Approach To Influence Of VEGF On The Endothelialisation Of Dendritic Cells

BackgroundDendritic cells (DC) are the best professional antigen presenting cells (APC) known so far, which are uniquely able to activate a naive T-cell response. They play the central role in initiating, regulating and maintaining immune responses. The T-cell mediated immune reaction actived by dendritic cells play a key role in anti-tumor immune responses. The defection on numbers and functions of DCs in tumor-bearing mice and in cancer patients is the main reason for the tumor cells to escape initial immune recognition. Therefore, the biological function of DCs is closely connected with the specific anti-tumor response and plays a key role in it.Vascular endothelial growth factor (VEGF) is one of the most potent direct-acting angiogenic molecules secreted by tumor cells, which is a highly effective specific mitogen for endothelial cells. It plays a key role on tumor angiogenesis and metastasis. Reports have proved that VEGF from tumor cells could inhibit the activation of NF-κB , then affected the differentiation development and function of DCs in tumor bearing host. The inhibition could induce abnormality of the host's immune system, and promote the tumor growth.A recent research reported a novel leukocyte subset within ovarian carcinoma that coexpressed endothelial and dendritic cell markers. It has been proved that murine bone marrow-derived DCs could undergo endothelialisation in vitro after incubation in tumor-conditioned medium containing high levels of VEGF favours. These results suggest that VEGF may contribute to the endothelialisation of DCs in tumor microenvironment. It indicates that by promoting the endothelialisation of DCs, the tumor might not only ensure a sufficient blood supply but also prevent these DCs from initiating an immune response against tumor antigens. In this case, therapeutic targeting of this pathway could both reduce tumour growth and promote immune attack. There are many reports about the use of intratumoural DC inoculation as a means for tumor vaccination in the world, but research on endothelialisation of DCs is still limited. The effects of this phenomenon also need further investigation.Experiment aimThe purposes of our study is to incubate human monocyte-derived DCs under the induction of VEGF, and observe the degree of endothelialisation.MethodsPrecursors of DC were separated from peripheral blood monouclear cells (PBMC) divided from healthy volunteers' peripheral blood by Ficoll density gradient centrifuge. After 3 hours incubation, remove non-adherent cells. The left precursors were cultured at 3×10⁶/ml with RPMI1640 medium containing GM-CSF, IL-4. And different concentration rhVEGF(10ng/ml, 100ng/ml) were added to DCs on the 3_rd,7_th days of culture. Observed the appearance character of DCs with microscope during the culture process. Normal control DCs without induction of VEGF were analysed the phenotype by Flow cytometer at l_st, 3_rd, 7_th, 10_th, 14_th days of culture. The experimental group cells were collected after 7 days induction, then we analysed the expression of CDla,CD34,CD31 by Flow cytometer , and detected the expression of vWF by immunocytochemistry and immunofluorescence . All data were expressed as mean value±S.D., and analyzed by statistical software SPSS12.0. P-value of < 0.05 was considered to be statistically significant.Results1. The Morphologic changes of normal control cells were coincide with the typicalDCs' reported. The experimental cells' growth speed was slower than the control cells', and the cell density was lower.2. The expression of CD34 and CD31 in normal DCs reduced gradually, and the expression of CD1a elevated along with the culture time lengthening. The expression of CD1a was suppressed in experimental cells induced 7days by VEGF. The coexpression of endothelial and dendritic cell markers was higher than normal control cells'.3. Immunocytochemistry and immunofluorescence results suggested the expression of vWF in experimental cells induced by VEGF, and the Aera% , IOD of experimental group cells were higher than normal control cells'.ConclusionDCs derived from human monocyte could appear endothelialisation in vitro under the induction of VEGF, and in certain ranges showing a dose-dependent.