Purification and in vitro functional validation of exosomes from 293T cells with over-expressed membrane-localized IL-3
10.16352/j.issn.1001-6325.2024.07.0947
- VernacularTitle: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
- Author:
Lu GAO
1
;
Menghua CAI
;
Yi XU
;
Wei HE
;
Hui CHEN
;
Jianmin ZHANG
Author Information
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
- Keywords:
interleukin-3(IL-3);
exosome;
mouse brain microglial cell line(BV-2);
Alzheimer's disease
- From:
Basic & Clinical Medicine
2024;44(7):947-953
- CountryChina
- Language:Chinese
-
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.
