The proangiogenic effects of extracellular vesicles secreted by dental pulp stem cells derived from periodontally compromised teeth
The proangiogenic effects of extracellular vesicles secreted by dental pulp stem cells derived from periodontally compromised teeth
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牙周受损牙齿牙髓干细胞分泌的细胞外囊泡的促血管生成作用
DOI：
10.1186/s13287-020-01614-w
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发表时间：
2020-03-06
影响因子：
7.5
通讯作者：
Chen, Fa-Ming
中科院分区：
文献类型：
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作者：
Zhou, Huan;Li, Xuan;Chen, Fa-Ming
背景虽然从牙周受损牙齿（P-DPSC）中分离的牙髓干细胞（DPSC）已被证明保留多能性和再生潜力，但它们作为治疗剂的用途仍然很大程度上尚未被探索。在这项研究中，我们使用体外和体内测试模型研究了 P-DPSC 分泌的细胞外囊泡 (EV) 的促血管生成作用。方法 使用源自牙周健康牙齿的患者匹配 DPSC（H-DPSC）作为 P-DPSC 的对照。制备源自 H-DPSC 和 P-DPSC（H-CM 和 P-CM）的条件培养基（CM）、源自用 EV 分泌阻滞剂 GW4869 预处理的两种细胞类型的 CM（H-GW 和 P-GW）以及 H-DPSC 和 P-DPSC 分泌的 EV（H-EV 和 P-EV），以测试它们对内皮细胞（EC）的促血管生成作用。分别使用细胞计数试剂盒-8 (CCK-8)、transwell/划痕伤口愈合和基质胶测定评估细胞增殖、迁移和管形成。具体来说，使用定量逆转录酶-聚合酶链反应（qRT-PCR）和蛋白质印迹分析来检查EC中血管生成相关基因/蛋白质对基于EV的孵育的反应的表达水平。最后，应用全层皮肤缺损模型来测试EV对伤口愈合和新血管形成的影响。结果 H-CM和P-CM均促进EC血管生成，但当EC在H-GW和P-GW中孵育时，促血管生成作用受到损害，其中EV分泌被GW4869预处理阻断。在基于 EV 的孵化中，虽然发现 H-EV 和 P-EV 都能增强 EC 的血管生成相关活性，但 P-EV 在刺激 EC 增殖、迁移和管形成方面发挥了更强大的潜力。此外，与 H-EV 相比，P-EV 导致 EC 中血管生成相关基因/蛋白的表达水平更高。同样，P-EV 和 H-EV 都被发现可以加速小鼠伤口愈合并促进皮肤缺损处的血管形成，但用 P-EV 治疗的伤口可以实现更快的愈合结果并增强新血管的形成。结论 本研究的结果提供了额外的证据，证明源自牙周病牙齿的 P-DPSC 是研究和治疗用途的潜在细胞来源。特别是，P-EV 的促血管生成作用表明 P-DPSC 可用于促进细胞治疗和再生医学中的新血管形成。
Background Although dental pulp stem cells (DPSCs) isolated from periodontally compromised teeth (P-DPSCs) have been demonstrated to retain pluripotency and regenerative potential, their use as therapeutics remains largely unexplored. In this study, we investigated the proangiogenic effects of extracellular vesicles (EVs) secreted by P-DPSCs using in vitro and in vivo testing models. Methods Patient-matched DPSCs derived from periodontally healthy teeth (H-DPSCs) were used as the control for P-DPSCs. Conditioned media (CMs) derived from H-DPSCs and P-DPSCs (H-CM and P-CM), CMs derived from both cell types pretreated with the EV secretion blocker GW4869 (H-GW and P-GW), and EVs secreted by H-DPSCs and P-DPSCs (H-EVs and P-EVs) were prepared to test their proangiogenic effects on endothelial cells (ECs). Cell proliferation, migration, and tube formation were assessed using the Cell Counting Kit-8 (CCK-8), transwell/scratch wound healing, and Matrigel assays, respectively. Specifically, quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and western blot analysis were used to examine the expression levels of angiogenesis-related genes/proteins in ECs in response to EV-based incubation. Finally, a full-thickness skin defect model was applied to test the effects of EVs on wound healing and new vessel formation. Results Both H-CM and P-CM promoted EC angiogenesis, but the proangiogenic effects were compromised when ECs were incubated in H-GW and P-GW, wherein the EV secretion was blocked by pretreatment with GW4869. In EV-based incubations, although both H-EVs and P-EVs were found to enhance the angiogenesis-related activities of ECs, P-EVs exerted a more robust potential to stimulate EC proliferation, migration, and tube formation. In addition, P-EVs led to higher expression levels of angiogenesis-related genes/proteins in ECs than H-EVs. Similarly, both P-EVs and H-EVs were found to accelerate wound healing and promote vascularization across skin defects in mice, but wounds treated with P-EVs resulted in a quicker healing outcome and enhanced new vessel formation. Conclusions The findings of the present study provide additional evidence that P-DPSCs derived from periodontally diseased teeth represent a potential source of cells for research and therapeutic use. Particularly, the proangiogenic effects of P-EVs suggest that P-DPSCs may be used to promote new vessel formation in cellular therapy and regenerative medicine.