Stathmin在人牙髓干细胞成牙本质向分化过程中的作用及分子机制研究

The formation of pulp-dentin complex of dental pulp stem cells is an important foundation for tooth regeneration, however,the molecule mechanism of odontoblast differentiation of dental pulp stem cells is remain unclear.Stathmin is an important microtublue-associated protein which is realted to mineralization of bone. It was blotted from normal and cariou dental pulp stem cells by proteomics, Human and rat osteoblast-like express stathmin and that this protein could play a role in regulation of the growth of these cells in response to various hormonal stimuli. There was report suggest that stathmin, which alters microtubule dynamics, plays an essential role in maintenance of postnatal bone mass by regulating both osteoblast and osteoclast functions in bone.However,there was no report about the relationship between Stathmin and the odontoblast differentiation of dental pulp stem cells. Our previous results indicated that the expression of Stathmin in normal dental pulp stem cells is higher than that in carious dental pulp stem cells, and the ability of mineralization and expression of ostoegenesis-related gene of dental pulp stem cells down-regulated after silencing the expression of Stathmin. This study was futher to identify the effect of Stathmin on the proliferation and odontoblast differentiation ability of dental pulp stem cells; to explore if Stathmin regulate odontoblast differentiation of dental pulp stem cells by Shh signaling pathway; to the protein interaction with Stathmin by Y2H. The study is very important to identify the relationship between Stathmin and dental pulp stem cells and possible molecule mechanisms.

牙髓干细胞成牙本质向分化是牙齿再生的关键环节，然而分化机制目前尚未明确。我们已经证明深龋牙髓干细胞较正常牙髓干细胞具有较强的增殖与骨向分化能力，为寻找正常和深龋牙髓干细胞生物学特性差异的原因，并深入探讨牙髓干细胞成牙本质向分化的机制。本课题组利用荧光双向凝胶电泳从正常和深龋牙髓干细胞筛选并鉴定出18个差异表达蛋白，其中Stathmin作为微管蛋白与骨矿化密切相关。进一步实验揭示:上调Stathmin可促进牙髓干细胞成牙本质向分化，下调Stathmin可抑制牙髓干细胞成牙本质向分化。由此，本课题拟进一步采用病毒转染、Co-IP、酵母双杂交等技术，旨在阐明Stathmin对牙髓干细胞生物学特性的影响，探讨Stathmin调控牙髓干细胞成牙本质向分化的分子机制，筛选并鉴定与Stathmin相互作用的蛋白。本课题将为探究牙髓干细胞成牙本质向分化机制提供新的思路,为构建组织工程牙齿提供新的理论依据。

微管蛋白 Stathmin 是本研究团前期筛选出的在正常和深龋牙髓干细胞中表达存在差异的蛋白，与骨组织 矿化关系密切。Sonic hedgehog(Shh)作为调节牙齿发育的经典信号通路，在牙髓干细胞成牙本质向分化及增殖中发挥重要作用。目前尚未有研究报道 Stathmin 在人牙髓干细胞增殖与成牙本质向分化中发挥的作用及作用机制，将沉默Stathmin组的牙髓干细胞进行矿化诱导，14d后矿化结节生成能力与ALP、BSP、OCN、DSPP mRNA表达量较Stathmin- Ctrl组明显降低;CCK8检测Stathmin-shRNA组细胞增殖能力较Stathmin-Ctrl组明显降低。Stathmin-shRNA组Shh及其下游信号通路分子PTCH1、SMO、GLI1 mRNA与蛋白表达量较Stathmin-Ctrl组明显降低。在Stathmin-shRNA组人牙髓干 细胞中加入激活剂purmorphamine后激活Shh信号通路,Realtime PCR、Western blot检测shRNA-Stathmin+PM组PTCH1、SMO、GLI1 表达量较shRNA –Stathmin 组显著增加。shRNA-Stathmin+PM组矿化诱导14d后矿化结节生成能力与ALP、 BSP、OCN、DSPP mRNA表达量较shRNA-Stathmin组明显升高;CCK8检测 shRNA-Stathmin+PM组细胞增殖能力较Stathmin-shRNA组明显增加。将沉默Stathmin的牙髓干细胞和对照组进行转录组测序分析，共筛选出3214个基因，其中1644个下调，1570个基因上调，并对其中感兴趣的基因进行PCR验证，结果与测序结果一致。本课题为探讨Stathmin调控牙髓干细胞分化及分子机制提供了新的思路。