摘要
由炎症、损伤、肿瘤、先天畸形和增龄等原因引起的牙槽骨吸收与颌骨缺损是口腔医学领域中最常见的临床表现之一，不仅影响咀嚼、发音、进食等功能，而且还影响颜面美观以及进一步的义齿修复。因此，如何调控骨重塑、促进骨修复成为临床和基础研究的重点。研究骨形成与骨吸收的分子机理不仅对阐明骨缺损和骨代谢性疾病的发病机制具有重要的理论意义，根据这些分子机理或机制、通过调控骨形成与骨吸收等过程，也是治疗骨破坏性疾病的主要策略。
骨组织是终生不断进行代谢的组织，成骨细胞介导的骨形成与破骨细胞介导的骨吸收构成骨重塑，持续进行的骨重塑可维持矿化平衡及骨组织自身结构的完整。以往的研究表明，RANKL/RANK轴是维持骨稳态的重要信号通路，成骨细胞产生的RANKL作用于破骨细胞前体细胞上的相应受体RANK，进而促进破骨细胞的分化和功能。近年来，国内外学者们相继发现，在骨重塑过程中，还通过ephrin/Eph轴传递破骨细胞和成骨细胞之间的相互作用。Ephrin配体主要表达于破骨细胞，Eph受体主要表达于成骨细胞。研究表明，由ephrinB到EphB的正向信号促进成骨细胞的分化，而由EphB到ephrinB的逆向信号抑制了破骨细胞的形成，其相互作用在骨重塑过程中发挥重要的作用。
最近，研究者发现促红细胞生成素（EPO）能够促进骨折愈合和加快下颌骨的牵张成骨速度；EPO在体外能刺激骨髓造血干细胞表达BMP-2和BMP-6，腹腔内注射rhEPO能促进HSCs表达BMPs。但是目前尚未见EPO对成骨/破骨细胞分化和功能的作用的详细研究。我们将围绕ephrinB2/EphB4轴，研究该轴介导的EPO对成骨/破骨细胞分化和功能的影响及其机理。进一步以此为靶点，通过体内动物实验，研究EPO对颌骨损伤修复的作用。研究目的
（1）通过体外诱导ST2细胞向成骨细胞分化实验，初步观察EPO对成骨细胞EphB4表达的影响，研究EPO对成骨细胞分化的作用；通过体外诱导RAW264.7细胞向破骨细胞分化，初步观察EPO对破骨细胞ephrinB2表达的影响，研究EPO对破骨细胞分化的作用；
（2）应用ephrinB2-Fc蛋白，模拟成骨细胞与破骨细胞共培养体系，观察和比较EPO在单因素条件下对成骨细胞分化和功能的影响，研究ephrinB2/EphB4信号介导的EPO促成骨细胞分化作用及其主要机理；采用基因沉默实验，进一步验证EPO通过EphB4/ephrinB2信号对骨重塑的调控作用；
（3）利用大鼠切牙拔除后剩余牙槽骨吸收模型，体内观测EPO在牙槽骨骨改建中的作用。
研究方法
（1）采用小鼠骨髓基质细胞系ST2细胞作为成骨细胞前体细胞，在成骨条件诱导液（含抗坏血酸50mg/L、β-甘油磷酸钠10mmol/L、地塞米松10-8mol/L）中培养，经real-time PCR检测EPO对成骨细胞相关基因和EphB4表达的影响，通过组织化学染色检测ALP活性和钙结节数量比较EPO对成骨细胞功能的影响；诱导小鼠单核/巨噬细胞系RAW264.7细胞向破骨细胞分化（RANKL50ng/ml），经real time PCR检测EPO对破骨细胞相关基因及ephrinB2表达的影响，通过TRAP阳性细胞数和骨吸收陷窝数量变化，观察破骨细胞分化过程中EPO对破骨细胞数量和功能的影响；
（2）在诱导成骨细胞分化前加入ephrinB2-Fc模拟成骨细胞破骨细胞共培养体系，分别通过real time PCR，ALP活性和钙结节染色实验，检测EPO在成骨细胞分化中作用；通过ephrinB2siRNA和EphB4shRNA沉默破骨细胞或成骨细胞中ephrinB2或EphB4基因，通过real-time PCR检测相关基因的表达情况，观察阻断EphB4/ephrinB2信号后EPO对成骨/破骨细胞分化的影响；
（3）体内建立大鼠拔牙模型，通过软X线测量牙槽嵴高度探讨EPO对剩余牙槽嵴骨吸收的作用，通过骨密度测量和组织学观察EPO对骨修复能力的影响。
研究结果
（1）成骨细胞诱导分化实验中，与对照组相比，EPO可以增加Runx2、Sp7和ColⅠ表达，增强ALP活性和增加钙结节数量，同时EPO增加了EphB4的表达；破骨细胞诱导分化过程中，EPO先通过增加c-fos和NFATc1表达增加了破骨细胞数量，随后下调了Ctsk和MMP9的表达，抑制了破骨细胞活性，TRAP染色和骨吸收陷窝实验进一步证实了上述结果，同时EPO增加了ephrinB2的表达；
（2）在模拟共培养体系中，EPO可以通过增加的ephrinB2上调成骨细胞相关基因的表达，促进成骨细胞的分化和骨形成功能，提示EPO可能通过EphB4/ephrinB2信号调控骨重塑；基因沉默实验证实，阻断EphB4基因转录后EPO促进成骨细胞分化的作用被减弱，提示EPO通过促进骨形成、抑制骨吸收以调控骨重塑可能是由EphB4/ephrinB2信号介导的；
（3）体内实验结果显示，应用EPO牙槽嵴骨密度和骨形成量均高于对照组；与对照组比较，EPO降低了剩余牙槽骨的吸收量，促进了牙槽骨损伤的修复。
研究结论
（1） EPO可以促进成骨细胞分化及其骨形成；
（2） EPO虽然能增加破骨细胞形成，但所增加的破骨细胞缺乏骨吸收功能；
（3） EPO通过ephrinB2/EphB4双向信号调控骨重塑；
（4） EPO促进了牙拔除后剩余牙槽骨的愈合。
Bone repair or bone regeneration has become an important strategy in thetreatment of bone deformity and bone defects caused by trauma, tumor, andinflammation. Bone regeneration is a complex process of balance betweenbone-forming activity of osteoblasts and bone-resorbing activity of osteoclasts. Manymolecules are involved in the communication between osteoblasts and osteoclasts. Itwas believed that some proteins and signal pathways could affect both osteoclast andosteoblast at the same time to balance bone regeneration, such as ephrinB2/EphB4signal pathway. It was found that ephrinB2, which is synthesized in osteoclasts andbecome a transmembrane ligand in osteoclasts, and EphB4, a tyrosine kinase receptorin osteoblasts, are involved in the control of bone regeneration. Erythropoietin (EPO)is not only involved in red blood cell production, but recently was also found to beinvolved in many other biological functions, such as inducing bone regeneration.However, little is known about how EPO regulates bone regeneration. In this study,we hypothesized that EPO might be the double-edged protein that plays interestingroles by ephrinB2/EphB4in bone regeneration.
Objective:
(1) The role of EPO in the osteoblast/osteoclast differentiation.
(2) In order to observe the roles of erythropoietin in bone regeneration throughephrinB2/EphB4signal pathway. We used ephrinB2-Fc to partially mimic theinteractions of osteoclasts and osteoblasts and gene knockdown assays.
