BMP9联合TGF-β1及其下游靶分子HSP47诱导成骨的调控方案研究

Trauma caused large segment bone defeat is always difficult problem for skeleton system recovery. In previous study, we proved that TGF-1 is a fundamental co-regulated factor in promoting BMP9-induced osteogenic differentiation. Our results indicated that TGF-1 combined synergistically with BMP9 can improve the quality of bone formation, which has potential clinical application. However, the detailed molecular pattern and mechanism of TGF-1 synergistical function in osteogenesis remain to be elucidated. In recent research, HSP47, the downstream target gene of TGF-1, is worth to be concerned. HSP47 is the chaperone of collagen and play roles in osteogenesis imperfecta might be caused by defect of mature collagen I. In this project, we use recombinant BMP9 and TGF-1 induced osteogenic differentiation of mice mesenchymal stem cell line in vitro and in vivo; investigate the effect and molecular mechanism of TGF-1 and its signaling feedback mode, dose-timing mode underlying BMP9-induced differentiation. Furthermore, we evaluate the gene expression profile of TGF-1 and focus on the function and regular pattern of downstream HSP47. We preliminarily evaluate the healing effect of TGF-1 and HSP47 on bone defect animal mode in order to establish an efficient cocktail program.

外伤致大段骨缺损始终是骨骼系统损伤后难以恢复的常见难题。联合细胞因子、种子细胞和基质材料进行骨组织工程修复是当前公认的研究主要方向。本项目已初步证实TGF-1是能够促进BMP9诱导成骨的基本协同因子，其特定作用方式、规律、机制有待阐明。TGF-1下游靶基因HSP47在成骨分化中具有重要意义，HSP47是胶原特异的分子伴侣，OI表现型可能由于成熟Ⅰ型胶原分子缺陷引起，值得高度关注。本项目研究以重组腺病毒法建立BMP9联合TGF-1定向诱导鼠间充质干细胞成骨分化模型，通过体外与体内实验深入揭示TGF-1在BMP9诱导成骨分化中的作用规律与分子调控机制；认识其细胞信号转导反馈调节基本方式，在此基础上分析和掌握TGF-1协同处理的量效时效关系；进而，探讨TGF-1在BMP9诱导成骨分化中作用所引起的基因表达谱变化，重点针对下游靶基因HSP47的功能、表达规律进行鉴定；以骨缺损动物模型初步观察TGF-1协同BMP9诱导成骨分化的修复治疗中HSP47的作用，探索建立有效的调控成骨的“鸡尾酒”方案。

项目经过课题设计和实施，基本在预定期限内完成了研究目标。我们利用重组腺病毒感染法测定了TGF-β1病毒感染效率与目的蛋白浓度的量效关系，明确了腺病毒的使用规律；利用TGF-β1和BMP9腺病毒从体外、体内实验均验证了两者的促成骨协同效应；确定了HSP47作为胶原伴侣蛋白介导促进TGF-β1成骨的分子机制，抑制HSP47会影响TGF-β1成骨效能；意外的发现TGF-β1促进成骨的信号通路并不是经典的TGF-β/Smad信号通路，利用蛋白芯片抗体筛查明确了非经典信号通路JAK/STAT和PI3K/AKT/mTOR的激活，为今后的研究指明了方向。以上的研究结果，为联合应用细胞因子诱导骨髓间充质干细胞成骨，建立有效的调控成骨的“鸡尾酒”方案提供了更多的理论和实验基础。相关文章及成果将在近期接收。