PTH调节COX-2/PGE2-血管-骨偶联的促骨合成新机制研究

Osteoporosis in China has been reported poorly controlled with highly incidence of fracture due to the bone loss, which probably closely related with angiogenesis defect in bone and the vascular endothelial progenitor cells decrease and disfunction. Our previous study indicated that COX-2 expression was reduced by 65% to 75% in fracture bone from aged mice compared with young mice. Local administration of an EP4 agonist CP73 of the receptor of PGE2 to the fracture repair site in aged mice enhanced the rate of bone formation to levels observed in young mice. However, COX-2-/- mice had fracture healing phenotypes that are similar to aged mice, showing delayed initiation and impaired bone repair accompanied by slower rate of chondrogenesis, chondrocyte maturation, and severe angiogenesis deficiency and reduced bone formation. The EP4 agonist markedly improved the impaired healing in COX-2-/- mice by restoration of bony callus formation and an approximately 70% improvement of angiogenesis in the COX-2-/- bone callus, suggesting that the expression of COX-2 regulates critical subsequent events including osteogenesis and angiogenesis. We also found that PTH induced mesenchymal stem cells differentiating into vascular endothelial cells and promoted endothelial cells maturation in vitro, and enhanced vascular formation in callus in vivo, especially small vessel formation, which indicated that PTH can promote angiogenesis. In addition, PTH administrated intermittently increased bone mass and trabecular bone number and trabecular bone thickness with enhanced expression of COX-2 in both trabecular bone and mesenchyme of bone tissue. PTH is the unique bone anabolic drug at present; however the mechanisms to promote osteogenesis are still unclear. We will confirm the novel mechanisms of PTH enhancing osteogenesis based on angiogenesis through COX-2/PGE2 pathway in vitro and in vivo on this study, which will be of great importance to promote the rational use of bone anabolic drugs and to develop targeted medicine. So our aims are to determine, (1) whether the quantity and function of endothelial progenitor cells in peripheral blood in elderly are correlated with osteoporosis and fragility fracture; (2)The regulation of PTH on endothelial progenitor cells, especially on its paracrine function, can stimulate the differentiation of mesenchymal stem cells through COX-2/PGE2 pathway; (3)The mechanisms of PTH induced osteogenesis mediated by bone angiogenesis.

老年性骨质疏松及骨折发病率高、危害性大，目前防治效果不佳。申请者研究组前期研究发现骨组织血管生成减少与骨合成降低关系密切，在老年人骨质疏松发病中意义独特，COX-2/PGE2轴介导骨血管生成-骨合成偶联。我们还发现甲状旁腺素（PTH）在体外体内均有效促进骨血管生成，并可能涉及COX-2/PGE2轴。PTH是目前唯一促骨合成药物，但其促骨合成机制仍未充分阐明，我们拟通过体外和体内实验证实PTH调节COX-2/PGE2轴促骨血管生成作用是其促骨合成的新机制，这一机制的阐明将对临床合理使用促骨合成药物及开发其他靶向药物有重要意义。我们的研究内容：（1）老年人外周血内皮祖细胞数量功能异常与骨质疏松及骨折的相关性；（2）PTH通过调节内皮祖细胞旁分泌功能影响COX-2/PGE2对骨髓间质干细胞诱导分化的作用及其机制；（3）PTH促骨合成作用是否通过其促血管生成介导，两者之间的关联机制。

老年骨质疏松及骨折发病率高、危害性大，目前防治效果不佳。申请人前期研究发现骨血管生成减少与骨合成降低关系密切，在老年人骨质疏松发病中意义独特，甲状旁腺素（PTH）可能促进骨血管生成，并与其促骨形成作用相关，但仍不确切。通过本研究我们发现：（1）与非骨质疏松老人相比，脆性骨折老人外周血及骨髓中内皮祖细胞数量明显降低，细胞迁移率和小管形成功能明显降低，内皮祖细胞数量与股骨颈和全髋的骨密度呈正相关，与成骨功能、血管内皮功能呈正相关。在体外，成骨前体细胞和血管内皮细胞共培养产生对话促进彼此的成熟分化和活性；这部分结果证明，骨组织的血管发生与骨发生相互调节，血管异常直接影响骨质量。（2）血管内皮细胞可检测到PTH受体PTH1R，人重组PTH1-34（10-7M）促进内皮细胞的成熟分化、促进迁移率和小管形成，抑制其凋亡老化；但PTH1-34对内皮细胞COX-2/PGE2信号通路的作用仍不明确，故其作用机制还有待于进一步深入研究。在动物实验，间歇性人重组PTH1-34干预（40ug/kg/d）有效促进骨折部位骨痂毛细血管的形成，加速骨折愈合并改善骨痂骨密度和力学强度，PTH还具有促进外源性间充质干细胞向骨痂部位迁移募集的作用，从而与间充质干细胞协同促进骨折愈合提高骨质量，提示PTH1-34临床上可用于骨折不愈合、延迟愈合、骨不连接的患者，还可用于降低老年人骨、关节手术后因骨质量下降引起的手术失败率、返修率和假体松动率。（3） 为了深入研究PTH促进血管生成的机制，构建了Tie-2 Cre ERT2和PTH1R Loxp/Loxp转基因小鼠，并繁育得到组织特异性受体敲除的Tie-2 PTH1R-/-小鼠，完善了课题组动物实验平台，为后续表型鉴定和功能机制研究创造了条件。（4）比较骨质疏松和非骨质疏松老人骨髓间充质干细胞基因表达谱，通过二代测序建立了老年人间充质干细胞非编码RNA表达库，统计分析发现31个非编码RNA有显著性差异，扩大样本量验证后得到显著差异的6个microRNA和14个piwiRNA，对其进行初步功能研究发现某microRNA家族可明显促进间充质干细胞向成骨分化，初步机制研究提示其参与BMP2/wnt-b-catenin信号通路调节。