Effects Of HCC Exosomes On The Maturation And Function Of Human DC And The Regulating Effect Of Improved Aitongxiao Prescription

ObjectiveTo explore an exosome extraction method with high yield and few impurities by improving ExoQuick-TC kit.To investigate the effects of HepG2-derived exosomes(HepG2-EXO)on the maturation,cytokine secretion and the capacity of stimulating allogeneic T cell proliferation of human dendritic cell(DC).To investigate whether improved AITONGXIAO prescription(I-ATXP)can reduce the level of exosomes secrected by HepG2 cells and the effect of exosomes derived from I-ATXP-treated HepG2 cells(ATX-EXO)on the maturation and function of human DCs.Methods(1)Extraction of exosomes by modified Exoquick-TC kit method:The HepG2 cells were cultured for 24h in exosome-free serum medium,supernatant was collected,filtered with a 0.22?m filter membrane,centrifuged at 10,000 g for 30min to remove impurities with a density and volume greater than that of exosomes,and then filtered with a 10 kb ultrafiltration tube to remove small molecular impurities and concentrate the sample volume.Exosomes were finally extracted according to the ExoQuick-TC kit instructions.(2)Exosomes identification:Western Blot was used to detect the exosome marker protein CD63,ALIX and cellular component protein cytochrome C,and Transmission Electron Microscope(TEM)was used to observe the morphology structure and size of exosome,and Nano FCM was used to analyze the particle size distribution and particle concentration of exosomes.(3)Induction of immature dendritic cells(imDC):Fresh anticoagulant was taken from healthy donors,PBMCs were obtained by Ficoll density gradient centrifugation,and CD14~+cells were isolated by immunomagnetic bead method.ImDCs were obtained through culturing the CD14~+cells in the presence of GM-CSF 20 ng/mL,IL-4 10 ng/mL and IFN-b10~5 U/mL were used for 24 hs.(4)Intervention method of exosomes on DC maturation:LPS(10 ng/mL)was used for DC maturation.At the same time,HepG2-derived exosomes(50,100,200?g/mL)were added.Seventy-two hours later,cells and supernatants were collected,respectively.using Flow cytometry was used to detect the expression levels of CD80,CD83,CD86 and HLA-DR,ELISA was used to detect the level of IL–12p70 in the culture supernatant,CCK8 kit was used to detect the proliferation of allogeneic lymphocyte stimulated by DCs.(5)Intervention method of I-ATXP on HepG2:HepG2 cells were treated with 0.125,0.25,0.5,1.0(mg/mL)of I-ATXP for 24 h.Cell proliferation level was detected by CCK8 kit,and the supernatants were collected for extracting exosomes.The amount of exosomes was measured by coomase bright blue(Bradford method)and Acetylcholinesterase assay(AchE assay).(6)The maximum I-ATXP dose(0.5 mg/mL)with no effect on cell survival was selected to intervene HepG2 cells,and ATX-EXO was extracted to intervene imDC according to method(4),and the same index of DC was compaired.Results(1)Western Blot analysis showed that the expression levels of CD63 and ALIX in exosomes extracted with modified Exoquick-TC kit method were higher than those extracted with the Exoquick-TC kit method,none of them expressed cytochrome C indicates that the extracted samples don't contained cells.Transmission electron microscopy showed that the exosome samples extracted by the Exoquick-tc kit modified method were clearly visible as circuar vesicles with a diameter of 30-100 nm,which was consistent with the characteristics of exosomes and had fewer impurities than those extracted by the Exoquick-tc kit method.Nano FCM results show that the average diameter of HepG2-derived exosomes was 75.81±26.47(mean±s.d.,nm),consistent with the results of transmission electron microscopy.In addition,four other types of cancer or non-cancer hepatocellular exosomes extracted by the modified Exoquick-TC kit method also showed similar particle size distribution,indicating that this method was relatively stable.(2)Compared with the control group,the percentage of CD80-and CD83-positive DCs were decreased in HepG2-derived exosomes-treated DCs,while the mean fluorescence intensity(MFI)of CD86 and HLA-DR were not significantly different;the level of IL-12p70 in the culture supernatant was significantly lower in DC treated with 200?g/mL HepG2-EXO;in addition,the capacity of stimulating allogeneic lymphocyts proliferation was also decreased in 200?g/mL HepG2-EXO-treated DCs.(3)HepG2 cells(5×10~5 cells/mL)treated with 0.5 mg/mL of I-ATXP for24hs reduced exosomes production but no significant change on cell proliferation.(4)Compared with the HepG2-EXO intervention group,the expression levels of CD80,CD83,CD86,HLA-DR,the secretion levels of IL-12p70 and the ability to stimulate allogeneic T cells proliferation were no significant difference in ATX-EXO-treated groups.Conclusionsmodified Exoquick-TC kit method can extract the high production with less impurity exosomes.HepG2-derived exosomes can down-regulate the CD80and CD83 expression levels on LPS-matured DCs,reduce IL-12p70 levels secrated by LPS-matured DC,and reduce their ability on allogeneic lymphocytes stimulation.I-ATXP can reduce the amount of exosomes secreted by HepG2 without affecting its survival rate.However,I-ATXP did not changed the inhibitory activity of HepG2-derived exosome on DCs,that is,it may not cause the change of exosome constitution.