炎性牙周膜干细胞多向分化的ERK/Wnt信号通路调控机制研究

Periodontal ligament stem cells (PDLSCs) are an ideal cell source for periodontal regeneration, while their normal commitment to wanted tissue is pivotal to the final tissue regeneration result. We previously have found that there are stem cells existing in the inflammatory periodontal ligment, named as inflammatory periodontal ligament stem cells (I-PDLSCs), which show similar characteristics to PDLSCs but lower osteogenic differentiation potential. It's reported that the stem cells isolated from other inflammatory tissue show higher angiogenic differentiation potential, hinting that the inflammatory conditon may enhance the angiogenic differentiation potential of I-PDLSCs and thereby down-regulating their osteogenic differentiation potential. Hence, we suggest that the abnormal differentiation potential of I-PDLSCs may be related to the vascular endothelial growth factor (VEGF) in the inflammatory condition. VEGF may enhance the angiogenic differentiation ability of I-PDLSCs through ERK, and simultaneously it can enhance Wnt/β-catenin through ERK thereby inhibiting their osteogenic differentiation. In this study, cellular and molecular biological experiments will be conducted to compare the differentiation potential of PDLSCs isolated from normal and inflammatory tissues and uncover the mechanism underlying the attenuated osteogenic differentiation potential of I-PDLSCs, laying down basis for regulating the differentiation of I-PDLSCs for future clinical application.

牙周膜干细胞（PDLSC）是牙周组织再生的理想来源，而其正常分化是保证组织再生效果的前提。我们前期研究发现炎症牙周膜中也有干细胞存在（I-PDLSC），其基本特性均与PDLSC相似，但成骨分化能力降低。文献报道其他炎症环境来源干细胞具有成血管分化能力增强的特点，提示是否因炎症环境使I-PDLSC的成血管方向分化增强而影响其成骨分化。由此我们提出I-PDLSC分化方向能力的异常与炎症环境中血管内皮生长因子（VEGF）有关，并提出VEGF通过上调ERK促进了I-PDLSC成血管分化能力，同时ERK又通过上调Wnt通路中关键信号B-catenin的表达而下调了I-PDLSC成骨分化能力的信号机制。本课题拟采用细胞和分子生物学技术比较不同来源PDLSC的多向分化能力，探明I-PDLSC成骨分化能力减弱的影响因素及调控机制，为调控I-PDLSC的正常分化并应用于临床治疗提供分子基础。

我们前期研究发现炎症环境中PDLSC成骨分化能力降低。提出炎症环境干细胞（P-PDLSC）分化能力的异常与炎症环境中血管内皮向分化可能相关。.观察了慢性牙周炎时牙周组织中血管化表达变化、TNF-α与SDF-1α/CXCR4轴对牙周膜干细胞内皮向分化的影响；证明了PDLSC的成血管过程受到ERK通路的调节；自行构建壳聚糖温敏凝胶与异种骨及合成材料的复合支架；并体内外比较了载antimiR-138的壳聚糖/三聚磷酸钠/透明质酸纳米粒的理化性能、生物学性能、对大鼠颅骨极限缺损及犬的牙周骨下袋缺损模型的修复效果。.结果显示CD31、VEGF、SDF-1、MVD等血管向分化指标在炎症浸润部位表达明显增强。P-PDLSCs阳性表达VEGF、CD31、VE-cadherin、vWF强度更高。Matrigel assay的分支节点数、管腔长度均高于H-PDLSCs。SDF-1α诱导后，P-PDLSCs的CD31、VWF mRNA表达水平明显升高；Matrigel assay管腔形成数量明显增多。TNF-α刺激下H-PDLSC中SDF-1α及CXCR4表达与患者来源的P-PDLSC表达趋势相同；CD31、VWF、KDR表达水平升高； 而加入CXCR4抑制剂后再经诱导，CD31、VWF、KDR mRNA表达水平明显降低。.PDLSC在内皮分化诱导过程中，存在ERK1/2通路的磷酸化水平升高，阻断ERK1/2磷酸化过程后，再行内皮分化诱导， CD31、VE-cadherin、VEGF水平及细胞表面阳性表达比例均明显降低；TNF-α刺激条件下，PDLSC的CD31、VE-cadherin、VEGF水平及细胞表面阳性表达比例明显升高；阻断ERK1/2磷酸化后，TNF-α刺激后PDLSC的CD31、VE-cadherin、VEGF水平及细胞表面阳性表达比例明显下降。.制备的纳米级CTH NPs理化性能稳定，对相应miRNA的包封及抗酶解能力明显提高；对MSCs转染效率稳定；无细胞毒性，细胞迁移能力正常；能有效控释SDF-1α；有明确的成骨基因（RUNX2、COL-1、OPN及OCN）高水平表达；体外矿化结节形成能力提高。.SD大鼠颅骨极限缺损，分组植入复合材料，实验组的修复效果均高于单纯材料组； Beagle犬下颌一、二、三壁骨缺损，实验组明显比对照组具有更多的新生骨、新生牙骨质、新