Separation and identification of the Exosomes derived from heat shocked mouse hepatoma cell line(H22) and preliminary investigation of its protein composition

Objective:To study the effect of heat shock on the production and protein composition of Exosomes derived from a mouse hepatoma cell line(H22) in order to provide theoretical basis for the improvement in production and immunol therapy of exosomes. Methods:Normal Exosomes and heat shocked Exosomes(HS-Exo) were purified by serial ultracentrifugation and sucrose density gradiant centrifugation,then were observed and identified by electron microscopy.The protein composition of Exosomes and HS-Exo were analysed by SDS-PAGE electrophoresis. And their immunological factors were detected by Western blot. Results:Under the electron microscopic view,HS-Exo had similar morphologic pattern to Exosomes,and the structure was complete,as vesicles with a diameter ranging from 20~90nm;The production was increased in heat shocked Exosomes.And the expressions of immunological factors such as HSP70,HSC70,HSP60,ICAM-1 in HS-Exo were significantly up-regulated than those in Exosomes. Conclusion:Serial ultracentrifugation and sucrose density gradiant centrifugation can be used to purify HS-Exo. HSP70,HSC70,HSP60,ICAM-1 were expressed more in HS-Exo than in Exosomes,which may suggest that Exosomes derived from heat-shocked H22 cells would have more effective immunostimulatory functions.