Inducing Mouse Embryonic Stem Cell To Vascular Endothelial Cell In Vitro By TGF-β₁ And VEGF

Objective:To observe the competence of embryonic stem cell directonal induced to vascular endothelial cell in vitro in different condition.Method: Adult mice provided by animal department of Medical College of NanChang University were pregnant by wild pairing. We used the 12.5～14.5ds'foetus mice to manufacture mouce embryonic fibroblast feeder layer. Embryonic stem cell was amplificated on the fibroblast feeder layer.The fine growth condition embryonic stem cells amplificated on the fibroblast feeder layer were trypsinized,then briefly blowed to become single cell suspension.We suspended the single ESCs in ESCs culture solution without leukaemia inhibitory factor.Cells were cultivated in hanging drops(1×104/ml) on the cover of cell culture dishs and cultured for 3 day.After 3 days,cells turned into embryoid bodies.Then chose the fine embryoid bodies for further study.The experiment is divided into three groups:induced by transforming growth factor-β1,induced by vascular endothelial cell growth factor and induced by combination of the tow growth factors.Result:①ES cells could grow, proliferate, and form colonies during in vitro culture in the presence of MEF with LIF . When mitomycin C-treated MEF was used as the feeder cells for co-culture with ES-cells, the ES cells could form nested colonies with clear edge and smooth surface as well as close arrangement within the colony. No sign of differentiation was seen in the culture system.②After 2 days,embryoid bodies of the 3 groups were all briefly spreaded and epithelioid cell appeared around them. Pebble appearance cells could be seen around them after 3-4 days.The pebble appearance cells then formed tubiform structure that grew radiat from the embryoid bodies after 6 days. Induced tow weeks approximately,endothelial cell could be seen in the there groups and blood vessel appearance construction could be seen emerged around the embryoid bodies.The most number of embryonic bodies around them emerging blood vessel appearance construction occured in the third group.③Pebble appearance cells of the three groups turned into Fusiform shape .Cells RNA was extracted and then analysised through RT-PCR, three specific amplification straps could be seen. Compared with the strap of DNAmark,position of the three gene expression amplification parts were matched.The amplificated cells were stained with immunohistochemistry,Ⅷfactor antibody response was positive.Conclusion:①The culture system of MEF with LIF could be used to probote the growth and proliferation of ES cells,maintain their undifferentiation and pluripotentiality. It will benefit to the maintain their undifferentiation application of ES cells in next study.②TGF-β1 and VEGF could be used to induce embryonic stem cell to eodothelial cell.③the combination use of the tow growth factors maybe raise the induction efficency.