肌切蛋白Adseverin介导细胞骨架修饰和胞外基质分泌调控促进牙本质形成的研究

Extracellular matrix (ECM) secretion is the key of reparative dentine formation.Previous studies have shown that adseverin is an actin-severing and -capping protein that is primarily expressed in secretory cells, where it regulates the filamentous actin cytoskeleton during cell exocytosis. Our pilot study revealed that adseverin was intensely expressed in secretory odontoblasts in tandem with the commencement of predentin during tooth development, and adseverin silenced odontoblast-like cells displayed round shape without any cellular processes where ECM secretion. Based on these work, the current project will clarify the molecular links between cytoskeletal reorganization and ECM secretion(Type I collagen and matrix vesicles) in vivo and in vitro by adseverin silence or overexpression. Furthermore, signaling pathways that involved in matrix vesicles secretion will be explored by comparative proteomics .All these information will enrich the research field of dentin formation via targeting on cytoskeleton reorganization and ECM secretion , which may supply new ways to study the mechanism of dentin repairing and to develop new pulp capping agents in future.

胞外基质分泌是修复性牙本质形成的关键。研究显示，肌切蛋白Adseverin可通过解聚丝状肌动蛋白改建细胞形态，参与调控细胞分泌。申请人前期研究发现，Adseverin特异性表达于大鼠牙胚分泌性成牙本质细胞层，瞬时沉默Adseverin可导致成牙本质样细胞变圆，基质分泌关键位点-细胞突起消失，提示其可能与成牙本质细胞胞外基质分泌密切相关。本课题拟建立Adseverin稳定沉默及过表达的hDPC-TERT模型，利用纳米颗粒跟踪分析、激光共聚焦显微镜、透射电镜等技术，通过体内外实验阐明Adseverin对成牙本质样细胞骨架及胞外基质（I型胶原及基质小泡）分泌的影响；借助比较蛋白质组学技术，筛选差异蛋白，进一步探索Adseverin调控基质小泡形成的关键信号通路。本课题的开展，将从细胞骨架及胞外基质分泌水平寻找调节牙本质形成的关键因子，为牙本质修复的机制研究及新一代盖髓药物的研制提供新的思路。

胞外基质（Extracellular matrix,ECM ）尤其是基质小泡（Matrix vesicles, MVs）的分泌是牙髓修复再生的关键。本项目以细胞骨架调节为切入点，首先通过破坏肌动蛋白F-actin稳态，明确其对牙髓细胞成牙本质向分化及基质小泡MVs分泌的影响，进而深入探讨肌切蛋白Adseverin对成牙本质细胞分化及MVs分泌的调控作用及相关机制。实验结果表明，Adseverin对人牙髓细胞HDPCs和鼠成牙本质细胞MDPC-23的增殖及迁移作用相似，但对细胞矿化发生及MVs分泌则具有截然相反的调控作用，推测细胞的固有形态的改建可能影响其分化方向。差异蛋白质组学分析亦表明，结构蛋白、离子通道蛋白、钙离子结合蛋白及骨架相关蛋白为主要的差异蛋白，参与蛋白酶通道，核糖体通道及钙离子信号通道及代谢信号通道，该研究为后续进一步探索MVs分泌调控密切相关蛋白提供了理论依据。本项目的实施，有助于从细胞骨架改建角度探索促进牙髓修复再生的人工干预新方式。