白聚集和发生剪切修饰。结论应用慢病毒质粒系统成功构建了高效稳定表达GFP-LC3蛋白的THP-1细胞系,从而为后续利用GFP-LC3融合蛋白研究自噬变化提供了细胞模型。
Objective To establish a stable GFP-LC3-expressed human acute monocytic leukemia cell line(THP-1).Methods The lentivirus plasmid system(pCDH-CMV-GFP-LC3-EF1α-puro)was constructed and transfected into 293T cells with transfection reagent.THP-1 cells were infected with lentivirus and then screened by puromycin.The GFP-LC3 protein expression in THP-1 cells was analyzed by Western blot assay and flow cytometry.Starvation and rapamycininduced autophagy were detected by confocal microscope and Western blot assay.Results The THP-1 cell line with stable expressing GFP-LC3 protein showed visible green fluorescence under inverted fluorescence microscope,as demonstrated by Western blot assay and flow cytometry.Moreover,starvation and rapamycin both could induce the formation of autophagosomes,which included LC3 aggregates and LC3 modification.Conclusion The THP-1 cell line with stable expression of GFP-LC3 protein is constructed efficiently by the lentiviral plasmid system,which provides a cell model for autophagy detection in monocyte-macrophage.
作者
雷蕾
黄珊
于明航
刘志强
刘师伟
李婷
赵秀娟
李泽兴
王玺
LEI Lei;HUANG Shan;YU Ming-hang;LIU Zhi-qiang;LIU Shi-wei;LI Ting;ZHAO Xiu-juan;LI Ze-xing;WANG Xi(Department of Cell Biology,College of Basic Medicine,Tianjin Medical University,Tianjin 300070,China;Department of Histology and Embryology,School of Integrative Medicine,Tianjin University of Traditional Chinese Medicine;Department of Internal Medicine,Xianshuigu Hospital of Jinnan District)
出处
《天津医药》
CAS
北大核心
2018年第4期341-344,共4页
Tianjin Medical Journal
基金
国家自然科学基金资助项目(31600693
81500170)
