炎症刺激下NBD多肽对成骨细胞分化作用的机制研究

At present there is a little literature about effect of the inflammation on osteoblasts differentiation, and how intervenes this effect have not been reported. In the previous study which osteogenic cells derived from C2C12 cells with BMP2, we have proved that TNF-α inhibits osteoblast differentiation through the activation of NF-κB，and was able to effectively antagonize NF-κB activity in osteoclastogenesis. However the molecular events of osteoclasts in response to NBD peptide are still not well understood so far,because of involving or existing between two or more signal pathways in inflammatory response.Differential proteomics can be applied to evaluate the significant proteins in cellular metabolism, which would aid in better understanding of the molecular mechanisms of osteoclasts in response to inflammation and NBD peptide。we quantitatively and qualitatively analyzed the differentially expressed proteins in osteoclasts under inflammatory response after NBD polypeptides in an in vitro osteoclast culture modal by two-dimensional gelelectrophoresis, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS). The aim of our study was to find out the major proteins involved in osteoblast formation and find out a new target in the treatment with NBD polypeptides.

炎症刺激如何影响成骨细胞分化，如何改善炎症刺激对成骨细胞分化的影响，目前国内外相关研究较少。我们在前期实验以BMP2诱导肌源细胞C2C12分化为成骨细胞为模型的研究中发现：TNF-α能通过NF-kB信号通道抑制成骨细胞分化，同时NBD多肽能够特异性阻断该通道改善TNF-α对成骨细胞分化的影响。但考虑炎症反应涉及多个信号通道且存在通道旁通现象，为明确单一信号通道阻断是否有效，本课题拟用蛋白质组学技术研究成骨细胞分化在炎症刺激下差异蛋白的表达，筛选相关差异蛋白，并以改善炎症刺激抑制成骨细胞分化的兴趣蛋白作为潜在药靶，进行生物功能鉴定。我们拟根据前期研究中NBD多肽阻断NF-кB信号通路干预炎症刺激抑制成骨细胞分化，继续验证NBD多肽是否影响上述潜在药靶在炎症刺激下成骨细胞分化中的表达，为寻找可联合NBD多肽组成多靶点改善炎性刺激抑制成骨细胞分化的特异性和可药性药靶提供新思路和理论依据。

目前国内外对炎症刺激如何影响成骨细胞分化，如何改善炎症刺激对成骨细胞分化的影响研究较少。本课题以TNF-α作为刺激物、BMP-2诱导C2C12肌源细胞分化为成骨细胞，并且可以成功作为炎症刺激下成骨细胞分化的稳定细胞模型。同时初步验证了TNF-α能通过NF-kB信号通道抑制成骨细胞分化，而NBD多肽能够特异性阻断该通道改善TNF-α对成骨细胞分化的影响。本研究组基于iTRAQ技术筛选出炎症刺激干预成骨细胞分化的5个差异蛋白：S100-A4，ATPase inhibitor ，Cytochrome b5，SERCA3 及ELP2，并且对兴趣蛋白SERCA3与ELP2的表达趋势进行MRM, Western-blot及RT-PCR 的验证，初步研究显示沉默药靶蛋白基因ELP2可以改善炎症刺激对成骨细胞分化的抑制，并阐明了ELP2通过TNF/ELP2/STAT3/BMPR2/Smad参与炎症对成骨细胞分化的抑制，证实ELP2可以作为一个理想的药物靶点改善炎症相关性成骨分化抑制性疾病。进一步动物实验通过Norden造模法成功制备新西兰大白兔胫骨慢性骨髓炎模型并加以鉴定，并证实NBD多肽在兔胫骨骨髓炎模型中具有一定的抗炎促成骨效应；根据前期研究中NBD多肽阻断NF-кB信号通路干预炎症刺激抑制成骨细胞分化，验证了NBD多肽可以在炎症刺激下成骨细胞分化中的表达，为下一步寻找可联合NBD多肽组成多靶点改善炎性刺激抑制成骨细胞分化的特异性和可药性药靶提供新思路和理论依据。

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