p75神经营养因子对牙周膜干细胞在牙周组织缺损再生修复中的影响研究

PDLSCs plays an critical role during periodontal defects regeneration and repair process, but the complexity and heterogeneity of periodontal tissue remain to be bottleneck of applying PDLSCs to periodontal tissue engineering. p75NTR is the important cell surface marke to sorted pure cranial neural crest originated MSCs by flow cytometer. Previous results showed that using p75NTR could select the EMSCs of homogeneous morphology and favorable proliferation and differentiation capacity, its also positively regulated mineralized differentiation in MSCs. This study is to 1. recruit gene chip for detecting the biological characteristics difference among p75NTR+, p75NTR- and unsorted PDLSCs selected by flow cytometer. 2.Determine the intervention effects and underlying mechanism of regulateing p75NTR in PDLSCs. 3. Establish the animal periodontal defects model, transplant the before and after p75NTR sorting and regulating PDLSCs to periodontal defects,observe the regeneration and repair difference in periodontal tissue to provide pure and distinct biological characteristics seed cells and lay the experimental foundation and theoretical basis for periodontal tissue engineering.

PDLSCs在牙周缺损再生修复过程中起重要作用，但牙周膜组织结构的复杂性和异质性仍是PDLSCs应用到牙周组织工程发展的瓶颈。p75NTR是颅神经嵴源性MSCs的重要表面标志物，可以通过流式分选纯化细胞，筛选相对单纯MSCs。前期研究表明，利用p75NTR可以筛选出形态均一，增殖分化能力良好的外胚间充质干细胞，其能正向调控细胞的矿化分化能力。本项目（1）利用p75NTR流式筛选PDLSCs，对比p75NTR+、p75NTR-及未筛选PDLSCs生物学特性，基因芯片检测三组细胞内在差异并探讨可能存在的机制；（2）调控p75NTR，以明确p75NTR对PDLSCs生物学特性的干预作用及相关机制；（3）建立牙周缺损动物模型，异体移植p75NTR分选及调控前后的PDLSCs，观察其对牙周组织再生修复差异，为牙周组织工程提供单纯及生物学特性优良的种子细胞奠定理论依据及实验基础。

完成了体外细胞模型PDLSCs的构建，在这基础上进行了PDLSCs的流式分选和鉴定，从形态学、mRNA及蛋白水平观察对比p75NTR+ PDLSCs、p75NTR- PDLSCs以及未分选的PDLSCs细胞生物学特性相关基因，并初步探讨各组细胞生物学特性差异，评价各指标的体外稳定以及各组细胞差异存在的分子机制；之后激活外源重组NGF，人为调控p75NTR的表达，检测p75NTR上调、p75NTR下调以及对照组的PDLSCs间充质干细胞体外扩增速度、细胞增殖活性、细胞凋亡情况、细胞表型，从形态学、mRNA及蛋白水平观察对比各组PDLSCs生物学特性相关基因，进而验证p75NTR人为干预后对PDLSCs生物学特性的影响。此外，本课题组在主要项目的基础上进行了横向延伸，选用p75NTR基因敲除(p75NTR−/−)小鼠和野生型(WT)仔鼠相对比，研究了P75NTR对小鼠牙槽骨发育的调节作用及对小鼠外胚间充质干细胞矿化潜能的影响，还试图探索与PI3K/akt/β-catenin通路相关的潜在机制。完成了体外细胞模型p75+EMSCs的构建，并探讨Mage-D1在p75+EMSCs体外矿化过程中的调控作用与机制研究，从牙周组织工程三要素出发，分别进行筛选优化种子细胞、构建支架材料及挑选生物活性因子的相关研究。最后，研究团队研究了糖尿病伴牙周炎的机制及治疗，进一步探讨了2型糖尿病伴牙周炎患者的人牙周膜干细胞成牙骨质分化能力的变化。