EphrinB2对TNF-α作用下的脐静脉内皮细胞与牙髓干细胞血管生成的影响

The effects of EphrinB2 on the angiogenesis of umbilical vein endothelial cells and dental pulp stem cells induced by TNF-α

目的:研究EphrinB2对TNF-α作用下的人脐静脉内皮细胞(HUVECs)与牙髓干细胞(hDPSCs)共培养体系体外成血管的影响。方法:按1∶5比例共培养hDPSCs与HUVECs,TNF-α(0~80 ng/mL)处理共培养细胞,检测EphrinbB2蛋白表达水平。用特异性沉默EphrinB2基因的慢病毒载体转导hDPSCs,经不同浓度TNF-α处理后,在共培养第9、12小时检测小管形成情况及EphrinB2通路蛋白表达。结果:Western blot结果显示20 ng/mL的TNF-α显著促进共培养体系EphrinB2的表达(P<0.001)。体外小管形成实验结果显示在第12小时,HUVECs+EphrinB2-shRNA-hDPSCs组小管相对分支点数较少(P<0.01),相对长度较短(P<0.01)。Western blot结果显示EphrinB2和ephb4表达在TNF-α+HUVECs+hDPSCs组最高(P<0.01),而在HUVECs+EphrinB2-shRNA-hDPSCs组表达量最低(P<0.01)。结论:TNF-α可以通过激活EphrinB2/ephb4通路,逆转沉默hDPSCs的EphrinB2引起的小管裂解,从而促进小管形成。

Objective:To study the effects of EphrinB2 on the angiogenesis of human umbilical vein endothelial cells(HUVECs)and human dental pulp stem cells(hDPSCs)induced by TNF-αin vitro.Methods:hDPSCs and HUVECs were co-cultured in vitro at a ratio of 1∶5.The cells were treated with TNF-α(0-80 ng/mL),hDPSCs was infected with lentivirus vector specific to silencing EphrinB2 gene.The cells were treated with and without TNF-αrespectively.Matrigel assay were performed on the co-culture system to detect tubule formation.Relative EphrinB2 expression was measured by Western blot.Results:Western blot showed that TNF-αat 20 ng/mL significantly promoted the expression of EphrinB2 in co-culture system(P<0.001).At the 12th hour of treatment,the number of relative branches of tubules in HUVECs+EphrinB2-shRNA-hDPSCs group was less than that in the other 2 groups(P<0.01),and the relative length was shorter than that in the other 2 groups(P<0.01).Western blot results showed that the expression of EphrinB2 and ephb4 in the TNF-α+HUVECs+hDPSCs group was higher than that in the TNF-α+HUVECs+EphrinB2-shRNA-hDPSCs group(P<0.01),while the expression of EphrinB2 and ephb4 was the lowest in HUVECs+EphrinB2-shRNA-hDPSCs group(P<0.01).Conclusion:TNF-αcan reverse the tubular cleavage induced by silencing EphrinB2 of hDPSCs,and promote the tubular formation.