脐静脉内皮细胞与牙本质浸提液诱导的牙髓干细胞共培养成血管的研究

目的:探讨共培养牙本质浸提液(dentin extract, DE)诱导的人牙髓干细胞(human dental pulp stem cells, hDPSCs)和人脐静脉血管内皮细胞(human umbilical vein endothelial cells, HUVECs)在成血管方向的潜能。方法:制备人牙本质浸提液,分别诱导培养hDPSCs 7 d和14 d后,Real-time PCR和Western blot检测诱导后hDPSCs中牙本质涎蛋白(DSP)、牙本质涎磷蛋白(DSPP)和牙本质基质蛋白1(DMP-1)的表达;血管形成实验共分为5组,分别是:HUVECs组、DE诱导后的hDPSCs组以及按5∶1、5∶5、5∶10比例共培养HUVECs和诱导后hDPSCs组,分别在第3、6、9h检测各组小管形成的相对长度和节点数目。结果:DE诱导的hDPSCs 在mRNA和蛋白水平上表达的DSP、DSPP和DMP-1显著提高(P＜0.05);体外小管形成实验结果示共培养组较早形成稳定的网状结构,在3 h时,HUVEc与诱导后hDPSCs比例为5∶1组形成的小管相对长度和相对分支点数明显高于HUVECs组(P＜0.05),而5∶5组和5∶10组低于HUVECs组(P＜0.05)。结论:牙本质浸提液诱导后的hDPSCs与HUVECs按一定比例共培养可促进血管结构的形成。

Angiogenic Potentials of Coculture of Human Umbilical Vein Endothelial Cells and Dentin Extract-induced Human Dental Pulp Stem Cells.

Objective: To observe the angiogenic potential of coculture of dental pulp stem cells (hDPSCs) induced by dentin extract (DE) and human umbilical vein endothelial cells (HUVECs) during dental pulp regeneration. Methods: hDPSCs were induced by DE for 7 and 14 days. Real-time PCR and Western-blot assay were used to detect the expression of DSPP, DMP-1 and DSP in hDPSCs. Angiogenic assays were divided into five groups: HUVECs, induced hDPSCs and HUVECs cocultured with induced hDPSCs in the ratios of 5:1, 5:5 and 5:10. The tubular length and branching point number were detected after 3,6 and 9 h after the cells were seeded on the matrigel. Results: The expressions of DSPP, DMP-1 and DSP in induced group were up-regulated. Angiogenic assay showed that vessel-like structures formed more than other groups when HUVECs: induced hDPSCs cocultured ratio was at 5:1 at 3h (P＜0.05). While vessel-like structures in 5:5 and 5:10 coculture groups were less than those in HUVECs group at all the time points (P＜0.05). Conclusion: Coculture of hDPSCs induced by DE and HUVECs in a certain proportion can enhance the angiogenic potential of dental pulp tissues.