III型TGF-β受体介导强直性脊柱炎成纤维细胞成骨分化作用及其分子机制研究

Osteogenesis of central axis joints and spinal ligament is the final result and disabling risk of ankylosis of ankylosing spondylitis(AS), and fibroblast plays a crucial role during the ossification process. Previously we found expression of TGF-βR3(TβR3) significantly increased in the AS fibroblast, and its ligands, TGF-β1 and BMP-2, remarkablely induce osteogenesis of it. Hence, we surmise that TGFβR3 contributes to the ossification formation of AS fibroblast. To confirm this hypothesis, this study intends to further investigate around TGFβR3. Firstly, overexpression and RNA interference of TGFβR3 in fibroblasts were performed, to test whether TGF-β1 and BMP-2 influence it again. Secondly, biosensor and molecular docking methods were performed to unveil the interactions between TGFβR3 and its ligands, TGF-β1 and BMP-2. Finally, the changes of members involved in TGFβR3/Smad pathway were detected, to find the key downstream effectors. In summary, this investigation aims to unveil the molecular mechanism of osteogenesis of AS fibroblast mediated by TGFβR3, and further find new drug targets.

脊柱中轴关节及韧带骨化是强直性脊柱炎(AS)的最终结果和致残原因，成纤维细胞成骨分化在其中发挥重要作用。基于前期研究发现AS成纤维细胞III型TGF-β受体（TGFβR3）表达显著升高、其配体TGF-β1和BMP-2显著诱导细胞的成骨分化，提出“TGFβR3介导AS成纤维细胞成骨分化” 的假说。因此，本项目围绕TGFβR3开展工作：首先在细胞实验中采用过表达、RNA干扰研究TGFβR3表达改变对TGF-β1和BMP-2作用的影响，明确TGFβR3对细胞成骨分化的作用；其次采用生物传感器技术和分子对接探讨TGF-β1、BMP-2与TGFβR3蛋白的分子间相互作用，进一步确认靶点；最后研究TGFβR3/Smad通路分子在AS成纤维细胞成骨分化中的变化，寻找并确认下游关键效应分子。以期探讨TGFβR3介导AS成纤维细胞成骨分化的分子机制，为获得新的干预靶点奠定基础。

前期工作发现TGFβR3在AS棘上韧带中高表达、与AS成纤维细胞成骨分化有关，本项目拟在此基础上确认TGFβR3介导AS成纤维细胞成骨分化，并探讨其分子机制。首先，构建TGFβR3过表达载体并合成靶向siRNA，建立TGFβR3高表达和低表达工具细胞；在此基础上确认TGF-β1联合BMP2进一步诱导TGFβR3过表达细胞成骨分化，在体外实验确认TGFβR3介导AS成纤维细胞成骨分化。其次，完成TGFβR3蛋白的真核表达和纯化；进一步通过基本表面离子共振（SPR）原理的生物传感器技术，确认TGFβR3可特异性结合TGF-β1和BMP2；通过共定位实验和免疫沉淀，进一步确认TGFβR3可同时TGF-β1和BMP2，而TGF-β1促进TGFβR3结合BMP2，从而将BMP2递呈给其受体BMPR1A；通过分子对接模拟，探讨了TGFβR3同时结合TGF-β1和BMP2的结合方式和空间姿态，并分析了其分子互作细节。再次，采用TGFβR3过表达和低表达成纤维细胞，确认TGF-β1联合BMP2，同时上调TGFβR3过表达细胞的TGF-β1通路Smad2/3磷酸化、BMP2通路Smad1磷酸化、及两条通路的co-Smad—Smad4的表达，从而同时活化TGF-β1通路和BMP2通路，促进TGF-β1依赖的成纤维细胞增殖并上调RUNX2表达而促进成纤维细胞成骨分化；来源于AS患者的棘上韧带中，也发现p-Smad2/3、p-Smad1、Smad4和RUNX2的高表达。综上所述，本研究确认TGFβR3高表达介导AS成纤维细胞成骨分化，其机制为过表达的TGFβR3同时活化TGF-β1和BMP2通路，诱导AS成纤维细胞增殖和成骨分化。