天津医药

天津医药 ›› 2018, Vol. 46 ›› Issue (4): 341-344.doi: 10.11958/20180074

• 实验研究 • 上一篇    下一篇

稳定表达GFP-LC3蛋白的THP-1细胞系的构建

雷蕾 1，黄珊 1，于明航 1，刘志强 2，刘师伟 3，李婷 1，赵秀娟 1，李泽兴 1△，王玺 1△   

  1. 基金项目：国家自然科学基金资助项目（31600693，81500170） 作者单位：1天津医科大学基础医学院细胞生物学系（邮编300070）；2天津中医药大学中西医结合学院组织胚胎学教研室；3天津市津南 区咸水沽医院内科 作者简介：雷蕾（1986），女，硕士，助理实验师，主要从事细胞生物学和表观遗传调控研究 Δ通讯作者 李泽兴 E-mail: lizexing@tmu.edu.cn；王玺 E-mail: wangxi@tmu.edu.cn
  • 收稿日期:2018-01-15 修回日期:2018-03-07 出版日期:2018-04-15 发布日期:2018-04-15
  • 通讯作者: 雷蕾 E-mail:ll126com@126.com
  • 基金资助:
    一氧化氮相关蛋白SAP对巨噬细胞天然免疫信号通路调控的分子机制;组蛋白去甲基化酶FBXL10在人弥漫性大B细胞淋巴瘤中的作用及机制研究

The construction of THP-1 cell line stably expressing GFP-LC3 protein

LEI Lei1, HUANG Shan 1, YU Ming-hang1, LIU Zhi-qiang2, LIU Shi-wei3, LI Ting1, ZHAO Xiu-juan1, LI Ze-xing1△, WANG Xi1△   

  1. 1 Department of Cell Biology, College of Basic Medicine, Tianjin Medical University, Tianjin 300070, China; 2 Department of Histology and Embryology, School of Integrative Medicine, Tianjin University of Traditional Chinese Medicine; 3 Department of Internal Medicine, Xianshuigu Hospital of Jinnan District △Corresponding Author LI Ze-xing E-mail: lizexing@tmu.edu.cn; WANG Xi E-mail: wangxi@tmu.edu.cn
  • Received:2018-01-15 Revised:2018-03-07 Published:2018-04-15 Online:2018-04-15
  • Contact: Lei LEI E-mail:ll126com@126.com

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摘要： 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pCDH-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。

关键词: 自噬, GFP-LC3, THP-1细胞, 稳定细胞系, 雷帕霉素

Abstract: Abstract: 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 rapamycin induced 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.

Key words: autophagy, GFP-LC3, THP-1 cell, stable cell line, rapamycin