A Research About Iducing Effect Of Indirect Co-culture With C6Glioma Cells In Differentiation Of Rat Bone Marrow Mesenchymal Stem Cells Into Endothelial-Like Cells In Vitro

ObjectiveThe present study aims to study the differentiation of bone mesenchymal stem cells (BMSCs) impact by tumor micro-environment, clear the effects of bone marrow mesenchymal stem cell in the tumor microenvironment is affected by the cytokines, signal molecules and their differentiation, discusses indirect co-culture with glioma cells induced bone marrow mesenchymal stem cells to endothelial cell differentiation, thus further reveal the mechanism of malignant glioma angiogenesis source of endothelial cells and malignant glioma cells secrete factors on the influence of mesenchymal stem cells into endothelial cells, tumor local microcirculation rich set up mode of related theory.MethodsTake2-3weeks old SD rats, through the whole bone marrow adherence method to obtain the primary BMSCs were amplified, purified, draw the growth curve of the third and fourth generation BMSCs MTT method. Cells were observed by inverted phase contrast microscope morphology by optical microscopy, and cell phenotype was identified by flow cytometry BMSCs cell surface antigen CD44, CD90, CD34and CD45. C6glioma cells have the same culture medium with BMSCs. BMCS induced by C6glioma cells experiments, according to the different induction time were divided into3groups:(1)7d co-culture group:7d induced by C6glioma cells were inoculated with density2×105/hole in Transwell cell layer, BMSCs, the density of2×105/inoculation of fourth holes of the lower7d Transwell in the control group; the upper and lower chamber with density of2×105/hole with fourth generation BMSCs, BMSCs were seeded on coverslips preset lower six-well culture plate, every3days for a liquid.(2)14d co-culture group:14d induced by C6glioma cells were inoculated with density1×105/hole in Transwell cell layer, generation of BMSCs with density inoculated fourth1×105/hole layer;14d group Transwell chamber upper lower in density with fourth1×105/hole BMSCs, BMSCs in the preset coverslips six-well culture plate in the lower layer, every3days for a liquid.(3)21d co-culture group:21d induced by C6glioma cells were inoculated with density5×104/hole in Transwell cell layer, generation of BMSCs with density inoculated fourth5×10/hole layer;21d group Transwell chamber upper lower in density with fourth5×104/hole BMSCs, BMSCs in the preset coverslips six-well culture plate in the lower layer, every3days for a liquid. Co-culture after remove slides, co-culture with immunohistochemical assay of BMSCs von Willebrand factor (von Willebrand factor, vWF), endothelial cell specific marker expression of antigen CD31.Results1. The morphologic feature showed that the rat BMSCs had the general appearance of stem ceils, larger nuclei and minor plasm.The obtained rat BMSCs appear spindle-shape or platypelloid shape, but when they are enhanced to cover the entire bottle wall, they show swirl and radiated arrangement.The Cell growth curve were representative. The cell surface antigen CD44positive identification (the average positive rate98.4%), CD90positive (positive rate98.7%) on average, CD34negative (positive rate2.5%) on average, CD45negative average positive rate (4.5%).2. Indirect with C6glioma cells after cocultivation, BMSCs differentiation into endothelial cells, and immunohistochemical testing results show that the contact between trained to C6glioma cells, with the increase of induction time, BMSCs vWF and CD31expression increased, BMSCs morphology changes, differentiation into endothelial cells.Conclusions1. The rat BMSCs can be obtained easily through using passage adherence screening method in vitro, and the BMSCs appear spindle-shape or platypelloid shape. The Cell growth curve were representative.2. Indirect co-culture with C6glioma cells can induce BMSCs to differentiate into endothelial cells, the performance of the BMSCs increase the expression of endothelial cell specific protein vWF and CD31expression, and changes in cell morphology.