The Study Of Endothelial-like Differentiation Tendency Of Dendritic Cells In The Supernatant Of Sw620

BackgroundDendritic cells (DCs) are the best professional antigen presenting cells (APC) known so far, which are obtained further understanding and attention, also play a key role in the body immune response as a cell group in recent years. DCs not only are uniquely able to activate a naive T-cell response, but also can produce stimulation molecules that make dormat T cells to enter cell cycles and stimulate their differentiation. However, most studies have shown that both the phenotype and presenting function of tumor infiltrating dendritic cells(TIDC) decreased. The tumor tissue secretes kinds of immune factors, which can inhibit maturation of DCs, such as IL-10, VEGF or IL-6 and M-CSF, and so on. Therefore, the biological function of DCs is closely connected with the specific anti-tumor response and plays a key role in it.Vessel formation is an important hallmark of rapidly growing tumours. Classically, vascularization has been considered to occur through the processes of angiogenesis, which involves outgrowth of new endothelial cells from mature preexisting endothelial cells. More recent studies have suggested that vasculogenesis, which occurs moreprominently during embryonic development, can contribute to vessel formation in adults. Vasculogenesis is the development of vessels from precursor cells that migrate to the site of vascularization. Vasculogenesis may also occur concomitantly with angiogenesis. Recent studies have shown that the tumor vasculogenesis relates to bFGF, VEGF, HGF and so on.On 2004 year, Coukos G proved a novel leukocyte subset within ovarian carcinoma of rice and human that coexpressed endothelial and dendritic cell markers in Nature Medicine. Until recently, some researchers reported additional incubation of tumour-associated dendritic cells (TADC) with pro-angiogenic factors, such as vascular endothelial growth factor and oncostatin M, led to transdifferentiation into endothelial-like cells. These studies indicates DCs were inhibited in anti tumor immunity and some DCs differentiate endothelial-like cells in tumor microenvironment. These endothelial-like cells maybe partake tumor vessel formation and conduce to tumour growth. There are many reports about the use of presenting antigens function of DCs how to against tumor growth in the world, but research on endothelialisation of DCs is still limited. The effects of this phenomenon and mechanisms also need further investigation.Experiment aimTo study the influence of microenvironment created by dissoluble cytokines derived from colon carcinoma cells SW620 on the differentiations of different stages dendritic cells (DCs) from peripheral blood monouclear cell (PBMC), and observe the degree of endothelialisation.Methods1. The supernatant of SW620 was prepared.2. Precursors 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 (100 ng/ml), IL-4(5 ng/ml). In addition, the supernatant of SW620 cells were added to DCs on the 2^nd, 7^th days of culture in experimental group. And half changed the medium every other day and every group is cultured to continue 7 days. The DCs (DCs cultured with normo-medium) and experimental dendritic cells (DCs cultured with supernatant of SW620) were collected at 10^th, 14^th for further study.3. Observed the appearance character of DCs with microscope during the culture process. The collected cells were analysed the phenotype as CD86, CD1a, CD11c by Flow cytometer, and detected the expression of vWF by immunofluorescence . At last, we detected the expression of gene CD1a, vWF and CD144 of every group cells with RT-PCR. Viewed every group cells form network-like structures on fibronectin-coated flasks with microscope.4. Cultured masculine groups of HUVEC with trypsase and ECM.All data were expressed as mean value±S.D., and analyzed by statistical software SPSS12.0. P< 0.05 was considered to be statistically significant.Results1. The Morphologic changes of normal control cells were coincide with the typical DCs' reported. The 2d experimental cells' growth speed was slower than the control cells', and the cell density was lower. However, there was no significant difference between 7d groups and control groups.2. Compared with the normal control groups cells, the expression of CD86, CD1a and CD11c in 2d immature DCs were reduced gradually by the tumor-conditioned medium, and there was statistics disparation between 2d groups and control groups. Nevertheless, 7d groups and its control groups were resemble.3. Immunofluorescence results suggested the expression of vWF in 2d experimental cells induced by the tumor-conditioned medium, and 7d experimental cells were absent, compared to the normal control cells.4. In RT-PCR analysis, only the CD1a-positive fraction showed in the normal control groups. a clear vWF and CD144 fraction were expressed in 2d DCs, and CD1a was lost. But respective gene fractions of 7d DCs were similar to that of control DCs.5. The 2d groups changed their morphology into spindle-like cells and formed network-like structures as well as HUVEC. There were missing in the network-like structures of normal control groups and 7d groups. Conclusion1. The supernatant of SW620 can inhibit the growth of DCs and its expression of relative antige, which may be one of the mechanisms on tumors immune escape.2. Under the induction of the supernatant of SW620, the expression of CD144 and vWF are increased in immature DCs, compared with that in mature DCs. Meanwhile, the network-like structures of endotheliocyte is liable to form in immature DCs.