14-3-3η蛋白通过调控MAPK/Smad1信号通路影响强直性脊柱炎患者新骨形成的机制研究

Inflammation and new bone formation are the main characteristcs of ankylosing spondylitis(AS). Excessive osteogenesis is the primary cause of disability in AS. It is not completely clear that how inflammation results in new bone formation in AS. 14-3-3 η protein was a new proinflammatory mediator which was investigated to upregulate cytokines and was in association with bone erosion in rheumatoid arthritis. There were no previous reports about the influence of 14-3-3 η protein in the pathogenesis of inflammation and new bone formation in AS, also no any investigations published about associations between 14-3-3 η protein and signal pathway of osteogenesis in AS. We will verify whether inflammatory cytokines such as IL-17, IL-23, TNF-a can stimulate the expression of osteogenesis related molecules, including bone morphogenetic protein-2, Smad1, β-actenin, DKK1, RANKL and OPG detecting by QRT-PCR and Western-Blot, in bone marrow mesenchymal stem cells (BMSCs) by inducing 14-3-3 η protein, whether 14-3-3 η protein can influence the expression of osteogenesis related molecules in BMSCs or peripheral blood mononuclear cells from AS patients and proliferations of bone cells by transfection or gene silencing technology. We also investigate whether 14-3-3 η protein can inhibit osteogenesis by directly combining with MAPK and subsequently resulting in Smad1 phosphorylation degradation. We will also detect the plasma levels of 14-3-3 η protein and 14-3-3 η protein autoantibody from 30 cases with early-stage AS and 30 cases with established AS by Western-Blot and ELISA and also changes of plasma levels of 14-3-3 η protein and 14-3-3 η protein autoantibody after 2-years’ follow-up. Associations between 14-3-3η protein/14-3-3 η protein autoantibody levels and inflammation (clinical inflammatory manifestations and radiographic inflammatory features on MRI) as well as radiographic progression of new bone formation in spine (mSASSS score) will be analysised. Expected results would be verified in our study that 14-3-3 η protein, which could upregulate inflammation, might elevate in early stage of AS, while 14-3-3 η protein autoantibody levels would increase in established AS and promote radiographic progression of new bone formation in AS by inhibiting MAPK/Smad1 signal pathway.

强直性脊柱炎(AS)以炎症和新骨形成为主要特点，成骨过度是其致残的主要原因，但炎症导致AS新骨形成的机制尚不完全清楚。14-3-3η蛋白是一种新的促炎介质，其在AS炎症和新骨形成中的机制如何？是否与AS的成骨信号通路有关，尚未见报道。本研究将通过体外实验（转染和基因沉默技术）探明炎症细胞因子是否通过14-3-3η蛋白影响BMSCs成骨相关蛋白的表达、14-3-3η蛋白能否影响BMSCs和AS患者PBMC成骨相关蛋白的表达和骨细胞的增殖、14-3-3η蛋白是否通过对MAPK/Smad1的作用影响成骨研究；同时测定早期与非早期AS患者随访2年前后外周血14-3-3η蛋白及14-3-3η抗体水平变化，分析其与AS影像学进展间的关系；从而验证早期AS患者以14-3-3η蛋白升高为主，与AS炎症有关；非早期AS患者14-3-3η蛋白抗体水平升高，并通过抑制MAPK/Smad1信号通路促进其新骨形成。

强直性脊柱炎主要特点是炎症和新骨形成，炎症导致新骨形成的机制是研究的热点和难点，我们的研究结果主要有：.1.14-3-3η蛋白水平可作为SpA疗效评价之一，对炎性关节病患者具有诊断识别意义。14-3-3η蛋白与病程、枕墙距、胸廓扩张度、腰椎活动度、SPARCC评分呈正直线相关。随访2年治疗后TNF-α、14-3-3η蛋白水平较治疗前下降，TNF-α、14-3-3η蛋白、血清IL-6水平改变与SPARCC评分、mSASSS均具有相关性。SpA组ROC曲线显示：血清14-3-3η蛋白截点值为0.447ng/ml时，灵敏度为86.9%，特异度为92.5%。.2.14-3-3η蛋白可能是通过MAPK/Smad1信号通路实现抑制骨髓间充质干细胞（BMSCs）的成骨分化作用。敲低14-3-3η蛋白能促进BMSCs细胞迁移，培养过程中成骨指标和其mRNA水平的表达逐渐上调。在低氧条件下诱导BMSCs成骨，发现14-3-3η与MAPK结合减少、miR-142-3P较在有氧正常对照组明显上调。在转染重组载体质粒Psi-14-3-3η 3’-UTR的试验中，相比于对照组miR-142-3P组荧光素酶活性表达出显著下降。说明14-3-3η基因是miR-142-3P直接作用的靶基因，并且miR-142-3P于14-3-3η基因的3'-UTR区域结合，在转录后水平对14-3-3η基因有直接抑制作用。.3.研究发现过表达miR-142-3P后14-3-3η蛋白、MAPK蛋白水平、14-3-3η的mRNA水平较对照组明显下调，成骨蛋白BMP-2、miR-142-3P的mRNA较对照组表达明显增高。抑制miR-142-3P表达14-3-3η蛋白和MAPK水平较对照组上调，成骨蛋白BMP-2较对照组表达下调；同时14-3-3η的mRNA水平较对照组上调，而miR-142-3P的mRNA较对照组下调。.4.模式动物实验结果显示：与对照组相比敲低14-3-3η或过表达miR-142-3P在体外能促进成骨，而过表达14-3-3η或敲低miR-142-3P在体外抑制成骨，同时与对照组相比同时过表达miR-142-3P和14-3-3η成骨较差，敲低miR-142-3P同时敲低14-3-3η成骨较好，说明miR-142-3P通过14-3-3η影响成骨。