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OACSTPCD

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.