瞬时受体电位通道M7对人牙髓干细胞成牙本质向分化的作用及其分子机制研究

The odontoblast differentiation of DPSCs is the key to the formation of dental pulp dentin complex, however, the mechanism is not yet clear. Our previous study confirmed that TRPM7 participates in the process of odontogenesis of DPSCs. The biological software indicated that miR-30a-5p might be the upstream regulatory factor of TRPM7. Inhibiting the expression of TRPM7, the RNA-seq showed that the expression of Wnt5a was down-regulated. The previous study suggested that miR-30a-5p and Wnt5a may be upstream and downstream regulatory factors of TRPM7. In order to further investigate the molecular mechanism of TRPM7 in odontogenesis, our group intends to further investigate the role of miR-30-5p-TRPM7-Wnt5a in the process of odontoblast differentiation by using animal experiments, lentivirus transfection, luciferase assay reporter, qRT-RCR and western blot. This study is aimed to clarify the possible specific signaling pathways of TRPM7 and find a potential target for intervention in odontoblast differentiation. It will provide new ideas for the construction of dental pulp dentin complex.

DPSCs成牙本质向分化过程是牙髓牙本质复合体形成的关键，然而分化机制尚未明确。本课题组前期研究证实TRPM7参与DPSCs成牙本质向分化过程。生物学软件预测miR-30a-5p可能作为TRPM7上游调控因子，抑制TRPM7表达，RNA-seq结果表明Wnt5a表达随之下调；提示miR-30a-5p和Wnt5a分别可能是TRPM7上游和下游调控因子。为深入探讨TRPM7调控DPSCs成牙本质向分化分子机制，本课题组拟采用动物实验、慢病毒转染，荧光素报告基因、qRT-PCR、Western blot等方法进一步探讨miR-30-5p-TRPM7-Wnt5a信号通路在调控DPSCs成牙本质向分化的作用，旨在阐明TRPM7对人DPSCs成牙本质向分化作用可能存在的具体信号通路，为寻找干预成牙本质分化作用寻找潜在靶点。本课题将为DPSCs构建牙髓牙本质复合体提供新的思路。

目的:课题组前期发现TRPM7可促进人牙髓干细胞成牙本质向分化，为探究其上下游分子机制，本研究探讨miR-22-3p/TRPM7通过PI3K/AKT/mTOR通路对人牙髓干细胞成牙本质向分化的影响。.方法:利用生物信息学软件和转录组测序结果预测TRPM7的靶向miRNA。通过双荧光素酶报告基因验证两者之间的相互作用，miR-22-3p的表达分别上调和下调。活/死染色分析细胞活性。采用QPCR、Western Blot、茜素红染色等方法验证TRPM7基因对人牙髓干细胞成牙分化的调控作用。通过下调miR-22-3p或抑制TRPM7的表达来验证PI3K/AKT/mTOR的表达变化。通过裸鼠体内皮下移植证实miR-22-3p/TRPM7对牙髓牙本质复合体形成的影响。.结果:miR-22-3p特异性靶向TRPM7;miR-22-3p antagomir转染牙髓干细胞增强增殖;qPCR、WesternBlot和茜素红染色结果显示， TRPM7和矿化相关基因表达增加，矿化结节数显著高于对照组(P<0.05)。下调miR-22-3p，抑制TRPM7的表达，可以调控PI3K/AKT/mTOR的表达。体内实验证实，下调miR-22-3p可促进牙髓牙本质复合体的形成，抑制TRPM7表达可抑制牙髓牙本质复合体的形成。.结论:miR-22-3p/TRPM7通过PI3K/AKT/mTOR通路促进人牙髓干细胞的成牙本质向分化。