压力环境下双调蛋白在人牙髓干细胞定向分化中的作用及其信号转导机制的研究

Mechanical pressure is often one of factors for tooth injury. However, the molecular mechanism of mechanical pressure during the process of dental pulp repair is not clear. Differentiation of Dental pulp stem cells (DPSCs) into odontoblast-like cell (OBLCs) is a key step in the recovery of the injured pulp. Our results showed that the expression of AREG is apparently upregulated, AREG siRNA inhibits the role of pressure stimulation in differentiation of DPSC regulated by pressure, which indicated that AREG was maybe involved in the differentiation of DPSCs regulated by pressure stimulation. The present project will try to establish DPSCs cell lines of gene overexpression or gene silencing of AREG, to investigate the role of pressure signaling and AREG in directional differentiation of DPSC, to ascertain the effect of AREG on directional differentiation of DPSC induced by pressure stimulation, to determine the interaction between transcriptional factors and downstream target genes for AREG under pressure stimulation, to detect the binding site of transcription factors in downstream target genes promoter. The aim of this project is to elucidate the role of AREG and underlying molecular mechanism in directional differentiation of DPSC into OBLC regulated by pressure stimulation. The study would be significant for elucidation the molecular mechanism of the directional differentiation of DPSCs into OBLC during the repair of the injured pulp. It also provides a new idea for the clinical conservative treatment of vital pulp and use of DPSC in tissue engineering in future.

压力是临床牙齿损伤常见因素之一，但压力下牙髓损伤修复机制仍不清楚。牙髓干细胞（DPSCs）定向分化为成牙本质细胞样细胞是牙髓损伤修复中的关键环节。我们预实验发现压力显著上调DPSCs双调蛋白 (AREG)表达，其siRNA抑制压力调控DPSCs分化，提示双调蛋白可能在压力环境下牙髓损伤修复中发挥重要作用。国内外关于AREG在DPSCs的作用尚未见报道。本课题拟构建AREG过表达和沉默的DPSCs系，先分别研究压力和AREG调控DPSCs分化及其信号通路；然后研究AREG在压力调控DPSC定向分化中的作用及相应信号通路，进一步通过免疫共沉淀和EMSA确定压力环境下AREG细胞内信号在下游靶基因的结合位点和相应转录因子。旨在阐明AREG在压力调控DPSCs分化中的作用及分子机制，对揭示压力环境下牙髓损伤修复的分子机制有重要意义，为将来临床牙髓保存治疗和DPSCs组织工程应用提供新思路。

压力是临床牙齿损伤常见因素之一，但压力下牙髓损伤修复机制仍不清楚。阐明力学刺激对人牙髓干细胞（human dental pulp stem cells,hDPSCs）分化的调控及其细胞内分子机制对进一步理解牙髓损伤修复有重要意义。课题组前期研究发现流体静水压对hDPSCs内AREG的表达有显著影响，后者反馈调控压力调控hDPSCs分化。本课题旨在阐明在压力环境下，AREG对牙髓损伤修复的影响及其分子机制。.本项目首先筛选了调控hDPSCs增殖和分化适宜的静水压力，结果显示30-120kPa是较适宜的静水压力,120Kpa促进细胞增殖和分化能力最强，150Kpa抑制细胞增殖。然后以碱性磷酸酶染色、茜素红染色、慢病毒转染、Western blot等观察流体静水压下hDPSCs 向 hOBLC 分化及AREG参与情况，以流式细胞术检测AREG对hDPSCs凋亡的影响；结果显示压力调控hDPSCs 向 hOBLC 分化，Intergrin、CAMII、Erk MAPK和PI3k参与压力调控hDPSCs，而GSK、p38 MAPK和JUNK MAPK信号通路不参与。通过构建AREG沉默或过表达的hDPSCs系，从体内和体外实验研究了AREG 调控hDPSCs分化及其细胞内信号途径；结果发现0.01-0.1ug/ml AREG促进hDPSCs分化，而0.1ug/ml AREG体内和体外实验均证实可促进hDPSCs细胞分化，进一步研究发现 AREG调控hDPSCs分化的细胞内信号途径是ERK MAPK、JUNK MAPK和PI3K，p38信号通路不参与。研究发现当AREG和压力共同作用hDPSCs时，AREG拮抗压力诱导的hDPSCs分化。观察了AREG对LPS调控hDPSCs分化和自噬的作用，结果发现AREG参与LPS调控hDPSCs分化和hDPSCs自噬。 . 本项目结果表明流体静水压调控hDPSCs分化和AREG表达，而AREG也调控hDPSCs分化，在压力和AREG调控hDPSCs分化分化过程中MAPK和PI3K信号通路都参与，但是两者共同作用时存在一定的拮抗作用。本项目由利于将来寻找合适的信号通路抑制剂调控压力引起的牙髓损伤修复，为牙髓保存治疗提供实验依据。