根尖牙乳头干细胞迁移介导的年轻恒牙牙髓再生研究

The loss of pulp tissue may lead to the loss of teeth, and may compromise the quality of life. How to regenerate dental pulp tissue is the main concern of current endodontic research. Existing effort in dental pulp regeneration has focused on cell transplantation. And this method needs excessive ex vivo manipulation, such as cells isolation, expansion, sorting, purification and storage processes, which are both time and energy-consuming. Direct cell homing of stem cells from dental apical papilla (SCAP) with the induction of chemotaxis may simplify the process of dental pulp regeneration. .The hypothesis of this study is that SCAP are target cells of stromal cells derived factor-1 (SDF-1). The expression level of SDF-1 receptor CXCR4 in SCAP on gene and protein level will be determined. By using uninfected and infected root canal models, migration of SCAP will be induced by SDF-1 to regenerate dental pulp tissue. Based on this, the global gene expression profile of regenerated and normal dental pulp tissue will be compared. Cells from the regenerated pulp tissue will be isolated to analyze the expression of stem cells markers and the multi-lineage differentiation ability, to further explore the characteristics and origins of regenerated pulp tissue. And the results may provide evidence for dental pulp regeneration in future clinical applications.

牙髓组织的丧失可导致牙齿的丧失，甚至影响生活质量。如何再生牙髓组织一直是学者们追求的方向。目前的研究主要集中在细胞移植介导的牙髓再生上，但是该方法需要体外扩增、筛选、纯化等操作，耗费大量的时间和精力。利用牙齿自身的根尖牙乳头干细胞在趋化因子作用下迁移至根管内，发生牙髓再生，将大大简化细胞移植介导的牙髓再生步骤。本研究拟从蛋白水平和基因水平考察犬根尖牙乳头干细胞SDF-1受体CXCR4的表达，确定根尖牙乳头干细胞是SDF-1的靶细胞；运用体外迁移实验明确根尖牙乳头干细胞在SDF-1趋化因子作用下的迁移运动；进一步利用体内实验在非感染根管、感染根管模型，运用SDF-1诱导根尖牙乳头干细胞迁移，形成牙髓再生。在此基础上，本研究将比较再生牙髓和正常牙髓的基因表达谱，分离培养再生牙髓组织内的细胞，分析其干细胞表面标志、多向分化等特性，深入探究再生组织的特性和来源，为牙髓再生的临床应用提供依据。

利用年轻恒牙自身的根尖牙乳头干细胞（SCAP）在趋化因子的作用下迁移至根管内，从而介导牙髓再生，将大大简化细胞移植介导的牙髓再生步骤。本研究分别从基因和蛋白水平考察了根尖牙乳头组织和SCAP中，基质细胞衍生因子1α（SDF-1α）的受体CXC趋化因子受体4（CXCR4）的表达，结果表明根尖牙乳头组织和SCAP均具有CXCR4表达，其中CXCR4主要表达在SCAP细胞内（57.49%-98.48%, n=4），仅有少数细胞表面可见CXCR4的表达（0.3%-2.34%, n=4）。SDF-1α浓度梯度可显著促进细胞表面CXCR4的表达。提示SCAP是SDF-1α的靶细胞。同时，研究采用体外迁移实验证实SDF-1α可显著促进SCAP的迁移，而加入抗CXCR4抗体，则可抑制该作用，且SDF-1α对SCAP的增殖并无显著影响，证实SCAP跨膜迁移确实为SDF-1α／CXCR4轴所介导。并且，三维胶原迁移实验表明SDF-1α可显著增加SCAP在胶原内的纵向迁移深度。在此基础上，课题组建立比格犬年轻恒牙的非感染根管和感染根管模型，运用SDF-1α诱导自体SCAP迁移，目前已获得在非感染根管中SDF-1α组牙根可正常发育的初步证据。为进一步探索SDF-1α/CXCR4轴介导SCAP发生迁移的机制，本研究考察了PI3K和PKC信号通路的参与可能性。结果表明SDF-1α可促进PI3K信号通路蛋白p85和PKC信号通路蛋白PKC的磷酸化，促进粘着斑相关成分（FAK、Paxillin和Vinculin）的磷酸化，促进粘着斑形成，细胞应力纤维重组，从而促进细胞迁移。而阻断PI3K和PKC信号通路则可抑制以上SDF-1α刺激所引发的变化，从而抑制SCAP迁移。综上，本研究结果为明确SDF-1α／CXCR4轴对SCAP的迁移作用及机制提供了重要证据，提示可进一步利用SDF-1α介导CXCR4阳性表达的SCAP迁移至根管内，从而实现内源性细胞迁移介导的牙髓再生。