ephrinb2ephb4介导epo对破骨细胞调控机理的研究word论文.docxVIP

摘要目的探讨促红细胞生成素（Erythropoietin,EPO）在破骨细胞分化中的作用机制，本实验拟以种系单一，性能稳定的小鼠单核巨噬细胞（RAW264.7细胞）作为破骨前体细胞，研究小鼠单核巨噬细胞在体外向破骨细胞分化过程中，促红细胞生成素与破骨细胞表面受体（Erythropoietinreceptor,EpoR）结合发挥作用的机理；进一步研究EphrinB2/EphB4轴参与EPO调控骨重塑中的作用。方法首先，EPO作用于EpoR受体不沉默和沉默的经诱导的小鼠单核巨噬细胞，观察TRAP阳性多核细胞数目变化；real-timePCR检测RAW264.7向破骨细胞分化过程中相关基因Epor、Nfatc1、Mmp9、EphrinB2基因的表达；然后，一组RA- W264.7细胞与EphB4-Fc蛋白共培养后，分别加入含或不含EPO的破骨条件诱导液培养；另一组RAW264.7细胞通过EphrinB2-siRNA沉默和不沉默表面EphrinB2加入含EPO的破骨条件诱导液培养。通过TRAP染色；以及RealTimePCR检测破骨细胞相关基因c-fos、Nfatc1、Mmp9及EphrinB2(由efnb2基因编码)的表达。结果首先，与对照组比较，EpoR受体沉没组4天时，Nfatc1、EphrinB2以及TRAPmRNA表达明显升高（P＜0.01），Mmp9mRNA的表达明显降低（P＜0.01）；当形成逆向信号后，破骨相关基因Nfatc1、Mmp9表达水平降低；再加入EPO后，Mmp9的表达更低；而沉默efnb2后再加入EPO，Mmp9的表达明显上升。结论EPO通过与破骨细胞表面受体EPOR结合，引发破骨细胞内相关基因表达改变，从而通过EphrinB2/EphB4信号通路调控破骨细胞数量和功能，进而影响骨重塑。关键词：促红细胞生成素；促红细胞生成素受体；RNAi；破骨细胞；siRNA；EphrinB2/EphB4-1-AbstractObjectives: The aim of this studywas to investigate howthe activation ofosteoclast can beregulated byEPO.Consideringthere aremanydifferences between primarycell and in vitro cells , invitro cell lineshavegood stabilityand is veryadvantageous, This study usingthe RAW264.7 mouse monocyte/macrophagecell line as an osteoclast precursor. Therole of erythropoietin (EPO) and Epo receptor (EpoR) signalingpathwaysfor production of red blood-cell has beenextensivelystudied. And EPOplaysa critical rolein the formation, proliferation and maturation ofbone cells. However, little is known about how EPO/EpoR signalingeffects on osteoclast differentiation .Inanotherwaythis study was to investigatehow the activation ofosteoclast can beregulated byEPO.Methods:SomeRAW264.7cellsweretreatedwithrhEpoandthereceptoractivatorof nuclearfactor-κB(RANK)ligand(RANKL)foranindicatedperiodoftime.Othercellswere treatedwithrhEPOandthereceptoractivatorofnuclearfactor-κB(RANK)ligand(RANKL) foranindicatedperiodoftime.Thenumbersoftartrate-resistantacidphosphatase(TRAP) stainingpositive,multinucleatedcellsafter5~9days.ThelevelsofEpor,Nfatc1,efnb2,matrix metalloproteinase-9(Mmp-9),cathepsinKw