Effect Of VEGF On Differentiation And Function Of Dendritic Cells Derived From CD34～+ Hematopoietic Progenitor Cells And Its Relationship And Correlative Mechanisms In Ovarian Carcinoma Immune

Part ⅠComparative studies on the properties of dendritic cells (DC) derived from human cord blood CD34~+ hematopoietic progenitor cells induced by different combinations of cytokinesObjective Comparison of amplification efficiency and function of DC generated from human cord blood CD34~+ hematopoietic progenitor cells induced by different combinations of cytokines, and providing a optimal DC culture condition. Methods After islolated from umbilical cord blood by using a high-gradient magnetic cell sorting system (MACS) , CD34~+ hematopoietic progenitor cells were induced and expanded with different combinations of cytokines composed of rhGM-CSF, rhTNF- α , rhSCF and rhFL. Kinetics analysis of cell proliferation were performed during the process of cell culture. Morphologic and functional studies were identified by invert microscope, electronic microscope, FACS, mixed lymphocyte reaction (MLR) and IL-12 secretion. Results Though typical features of DC were possessed in all culture conditions, the amplification efficiency and function of DC were dramatically different. Maximal expansion power of total cell number were yielded with the combination of GM-CSF/ TNF- α / SCF/ FL (615 ± 21.08 fold, P=0.0001), much higher than those of others. And this combination was theUmbilical cord blood; CD34~+ hematopoietic progenitor cells; Dendritic cells; CytokinesDendritic cells; Ovarian carcinoma; freeze-thaw antigen; Cytotoxicity T lymphocytesVascular endothelial growth factor; Ovarian Carcinoma Cells, Dendritic cells; CD34~+ hemopoietic progenitor cellscarcinoma cells effect on CD34~+ hematopoietic progenitor cells. Methods After islolated from umbilical cord blood by using MACS, CD34~+ cells were then stimulated by VEGF (50ng/ml) and SKOV3 culture supernatant (20%) to detect the tyrosine phosphorylation and nuclear translocation of STAT-3 and STAT-5 by Western Blot and immunocytochemistry methods. The expression of VEGFR-2/KDR on the membrane of CD34~+ progenitor cells was examined by immunocytochemistry. The specific neutralizing VEGF antibody was added into SKOV3 supernatant to detect whether the effect of SKOV3 supernatant was caused by VEGF. Meanwhile, ATWLPPR, an effective peptide screened from phage epitope library by affinity for membrane-expressed KDR and blocking the binding of VEGF to KDR was used to identify whether the activation of STAT pathway induced by VEGF and SKOV3 supernatant was blocked. Results Tyrosine phosphorylation of STAT-3 and STAT-5 was undetectable in unstimulated CD34~+ cells but was detectable in response to VEGF stimulation. VEGF resulted in a rapid and transient tyrosine phosphorylation of STAT-3 and STAT-5, and the maximal tyrosine phosphorylation was catched at 30 min and 45 min respectively. Neutralizing VEGF antibody can only partially inhibit the phosphorylation of STAT-3 and STAT-5 induced by SKOV3 supernatant. Immunocytochemistry confirmed that increased phosphorylated STAT-3 was translocated into the nuclei after stimulation with VEGF and SKOV3 supernatant. However, there is only increased phosphorylation of STAT-5 compared with unstimulated CD34~+ cells appeared mainly in cytoplasms, but no significant nuclear translocation after stimulation of VEGF and SKOV3 supernatant. The presence of VEGFR2/KDR was confirmed using anti-VEGFR2 antibody staining by immunocytochemistry. Moreover, phosphorylation of STAT-3 and STAT-5 were failed to be activated when ATWLPPR was added, suggesting that activation of the STAT pathway by VEGF and SKOV3 supernatant was specifically mediated by KDR in CD34~+ progenitor cells. Conclusions STAT3 participates in the signal transduction pathways activated by VEGF specifically mediated by VEGFR2 /KDR in human hemopoietic progenitor cells, and the aforementioned signaling pathway participated in the interaction of ovarian carcinoma cells and progenitor cells.