基础医学与临床2024，Vol.44Issue(7)：947-953,7.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
Purification and in vitro functional validation of exosomes from 293T cells with over-expressed membrane-localized IL-3
摘要
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-fluores-cence.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 purifica-tion of IL-3-exosomes.The results of immuno-fluorescence assay showed that IL-3-exosomes promoted the phagocy-tosis 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.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Key words
interleukin-3(IL-3)/exosome/mouse brain microglial cell line(BV-2)/Alzheimer's disease分类
基础医学引用本文复制引用
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):947-953,7.基金项目
国家自然科学基金(82071791,32300745) （82071791,32300745）
中国医学科学院创新工程(2021-I2M-1-035) （2021-I2M-1-035）
