EphrinB2和VEGF协同调控牙髓干细胞促进血管新生的实验研究

Rapid and overwhelming blood supply is of paramount importance in the survival and integration of high volume engineered tissues following in vivo implantation. The root canal space is encapsulated by dentin in the center of each tooth, with only a single blood supply from its apical end. This anatomical limitation poses even more difficulties for a speedy interconnection with the host vascular system. Therefore, it is absolutely fundamental to develop tissue engineering approaches aimed at accelerating blood vessel network formation and re-connectivity with the host tissues. Our previous study showed that dental pulp stem cells (DPSCs) enhanced tube formation when co-cultured with human umbilical vein endothelial cells (HUVECs), and led to stabilization of the preexisting vessel-like structures formed by HUVECs. It also increased the longevity of these structures. Our further study found that stem cells from apical papilla (SCAPs) increased secretion of hypoxia-inducible-factor-α (HIF-α), VEGF and ephrinB2 under hypoxic condition, which in turn significantly induced more tube formation in co-culture scenario of SCAPs and HUVECs, as they may occur in the implanted prevascularized tissue construct. SCAPs have similar characteristics to DPSCs for its stemness. These findings indicate that co-culture of DPSCs or SCAP swith ECs is a promising strategy for regenerative endodontics. It is rational to hypothesize that VEGF and ephrinB2 cooperatively regulate interactions between DPSCs and HUVECs, by stimulating endothelial cells proliferation, migration and assembly into vascular structures; and in another aspect, inducing DPSCs to differentiate into pericytes to stabilize the newly-formed vasculature. The objectives of the present proposal are to explore the role of VEGF and ephrinB2 in the crosstalk between DPSCs and HUVECs; to study the correlation of ephrinB2 expression and phosphorylation with angiogenesis; and to investigate the cooperative effect of VEGF and ephrinB2 on newly-formed vasculature and the detailed vascular structures. This study for the first time will unravel the role of ephrinB2 and VEGF in angiogenesis and in vivo anastomosis; The new knowledge will bring much needed insight into dentine/pulp regeneration and provide a molecular basis for pharmaceutical therapies acting on ephrinB2 and VEGF signaling, so as to orchestrate pericytes recruitment and assembly for dentine-pulp regeneration.

牙髓再生最简便的方法是将体外工程化的牙髓组织植入根管内，以期获得功能性的牙髓牙本质复合体。然而，植入根管内的组织只能通过根尖孔获得血液供应，远离根尖孔的组织会发生缺氧性坏死。因此，获得快速、充分的血液供应，是牙髓再生的关键。我们的研究已表明，牙髓干细胞能促进和稳定内皮细胞形成的血管网；在缺氧条件下上调VEGF和ephrinB2表达，进一步促进新生血管网的形成。因此，我们推测，牙髓干细胞与血管内皮细胞可能通过ephrinB2和VEGF的协同作用，促进内皮细胞增殖、移动和伸展，形成初级血管网，同时诱导牙髓干细胞分化形成周细胞，使新生血管成熟。我们将探讨牙髓干细胞与血管内皮细胞的相互作用；研究ephrinB2激活与血管新生的关系；探索VEGF和ephrinB2协同作用对血管形成和血管结构的影响，以及可能的调控信号。本项目如获得成功，将对牙髓根尖周病、以及其他缺血性疾病的治疗有明显的指导意义。

获得快速、充分的血液供应是牙髓再生的关键，旨在提高移植工程化牙髓组织的存活率以及与宿主整合。我们的实验研究已表明，体外及体内共培养牙髓干细胞DPSCs和人脐静脉内皮细胞HUVECs可以促进血管生成。然而迄今为止，调控DPSCs和HUVECs相互作用的血管生成机制尚不清楚。近来我们发现，人牙乳头干细胞SCAP在低氧条件下上调HIF, VEGF和EphrinB2基因及蛋白的表达。据文献报道，Ephrin2配体同其受体EphB4对血管发育和重构起关键调节作用。在本课题研究中，我们旨在探究EphrinB2/EphB4信号通路调控DPSCs 和HUVECs相互作用形成类血管结构的分子机制。以两个3-D共培养模型- Matrigel成血管实验和Fibrin gel磁珠实验为基础，我们发现EphrinB2/EphB4 信号通路在调控DPSCs和HUVECs形成血管状结构中起着至关重要作用。一方面，当二者共培养在Matrigel上时，EphrinB2/EphB4信号通路被激活，并且p-EphrinB2与p-EphB4磷酸化水平随着类血管结构的形成而变化。当加入EphB4受体抑制剂或者用shRNA下调EphrinB2或EphB4在DPSCs中的表达时，DPSCs的迁移能力减弱，其辅助HUVECs形成血管网的能力也显著降低。运用免疫缺陷小鼠模型进行体内实验结果显示，DPSCs-HUVECs 共培养组比单独培养组的类血管结构明显增多，然而EphB4受体抑制剂的加入显著地减少了血管网结构的生成以及CD31的表达。另一方面，Fibrin gel实验结果表明DPSCs促进并稳定HUVECs出芽式血管生成，并同时表达壁细胞相关的基因蛋白。一旦EphrinB2/EphB4通路被药物抑制或者EphrinB2及EphB4的表达被下调时，血管芽和血管网结构的生成也被显著抑制。此外，我们发现重组蛋白EphrinB2-Fc和EphB4-Fc刺激DPSCs导致VEGF分泌增多，从而加强了血管生成。本项目研究首次揭示EphrinB2/EphB4和VEGF 信号通路在牙髓干细胞和内皮细胞介导的成血管过程中的重要作用。本项目的实验结果将对牙髓根尖周病、以及其他缺血性疾病的治疗有明显的指导意义。