β-catenin“核浆穿梭效应”在牙髓干细胞多种应激防御修复反应中的作用及机制研究

Mineralization is the most common way of repair after dental pulp stem cells (DPSCs) stimulated. The forms of mineralization covers not only reparative dentin secreted in injury sites but calcification formed in the pulp cavity and root canals. The mechanisms of repair modes behind facade of mineralization remain unclear. Our previous study showed RhoA/ROCK activation or inhibition can regulate DPSC shift from stemness state to differentiation, and also appear diverse repair modes. The latest reports showed location and transformation of β-catenin in the cell membrane, cytoplasm and nucleus (nuclear-plasma shuttle effect) controlled the state of stemness and differentiation. The applicant hereby puts forward a new hypothesis that nuclear-plasma shuttle of β-catenin regulates a single mechanism of DPSC various defence and repair modes involved in RhoA/ROCK. The project applys a combination of molecular biological technique and immunohistochemistry, to study (1) The concrete manifestation of β-catenin nuclear-plasma shuttle when RhoA/ROCK activation or inhibition; (2) Effects of β-catenin nuclear-plasma shuttle in DPSC shift from state of stemness to differentiation; (3) The characteristics of diverse repair modes induced by β-catenin nuclear-plasma shuttle. The project contributes to clarify molecular mechanisms of in-situ repair and ectopic calcification, and to provide a new approach for exploring therapeutic schedule or agents of pulp protection.

牙髓干细胞受刺激后最常见的修复方式是矿化，表现为受损处的修复性牙本质或髓腔根管内的钙化等，矿化表象下多种修复方式的机制仍不清楚。课题组初步发现RhoA/ROCK通路活化/抑制时，可调控牙髓干细胞干性向分化转变并呈现不同修复模式，最新研究指出β-catenin核浆穿梭效应，即β-catenin在胞膜、胞浆、胞核的不同定位和转换可决定干细胞干性及分化状态。据此申请者提出β-catenin核浆穿梭效应是RhoA/ROCK调控牙髓干细胞多种应激防御修复反应单一机制的假说，联合使用分生和组化等技术研究（1）RhoA/ROCK通路活化/抑制时β-catenin核浆穿梭效应的特点（2）β-catenin核浆穿梭对牙髓干细胞干性-分化转换的调控效应（3）β-catenin核浆穿梭诱导牙髓干细胞多种应激防御修复的特点。为阐明牙髓应激防御中原位修复/错位钙化发生的分子机制，探索新的保髓治疗方案/试剂提供途径。

牙髓干细胞受刺激后最常见的修复方式是矿化，表现为受损处的修复性牙本质或髓腔根管内的钙化等，矿化表象下多种修复方式的机制仍不清楚。课题组立足前期研究基础及国内外最新研究进展，提出β-catenin核浆穿梭效是RhoA/ROCK调控牙髓干细胞多种应激防御修复反应机制的假说，本项目联合使用分子生物学、组织化学技术从体内、外研究发现（1）牙髓受损时，RhoA/ROCK通路可调控牙髓干细胞形成修复性牙本质及异位钙化，且LPA具有较好地促修复性牙本质形成的作用；（2）β-catenin核浆穿梭参与调控牙髓干细胞迁移与分化，且在调控牙髓干细胞迁移与分化过程中，Oct4、N-cadherin与β-catenin存在相互作用（3）β-catenin介导RhoA/ROCK调控牙髓干细胞的成牙本质向分化，但是其介导RhoA活化与ROCK抑制调控牙髓干细胞的分化中具有不同的作用机制。（4）线粒体氧化磷酸化途径激活对牙髓干细胞成牙本质向分化起正向调控作用，提示我们在研究牙髓损伤修复机制时应兼顾考虑能量代谢的调控作用，只有兼顾宏观及微观层面的调控机制才能促进细胞达到组织学上的精确修复。本研究阐明了牙髓应激防御过程中存在原位修复及错位钙化，洞悉牙髓应激防御修复反应的机制能够为避免异位钙化发生，促进修复性牙本质及结构规则的牙髓-牙本质复合体再生提供理论依据。