过表达膜定位IL-3的293T细胞外泌体的纯化及体外功能验证

doi:10.16352/j.issn.1001-6325.2024.07.0947

基础医学与临床 ›› 2024, Vol. 44 ›› Issue (7): 947-953.doi: 10.16352/j.issn.1001-6325.2024.07.0947

过表达膜定位IL-3的293T细胞外泌体的纯化及体外功能验证

高璐¹, 蔡孟华^1,2, 许依^1,2, 何维¹, 陈慧^1,2, 张建民^1,2*

1.中国医学科学院基础医学研究所北京协和医学院基础医学院免疫学系中国医学科学院T细胞与免疫治疗重点实验室重大疾病共性机制研究全国重点实验室,北京 100005;
2.常州西太湖细胞治疗前沿技术研究院,江苏常州 213000

收稿日期:2024-03-19 修回日期:2024-04-29 出版日期:2024-07-05 发布日期:2024-06-26
通讯作者: ^*jzhang42@163.com
基金资助:
国家自然科学基金(82071791,32300745);中国医学科学院创新工程(2021-I2M-1-035)

Purification and in vitro functional validation of exosomes from 293T cells with over-expressed membrane-localized IL-3

GAO Lu¹, CAI Menghua^1,2, XU Yi^1,2, HE Wei¹, CHEN Hui^1,2, ZHANG Jianmin^1,2*

1. Department of Immunology, Key Laboratory for T Cell and Immunotherapy CAMS, State Key Laboratory of Common Mechanism Research for Major Disease, Institute of Basic Medical Sciences CAMS, School of Basic Medicine PUMC,Beijing 100005;
2. Changzhou Xitaihu Institute for Frontier Technology of Cell Therapy, Changzhou 213000, China

Received:2024-03-19 Revised:2024-04-29 Online:2024-07-05 Published:2024-06-26
Contact: ^*jzhang42@163.com

摘要/Abstract

摘要： 目的体外验证过表达膜定位IL-3的293T细胞外泌体的功能,为在阿尔茨海默病模型动物的体内功能验证奠定基础。方法利用本课题组的专利结构,构建能定位于外泌体膜上的重组IL-3慢病毒载体,包装病毒感染293T细胞,筛选稳定表达细胞株。用流式细胞测量术和免疫荧光技术对IL-3的膜定位进行验证;超滤离心纯化IL-3外泌体,透射电镜观察外泌体形态;纳米流式测量术检测外泌体粒径分布及浓度;Western blot检测IL-3及外泌体相关标志蛋白质表达;免疫荧光技术检测其对小胶质细胞系BV-2吞噬Aβ淀粉样蛋白能力的影响。结果经过载体构建、病毒感染、嘌呤霉素筛选和验证,得到稳定过表达膜定位IL-3的293T细胞株;收集纯化外泌体,在透射电镜下可见直径50~100 nm的双层膜囊泡结构;免疫印迹结果显示CD63、ALIX、TSG101等多种外泌体标志蛋白质检测阳性,且与对照相比富含IL-3,提示IL-3外泌体纯化成功;免疫荧光技术检测结果显示IL-3外泌体能在体外促进BV-2细胞对Aβ淀粉样蛋白的吞噬作用。结论过表达膜定位IL-3的基因修饰293T细胞外泌体在体外兼具IL-3和外泌体的作用,能够促进小胶质细胞的吞噬作用,为阿尔茨海默病的临床治疗提供新的思路。

关键词: 白细胞介素3(IL-3), 外泌体, 小鼠脑小胶质细胞系(BV-2), 阿尔茨海默病

Abstract: Objective To verify the function of exosomes from 293T cells over-expressing membrane-localized IL-3 in vitro, so as to lay a foundation for in vivo function verification in animal models of Alzheimer′s disease. Methods Using the patented structure of the group, a recombinant IL-3 lentiviral vector was constructed and virus-infected 293T cells were packaged. Stable cell strain over-expressing IL-3 was screened. The membrane localization of IL-3 was verified by flow cytometry and immuno-fluorescence. Il-3-exosomes were purified by ultra filtration centrifugation, the exosmic morphology was observed by transmission electron microscope, the size distribution and concentration of exosomes were detected by nano-flow analysis, and the expression of IL-3 and exosome related marker proteins were detected by Western blot. The effect of BV-2 on the phagocytosis of Aβ amyloid was detected by immuno-fluorescence. Results Through vector construction, virus infection, screening and verification of puromycin, 293T cell strain with stable over-expression membrane-anchored IL-3 was obtained. The purified exosomes were collected and the structures of double-layer membrane vesicles with a diameter of 50-100 nm were observed under transmission electron microscope. Western blot results proved the presence of CD63, ALIX, TSG101 and other exosome marker proteins and these molecules were rich in IL-3 as compared with the control, that suggested the successful purification of IL-3-exosomes. The results of immuno-fluorescence assay showed that IL-3-exosomes promoted the phagocytosis of Aβ amyloid by BV-2 cells in vitro. Conclusions The gene modified 293T cell exosomes membrane-anchored expression of IL-3 can play a role of both IL-3 and exosomes in vitro, which promote the phagocytosis of microglia, there for provides a new idea for the clinical treatment of Alzheimer′s disease.

Key words: interleukin-3(IL-3), exosome, mouse brain microglial cell line(BV-2), Alzheimer′s disease

中图分类号:

R392.9

高璐, 蔡孟华, 许依, 何维, 陈慧, 张建民. 过表达膜定位IL-3的293T细胞外泌体的纯化及体外功能验证[J]. 基础医学与临床, 2024, 44(7): 947-953.

GAO Lu, CAI Menghua, XU Yi, HE Wei, CHEN Hui, ZHANG Jianmin. Purification and in vitro functional validation of exosomes from 293T cells with over-expressed membrane-localized IL-3[J]. Basic & Clinical Medicine, 2024, 44(7): 947-953.

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过表达膜定位IL-3的293T细胞外泌体的纯化及体外功能验证

Purification and in vitro functional validation of exosomes from 293T cells with over-expressed membrane-localized IL-3

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