(3) The effects of EPO resulted in promoting new bone formation and inhibitingbone resorption in the animal model of jaw bone destruction.
Methods:
(1) To further understand mechanisms of effects of EPO on bone formation,murine bone marrow stromal cell line ST2cellas the osteoblast precursor werecultured with osteogenic medium. We will study the effect of EPO on the expressionof EphB4and their down stream molecules in progress of osteoblast differention byReal time PCR, cytochemical staining (ALP) and calcium deposition. In order to prove that the role of EPO on bone remodeling is mediated by bi-directional signalingof ephrinB2/EphB4, human EPO protein was used in osteoclast precursor (RAW264.7murine cell line) in vitro. The effect of EPO on the osteoclast differention by Realtime PCR, cytochemical staining (TRAP), and bone resorption assays in vitro.
(2) In order to prove that the role of EPO on bone remodeling is mediated bybi-directional signaling of ephrinB2/EphB4, we will study the effect of EPO on theexpression of ephrinB2/EphB4and their downstream molecules in osteoclast andosteoblast co-culture system by Real time PCR and cytochemical staining, etc.Toobserve the effect of EPO on osteoclast/osteoblast differention by blockEphB4/ephrinB2signaling pathway by EphB4shRNA and ephrinB2siRNAin vitro.
(3) Identify the role and mechanism of EPO on bone remodeling mediated byephrinB2/EphB4axis, and observe the effect of EPO on the regeneration of residualalveolar bone following tooth extraction in rats by soft X ray, bone mineral density(BMD) and histology, which will settle a foundation for treatment of jaw bonedestruction by EPO.
Results:
(1) The process of osteoblast differention showed that EPO increased expressionof EphB4in osteoblast. In addition, EPO increased the expressions of Runx2, Sp7,and ColⅠ in osteoblast at various time points. And EPO also enhance the activity ofALP and calcium deposition. The process of osteoclast differention EPO increasedexpression of ephrinB2in osteoclast and the expressions of c-fos, NFATc1, MMP9and Ctsk at early time points in osteoclast, but later decreased expressions of MMP9and Ctsk. The data from TRAP staining and bone resorption assays showed that EPOdecreased bone resorption. These results suggest that EPO can increase theproliferation of osteoclast, but not increase the bone resorption function.
(2) EphrinB2-Fc interaction model was used as a stimulator to mimic theinteractions between osteoclast and osteoblast.EPO also increased expression ofosteogenicgenes and ALP positive osteoblasts and calcium deposition. The effects ofEPO in the osteoblast/osteoclast differention were attenuated by blockingephrinB2/EphB4gene transcription, suggesting that the regulation of bone remodelingby EPO might be through EphB4/ephrinB2signal pathway.
(3) In vivo animal assays, BMD analysis and measurement of relative height ofresidual alveolar ridgeand histological findings (H&E staining) clearly demonstrate that EPO can efficiently induce new bone formation in the alveolar bone regenerationmodel.Conclusions:
(1) EPO can promote osteoblast differentiation and the ability of bone formation;
(2) EPO can increase the number of osteoclasts to inhibite resorption ability;
(3) EPO directly stimulates ephrinB2expression in osteoclasts and EphB4expression inosteoblasts leading to the enhancement of bone formation while blacking the bone resorptionactivity of osteoclasts;
(4) EPO promotes bone formation in an alveolar bone regeneration model.
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