SFFV启动子高效驱动mCherry-GFP-LC3B双荧光体系在红系细胞中进行自噬检测

doi:10.16352/j.issn.1001-6325.2025.07.0866

基础医学与临床 ›› 2025, Vol. 45 ›› Issue (7): 866-873.doi: 10.16352/j.issn.1001-6325.2025.07.0866

SFFV启动子高效驱动mCherry-GFP-LC3B双荧光体系在红系细胞中进行自噬检测

任久强, 李静, 李卓, 刘雪会^*, 吕湘^*

中国医学科学院基础医学研究所北京协和医学院基础学院病理生理学系疑难重症及罕见病全国重点实验室,北京 100005

收稿日期:2025-03-27 修回日期:2025-04-03 出版日期:2025-07-05 发布日期:2025-06-24
通讯作者: ^*lvxiang@pumc.edu.cn; liuxuehui@ibms.pumc.edu.cn
基金资助:
中国医学科学院医学与健康科技创新工程(2021-I2M-1-019);国家自然科学基金(82170120);北京市自然科学基金(7252095)

A highly efficient SFFV promoter-driven mCherry-GFP-LC3B dual-fluorescence system for autophagy monitoring in erythroid cells

REN Jiuqiang, LI Jing, LI Zhuo, LIU Xuehui^*, LYU Xiang^*

State Key Laboratory for Complex, Severe, and Rare Diseases, Department of Pathophysiology, Institute of Basic Medical Sciences CAMS, School of Basic Medicine PUMC, Beijing 100005, China

Received:2025-03-27 Revised:2025-04-03 Online:2025-07-05 Published:2025-06-24
Contact: ^*lvxiang@pumc.edu.cn; liuxuehui@ibms.pumc.edu.cn

摘要/Abstract

摘要： 目的建立由造血细胞中高活性的脾焦点形成病毒(SFFV) 启动子驱动的mCherry-GFP-LC3B慢病毒表达体系,并实现红系终末分化细胞自噬流的高效动态示踪。方法构建pRSC-SFFV-mCherry-GFP-LC3B慢病毒质粒,包装慢病毒并感染人红系祖细胞系(HUDEP-2)及小鼠胎肝来源原代红系细胞;结合血清剥夺及氯喹干预模型,利用荧光成像、Western blot及流式细胞测量术分析红系终末分化细胞的自噬动态变化。结果 SFFV启动子在HUDEP-2(97% vs. 60%)及原代小鼠胎肝红系细胞(83% vs. 1%)中表达的阳性率均显著高于巨噬细胞病毒(CMV)启动子(P＜0.01,P＜0.001),且不影响正常分化。血清剥夺后,自噬溶酶体(红斑)数量增加,伴随LC3-Ⅱ/LC3-Ⅰ比值升高及p62蛋白下降;氯喹干预则导致自噬体(黄斑)累积(P＜0.001),且抑制效应呈剂量依赖性(r²=0.92)。结论 SFFV驱动双荧光体系可高效实时示踪红系细胞自噬流动态,为红系分化及相关贫血性疾病的机制研究提供了高灵敏度工具。

关键词: SFFV启动子, 自噬, mCherry-GFP-LC3B双荧光, 红系细胞

Abstract: Objective To develop a modified lentiviral expression system of mCherry-GFP-LC3B, driven by the hematopoietic-specific SFFV (spleen focus-forming virus) promoter, in order to perform an efficient and real-time monitoring of autophagy flux with in terminally differentiated erythroid cells. Methods The lentiviral plasmid pRSC-SFFV-mCherry-GFP-LC3B was constructed and packaged into lentiviruses for infection of human erythroid progenitor cell line(HUDEP-2) and mouse fetal liver-derived primary erythroid cells. Autophagy dynamics of the cells were then analyzed using fluorescence imaging, Western blot, and flow cytometry in serum starvation and chloroquine inter-vention models. Results The SFFV promoter rendered significantly higher reporter expression efficiency than CMV promoter in HUDEP-2 (97% vs. 60%)(P＜0.01) and in the primary mouse erythroid cells (83% vs. 1%)(P＜0.001), without disrupting normal erythroid differentiation. Serum deprivation increased autolysosomes (red puncta), elevated LC3-Ⅱ/LC3-Ⅰ ratios, and decreased p62 levels. Chloroquine treatment induced autophagosome (yellow puncta) accumulation (P＜0.001), showing a dose-dependent inhibition of autophagy flux (r²=0.92). Conclusions The SFFV-driven dual-fluorescent system enables robust and real-time monitoring of autophagy flux in erythroid cells, providing a sensitive tool for mechanistic study of erythroid differentiation and related disorders such as anemia.

Key words: SFFV promoter, autophagy, dual-fluorescent mCherry-GFP-LC3B, erythroid cells

中图分类号:

任久强, 李静, 李卓, 刘雪会, 吕湘. SFFV启动子高效驱动mCherry-GFP-LC3B双荧光体系在红系细胞中进行自噬检测[J]. 基础医学与临床, 2025, 45(7): 866-873.

REN Jiuqiang, LI Jing, LI Zhuo, LIU Xuehui, LYU Xiang. A highly efficient SFFV promoter-driven mCherry-GFP-LC3B dual-fluorescence system for autophagy monitoring in erythroid cells[J]. Basic & Clinical Medicine, 2025, 45(7): 866-873.

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SFFV启动子高效驱动mCherry-GFP-LC3B双荧光体系在红系细胞中进行自噬检测

A highly efficient SFFV promoter-driven mCherry-GFP-LC3B dual-fluorescence system for autophagy monitoring in erythroid cells

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