EphrinB2/EphB4信号介导的促红细胞生成素对骨重塑的作用及其机理研究

We know that interaction between ephrinB2 and EphB4 can regulate differentiation and function of osteoblast and osteoclast through forward and reverse signaling. We previously found that erythropoietin could affect the expression of some key molecules during the differentiation of osteoclast and osteoblast, with the change of expression of ephrinB2 and EphB4 on the surface of these cells. In order to prove that the role of EPO on bone remodeling is mediated by bi-directional signaling of ephrinB2/EphB4, we will study the effect of EPO on the expression of ephrinB2/EphB4 and their downstream molecules in osteoclast, osteoblast and co-culture system by Real time PCR, Western blot, and cytochemical staining, etc. and identify the role of EPO on the differentiation and function of osteoclast and osteoblast; then we will study the relationship between ephrinB2/EphB4 axis and the expression of osteoclastic and osteoblastic relating molecules under the influence of EPO by gain- and loss-of function through stimulation of gene expression and RNAi technology, etc., and identify the role and mechanism of EPO on bone remodeling mediated by ephrinB2/EphB4 axis; lastly, we will observe the effect of EPO on the regeneration of residual alveolar bone following tooth extraction in rats by Micro CT, histology and immunohistochemistry, etc., which will settle a foundation for treatment of jaw bone destruction by EPO.

已知ephrinB2-EphB4相互作用通过正向和逆向信号调控成骨和破骨细胞的分化与功能。我们发现EPO能影响破骨和成骨细胞分化过程中某些关键分子的表达，并伴随细胞表面ephrinB2和EphB4表达的改变。为了证明EPO对骨重塑的作用是由ephrinB2/EphB4双向信号介导的，采用RT-PCR、Western blot和细胞化学等方法研究EPO对破骨和成骨细胞单独培养或共培养中ephrinB2/EphB4及其下游信号分子表达的影响，明确EPO对破骨和成骨细胞分化及功能的作用；通过基因功能激活和RNAi等技术验证EPO作用下ephrinB2/EphB4同破骨和成骨细胞相关分子表达的关系，阐明EPO通过ephrinB2/EphB4信号对骨重塑的作用及机理；采用Micro CT和免疫组织化学等技术，观察EPO对大鼠切牙拔除后剩余牙槽嵴骨再生的作用，为应用EPO治疗颌骨破坏性疾病奠定基础。

最近研究表明，促红细胞生成素（EPO）在维系非造血系统生物学功能方面亦发挥重要作用。有学者研究指出，EPO可以影响骨代谢，然而关于其如何调控骨重塑、影响骨损伤修复的详细机制尚不清楚。本研究中，我们利用组织化学染色、Real Time PCR和Western Blot等细胞生物学技术和分子生物学技术检测了EPO作用后ephrinB2/EphB4、成骨细胞分化和功能标志物、破骨细胞分化和功能标志物的表达，观察其对骨重塑的影响。研究发现，EPO可以增加ST2细胞中EphB4表达，同时促进成骨细胞分化。然而，在RAW264.7细胞中EPO增加NFATc1和EphrinB2的表达却降低了MMP-9的表达，EPO增加了破骨细胞的数量但其骨吸收活性降低。进一步采用EphrinB2 FC实验和EphB4 shRNA基因沉默实验，证实ephrinB2/EphB4信号在EPO促进成骨细胞分化中具有重要作用。体内大鼠切牙拔除后剩余牙槽骨修复实验也清楚地表明，EPO通过有效诱导新骨形成、促进骨损伤修复。综上，这些结果表明ephrinB2/EphB4信号可能在EPO诱导骨形成中发挥重要作用。