| Objective:To contrast and investigate differential immunotherapy mediated by dendritic cell pulsed ovarian carcinoma antigen,we prepared antigen derived from ovarian cancer-exosomes;HS-exosomes; heat shocked protein;frozen tumor antigen(FTA).Methods:It was adopted the methods of overspeed and gradient centrifugation,To isolate and purify exosomes and HS-exosomes from the culture supernatants of ovarian cells-A2780 cells and SKOV3 cells,we analyzed their ultrastructure by electron transmission microscopy.Frozen and thawed ovarian cancer cells to get frozen tumor antigen.Lysis buffer of heat treated ovarian cancer cells was purified successively by chromatographic procedures.We identified its molecular weight by Western-blot.Results:Electron microscopy analysis exosomes derived from the culture supernatants of ovarian cancer cells.It is a double deck vesicles of 30-90nm in diameter.We consulted literature and it was confirmed that the vesicles was exosomes.Ovarian cancer cells lysis buffer purified were confirmed it including the molecular weight of HSP70 by SDS-PAGE and Western-blot.Conclusion:we could isolate and purify a double deck vesicles of 30-90 nm from culture supernatants of heat treated and untreated ovarian cancer cells.we analyzed their ultrastructure by electron transmission microscopy and detected them possessing representative shape of exosomes and HS-exosomes.Lysis buffer of heat treated ovarian cells included abundance HSP70. Objective:It was investigated the function of dendritic cells pulsed with ovarian carcinoma antigen and tumor-specific T cells induced them and its killing effect on ovarian cell.Methods:Mononuclear cells were isolated from healthy peripheral blood by Ficoll-Hypaque density gradient centrifugation, resuspended the cells in RPMI1640 containing rhGM-CSF and rhIL-4, On days 6thor 7th,the monocytes will been differentiated into DC.It was activated into Ag- DC when tumor antigen were put into cutivated system,DC and Ag-DC culture supernatant (IL-12/TNF-α)were detected using cytokine detected kit.We obtained T lymphocytes from healthy peripheral blood,and CTL were induced by Ag-DC.We detected T lymphocyte stimulating activities by MTT.At the same time,peripheral blood PMBC of healthy was activated into LAK/CD3AK/CIK by PHA,IL-2,IFN and anti-CD3AK. Mixed Ag-DC and these cells,as activated them into effective cells.Effectors:target cell ratio was 25:1,then added effectors into culture supernatants of target cells.Cytotoxicity test detected if effectors had powerful killing activity to target cells.Results:PMBC of Peripheral blood from healthy were cultured with GM- CSF,IL-4.After a week,The cells is irregulate,surface rough and tuber obvious,The cells is kidney shape and horse unguis shape. The cells was proved DC by FCM.DC and Ag-DC supematant (IL-12/TNF-α)were detected using cytokine detected kit.Its conclusion indicated:Quantity of cytokine secreted by DC pulsed the four kinds of antigen was obvious increased,it secreted by HS-exosomes-DC was the most,Multiplicating activity of T lymphocytes stimulated DC modified ovarian antigen was obvious difference from it Of T lymphocytes stimulated DC(p＜0.01) Multiplicating activity of T lymphocytes stimulated DC modified HS-exosomes was obvious difference from it of T lymphocytes stimulated other three kinds of antigens and it was no difference among them.T lymphocytes induced by DC modified ovarian antigen had strong killing role to tumor cells.T lymphocytes induced by DC modified heat shocked antigen was the strongest and obvious difference from it modified by frozen tumor antigen. There were no difference heat shocked protein and exosomes in killing tumor cells.Unactivated T lymphocytes had no killing to ovarian cells.Conclusion:In our experiment we prepared four kinds of antigen,and they could promote DC mature.Cytokine secreted DC modified by them and activity of T cells stimulated by them were obviously heightened.Even T lymphocytes stimulated by DC modified antigens had strong killing role to tumor cells... |
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