| Purpose To study the influence of ephrin-B2on human vein endothelial cells(HUVECs) at proteomic level, and explore their biological functions and the main signaling pathways that participated in and the possible mechanism of action. And then to explore the influence of ephrin-B2on the proliferation, migration, cell cycle of HUVECs, and the expression change of part proteins that involved in proliferation and survival. All results corroborated the proteomics research. Our research provides a new train of thought to explore the influence of ephrin-B2on the biological functions and signaling pathways of HUVECs. And also provides scientific basis to explore the importance of ephrin-B2in myocardial ischemia and become therapeutic candidate of angiogenesis research.Methods Using enzyme digestion rubbing method to extract the original generation of HUVECs and develop and extend. Using SILAC isotope technology for cultivating HUVECs labeling. Treated marked HUVECs with1μg/mL ephrin-B2/Fc or PBS for15hours respectively, and then1:1mixed protein which was extracted from treatment group and control group. Using liquid chromatography mass spectrometry detection technology to detect the change of the expression of protein in HUVECs. After that, IPA software was used for bioinformatics analysis, analysing the canonical signaling pathways which involved in, networks, and predicting its downstream effects; Using Western Blot detecting the expression changes of proliferation and survival related proteins; In order to observe its regulation of angiogenic on endothelial cells, we used CCK-8to measure the proliferation of HUVECs in given time points (1,3,5,7d) at different concentrations of ephrin-B2/Fc (0,0.01,0.03,0.1,0.3,1μg/mL)(n=6/group) and used flow cytometry technique to observe the effects of ephrin-B2/Fc on the cell cycle of endothelial cells. And then using transwell and scratch test to measure the migration of HUVECs at different concentrations of ephrin-B2/Fc (0,0.1,0.3,1,3μg/mL). Results According to the results of proteomic analysis, ephrin-B2/Fc significantly increased the expression of HUVECs’cytoskeleton proteins and activated multiple signaling pathways which were related to cell proliferation, survival, and migration. And results predicted it reduced the apoptosis and autophagy of cells, and promoted proliferation, migration and movement of cells. The analysis showed that ephrin-B2/Fc promoted the proliferation of HUVECs in a concentration dependence. When the concentration of ephrin-B2/Fc was between Oμg/mL and1μg/mL, the migration of HUVECs was concentration dependent, but when the concentration is3μg/mL, the migration promotion effect disappeared. Flow cytometry results showed that in1μg/mL ephrin-B2/Fc treatment group, the ratio of cells in GO/1phase decreased significantly (P<0.05) and the proportion of S phase increased (P<0.05). According to the results of Western Blot, ephrin-B2/Fc has promoted the expression of Bcl-2, c-Myc, and NPM which involved in cell proliferation and survival. And the expression of apoptosis related protein BAX was also raisedConclusion Ephrin-B2participated in cell survival, proliferation, migration and so on, and raised the expression of cytoskeleton proteins, proliferation and survival related proteins and activated multiple signaling pathways that involved in cell proliferation and migration. Ephrin-B2prompted cells which were in G0/G1phase enter into S phase. Certain concentration range of ephrin-B2/Fc promoted the proliferation and migration of human umbilical vein endothelial cells. But its effect on promoting migration disappeared when exceeded a certain concentration. |
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