组蛋白去乙酰化酶HDAC6通过调节自噬参与成牙本质细胞分化

Our previous studies showed that autophagy played important role in odontoblast survival and differentiation with inflammatory stimulation. Histone deacetylase 6 (HDAC6), a multifunctional cytoplasmic deacetylase, regulates actin and microtubule network to control autophagy. Our pilot experiment found HDAC6 and autophagy markers expressed during odontoblast, and they showed similar expression pattern. In this project, we explore the role of HDAC6 in regulating autophagy during odontoblast differentiation and the mechanisms. We first detect the expression of HDAC6, autophagy and odontoblast differentiation markers, analyze the relationship between HDAC6 and autophagy during odontoblast differentiation. To explore how HDAC6 regulate autophagy, the markers of autophagosome and lysosome are analyzed to observe the process of autophagy. Moreover, autophagy flux is detected with mRFP-GFP-LC3 plasmid, after inhibiting HDAC6 with tubacin or siRNA. Finally, the mechanisms of HDAC6 regulates autophagy will be studied: The substrates of HDAC6 (cortactin and tubulin) are analyzed in vitro and in vivo, after HDAC6 is inhibited or knockout. Discuss the role of HDAC6 in regulating F-actin and microtubule to control the fusion of autophagosome and lysosome to effect autophagy process. In summary, this project is helpful to elucidate the mechanism of odontoblast differentiation.

本课题组前期研究发现自噬在成牙本质细胞分化及炎性损伤修复中发挥重要作用。HDAC6定位于细胞质，对肌动蛋白、微管蛋白具有调控作用，从而与自噬密切相关。我们预实验发现成牙本质细胞分化过程中HDAC6和自噬有相似表达模式。本项目探讨HDAC6通过调节自噬参与成牙本质细胞分化，并深入研究其作用机制。利用成牙本质细胞分化模型，检测HDAC6、自噬表达，抑制剂或siRNA抑制HDAC6，检测自噬指标的改变，进行HDAC6与自噬的相关性分析。抑制／过表达HDAC6后，检测自噬体(LC3)与溶酶体(LAMP2)的改变，并使用mRFP-GFP-LC3报告质粒检测自噬流，分析HDAC6对自噬体与溶酶体融合过程的调节作用。进一步检测HDAC6底物Cortactin、Tubulin等的表达，分析HDAC6通过对肌动蛋白网和微管蛋白作用来调节自噬过程。该项目有利于加深自噬对成牙本质细胞分化调节机制的理解。

本课题组前期研究发现自噬在成牙本质细胞分化及炎性损伤修复中发挥重要作用。HDAC6定位于细胞质，对肌动蛋白、微管蛋白具有调控作用，从而与自噬密切相关。我们对HDAC6在成牙本质细胞分化过程进行了功能研究， 体外成牙本质细胞向分化及体内牙冠成牙本质细胞分化过程伴随着HDAC6表达的增高。而抑制HDAC6导致成牙本质细胞向分化能力的减弱。证明HDAC6通过调节自噬流参与成牙本质细胞分化过程。抑制HDAC6会阻断成牙本质细胞样细胞自噬流的进行，导致自噬体的聚集。揭示了HDAC6作为胞质中的去乙酰化酶，对细胞骨架tubulin进行去乙酰化，介导自噬元件的运输并与溶酶体融合。总的来说，我们揭示了HDAC6如何调控自噬过程调节成牙本质细胞的分化,加深了自噬在成牙本质细胞分化过程中的机制的理解。