The Study On Orientiatedly Incubating And Differentiating Human Marrow Mesenchymal Stem Cells Into Vascular Endothelium-like Cell

Objective To study the feasibility for inducing to differentiate mesenchymal stem cells(MSCs)into vascular endothelial-like cells which were isolated and purified in vitro, to observe the induced vascular endothelial-like cells whether to participate into wound surface angiopoiesis through animal experiment .Methods The purified bone marrow mesenchymal stem cells with 2 ml were obtained from patients without the severe diseases in clinical examination after Ficoll density gradient centrifugation and monolayer cultured of the collected bone marrow aspiration, these cells were cultured and passaged in vitro. They were observed with inverted phase contrast microscope for morphological observation and were identified by the flow cytometry,scanning electron microscope,transmission electron microscope,immunocytochemistry methods . We used L4(23)orthogonal experimental design to bolt induction factors vascular endothelial growth factor (VEGF) and basic fibroblast growth factor(bFGF), and incubated MSCs into vascular endothelial-like cells according to the bolting results . The morphological observation of induced cells were observed with inverted phase contrast microscope ,the induced cells were identified by scanning electron microscope,transmission electron microscope methods,the immunocytochemistry methods. Tissue engineering dermis surrogate was constructed with collagen protein sponge frame and vascular endothelial-like cells which were marked by 5-bromodeoxyuridine . The full-thickness skin defected wound surface in rabbit back were cured by the tissue engineering dermis surrogate. After 14 days, we used wound surface tissue skin to do the immunohistochemistry staining.Results The appearance of MSCs which were obtained by Ficoll density gradient centrifugation and monolayer cultured is fusiform , the short pachy microvilli of cell surface and cell matrix were saw by scanning electron microscope. Two architectures were corresponded to the different cell's function states by transmission electron microscope, flow cytometry showed that 85.35 percent of cells were found at G0,G1 phase. Expression of CD29,CD44 were positive on MSCs surface and CD34,CD45,â…§factor were negative . Compare theâ…§factor positive expression rate, VEGF induced group,bFGF induced group and VEGF+ bFGF induced group have disparity to MSCs group(P<0.05) , theâ…§factor positive expression rates of VEGF+ bFGF induced group are the highest in four groups(P<0.01). The appearance of MSCs induced by VEGF+ bFGF was oblate shape. We could find many the short fusiform and oblate shape cells in the induction medium. The Weibel-Palade body which were characteristics of vascular endothelium-like cells were found in differentiated cells by transmission electron microscope , microvilli and bridge pattern were found by scanning electron microscope , expression of CD34,â…§factor were positive in the induced cells surface by the bolted induction factors; Many differentiated cells were adhered in the tissue engineering dermis surrogate surface, which were observed by scanning electron microscope. The 5-BrdU positive cells located around the micrangium by the immunohistochemistry staining after 14 days .Conclusion The mesenchymal stem cells which were obtained by Ficoll density gradient centrifugation and monolayer cultured have the biology characteristics of stem cell in vitro. Both VEGF and bFGF can induce MSCs differentiate into vascular endothelial-like cells in vitro. The induction effects of VEGF were more obvious than those of bFGF . The induction effects which combined VEGF with bFGF were better than the single factor which was used. The appearance of vascular endothelial-like cells which were obtained through induction were familiar with the vascular endothelium cells in body, these vascular endothelial-like cells could also participate into the forming of micrangium.