FGF和Wnt协同增强低剂量BMP诱导成骨信号通路的研究

Using a very low amount of BMP to rapidly activate, enhance and prolong its-induced osteogenic signaling has become an main research trend in the field of large-volum bone defects repairing. In our previous in-vitro studies, we found that heterodimeric BMP could induce in-vitro osteoblastogenesis more rapidly with a lower concentration threshold and significantly higher dose-efficiency in comparison with homodimeric BMP. Our in-vivo experiment also corroborated that the low-dose heterodimeric BMP2/7 facilitated more rapid bone regeneration and more mature microstructure in peri-implant bone defects than the homodimeric BMPs. A recent study has indicated that Wnt and FGF could cooperatively enhance and prolong the low-concentration TGF-β/BMP signaling. Basing on these data, we put forward the hypothesis: Wnt and FGF can cooperatively enhance and prolong the very low-concentration BMP2/7-induced signaling transduction so as to accelerate and enhance osteogenesis through regulating the Smad4 level. In this study, we will first High throughput screen the suitable concentration for Wnt, FGF and BMP2/7 by checking differentiation markers. We will then identify the time-course and dose-dependent characteristics of their-induced osteogenesis of human osteoblasts. Thereafter, we will identify the intracellular signaling mechanisms of the combination using microarray, RNA silencing, and inhibitors, etc. Finally, we will test the osteoinductive efficacy of the combined Wnt, FGF and BMP2/7 in an in-vivo model.

以低剂量BMP快速开启并维持高强度成骨信号通路从而高效诱导成骨、降低副作用是修复大体积骨缺损的主要研究趋势之一。本课题组前期体外、体内研究发现：与BMP同二聚体相比，BMP 异二聚体在体外诱导成骨具有起效早、剂量低，作用快等优点，并能够促进骨微结构的改建和成熟。有最新的研究表明：在Wnt和FGF可以协同增强并延长更低剂量TGF-β/BMP所诱导的细胞内信号转导。我们提出假设：Wnt和FGF通过调节Smad4表达水平显著增强和延长更低剂量BMP2/7所诱导的成骨信号通路，从而显著加快和提高成骨效率。本项目拟先以高通量检测筛选Wnt、FGF促进BMP2/7诱导成骨的最适浓度范围，明确其诱导人成骨细胞成骨的剂效特征，利用microarray、RNA干扰、信号通路抑制剂等分子生物学初步揭示其内在机制，并在体内验证其效果。

以低剂量BMP快速开启并维持高强度成骨信号通路从而高效诱导成骨、降低副作用是修复大体积骨缺损的主要研究趋势之一。本课题组前期体外、体内研究发现：与BMP同二聚体相比，BMP 异二聚体在体外诱导成骨具有起效早、剂量低，作用快等优点，并能够促进骨微结构的改建和成熟。有最新的研究表明：在Wnt和FGF可以协同增强并延长更低剂量TGF-β/BMP所诱导的细胞内信号转导。我们提出假设：Wnt和FGF通过调节Smad4表达水平显著增强和延长更低剂量BMP2/7所诱导的成骨信号通路，从而显著加快和提高成骨效率。本项目通过检测各实验组ALP表达水平筛选出Wnt3a、FGF2及BMP2/7诱导人成骨细胞成骨分化的的最适浓度范围，明确其诱导人成骨细胞成骨的剂效特征，后对各细胞因子最适浓度作用下细胞成骨分化相关蛋白，细胞基质外矿化等多种指标进行检测；同时采用RNA测序的手段检测三种细胞因子单独或联合应用时基因表达的变化，筛选相关信号通路的差异基因后，RT-PCR进一步验证其基因的表达，同时检测信号通路抑制剂作用下Wnt信号通路，MAPK信号通路及FGF信号通路蛋白表达的变化，初步揭示其内在机制，课题组以体外细胞实验数据为依据，并在SD大鼠体皮下植入载有细胞因子的胶原海绵，18day后检测各组细胞因子诱导异位成骨的情况，micro-CT、硬组织切片染色及免疫组化染色结果均显示三因子联用组可以获得显著增多的异位骨形成。综上，研究结果显示，最适浓度的Wnt, FGF及BMP2/7三种细胞因子联合应用可以显著加快和提高成骨效率，为有效构建以BMP异二聚体为基础的组织工程化骨提供理论依据，促进其早期实现临床转化，修复大体积骨缺损。