The Experimental Study Of Effect Of CpG ODN On Differentiation,Maturation And Antitumor Of Human Peripheral Blood Dendritic Cells

Objective:To explore the effects of CpG ODN on the differentiation,maturation and antitumor of human peripheral blood dendritic cells.To investigate the regulation of CpG ODN to DC cultured with tumor cell supernatant.Methods:⑴After isolated from normal human peripheral blood,DC is examined its purity by S-100 protein immunocytochemistry staining.⑵The factorial design is applied to analyze the effect of different levels of CpG ODN concerntration,DC concerntration and culture time on DC growth,and the best condition of CpG ODN-stimulated DC in vitro is selected.⑶The cell morphology of DC that incubated with and without CpG ODN are observed by transmisson electron microscope,the expression of surface antigen HLA-DR,CD86 on them is analyzed by flow cytometry(FCM) and the effect of them on the proliferation of naive T cell in allogeneic mixed lymphocyte reaction(MLR) and antitumor experiment are detected by MTT test. ⑷The supernatant of tumor cell are collected and used as the conditional culture medium for DC freshly isolated from human peripheral blood. MTT colorimetric assay is used to detect the effect of different concerntration of tumor cell supernatant on the growth of DC at different time,and the expression of HLA-DR on DC cultured with and without supernatant is analyzed by FCM.⑸DC isolated freshly is incubated with 10%FCS RPMI1640 culture medium,CpG ODN(1μmol.l-1),tumor cell supernatant(30%)and CpG ODN(1μmol.l-1)+ tumorcell supernatant(30%)for 72h,respectively.The DC are characterized by proliferative and phenotypic analysis. Results:⑴S-100 protein immunocytochemistry staining analysis shows that DC's purity >90%.⑵The factorial test explains that CpG ODN concerntration has significant effect on the growth of DC(p<0.01).Also,with DC concerntration has a synergistic effect on it.The best condition of CpG ODN-stimulated DC:CpG ODN is 1μmol.l-1,DC is 6×104 well-1 and for 36h.⑶The morphological and functional alaysis of DC show that the surface of CpG ODN-stimulated DC has many thin,long and regular dendrites with numerous rough endoplasmic reticulum and scanty vacuole,in contrast to non-CpG ODN-stimulated DC. Meanwhile,CpG ODN up-regulates the expression of HLA-DR,CD86 on DC,improves significantly the proliferation of T cell in MLR and enhances the antitumor activity of DC,superioring to non-CpG ODN-stimulated group.⑷The phenotypic analysis shows that DC cultured in the presence of tumor cell supernatant express lower levels of HLA-DR,which is significantly lower than that of in control and CpG ODN+ tumor cell supernatant groups.Also,they display of suppressive capacity of the growth of DC.Conclusion:⑴CpG ODN may induce the differentiation and maturation of DC and increase the antitumor capacity of DC.⑵The tumor cell negatively regulates the growth and antigen expression of DC,while CpG ODN inhibits the down-regulation of tumor cell to those of DC.⑶CpG ODN has great immune stimulating capacity to DC induced in vitro and under microenvironment of tumor cell.This will supply theorial bases for CpG ODN as immune adjuvant is applied to immune regulation in vivo.