PDGF-BB功能化蚕丝水凝胶协同BMP9调控间充质干细胞成骨分化的机理研究

The addition of osteoinductive factors to tissue-engineered bone material can strongly promote osteogenic differentiation of mesenchymal stem cells and significantly improve the bone repair outcomes. Silk is becoming one of the most promising tissue-engineered bone materials due to its excellent biological properties. We previously remolded sikworm through transgenic technology and obtained functionalized silk of genetically engineered recombinant protein PDGF-BB. The purified protein and sericin hydrogel exhibit very low cytotoxicity, facilitate the proliferation of stem cells, and up-regulate the expression of Runx2, Bmp2 and PDGFRβ genes, which are related to the osteogenesis of mesenchymal stem cells. Recent studies demonstrated that BMP9 is the most potent osteogenic BMP. Therefore, we hypothesize that PDGF-BB silk sericin hydrogel is an effective osteogenic scaffold material and can synergize with BMP9 to induce more effective osteogenic differentiation of mesenchymal stem cells. This application, based on our previous work, will employ PDGF-BB silk sericin hydrogel as the osteogenic scaffold material and BMP9 for stimulation to evaluate their inductive effectiveness on the osteogenic differentiation of mesenchymal stem cells, and dissect the underlying synergistic inductive mechanism between PDGF-BB and BMP9.The project focuses on the mechanism of promoting the differentiation of mesenchymal stem cells using an innovative tissue-engineered bone material, should greatly improve the treatment effectiveness of bone defect or nonunion with certain clinical indications, and thereby possessing significant academic and translational potential.

在组织工程骨材料中添加成骨诱导因子能有力促进间充质干细胞（MSCs）分化并显著改善骨修复效果，蚕丝因其优异生物学特性成为极具潜力的组织工程骨候选材料。课题组前期利用转基因技术改造家蚕，获取了表达人重组蛋白PDGF-BB的功能化蚕丝，发现从其中纯化的蛋白及丝胶水凝胶的细胞毒性极小，可促进干细胞增殖和成骨分化相关Runx2、Bmp2、PDGFRβ基因表达；进一步研究发现BMP9促进MSCs成骨分化能力明显强于其他BMPs。因此本项目提出假设：PDGF-BB丝胶水凝胶是有效成骨支架材料、可协同BMP9增强诱导MSCs成骨分化；基于前期研究，将PDGF-BB丝胶水凝胶作为成骨支架材料，评估其协同BMP9诱导MSCs成骨分化效果，揭示PDGF-BB协同BMP9诱导MSCs成骨分化机理。本项目研究新型组织工程骨材料促进MSCs分化的机理，可显著提高骨缺损相关疾病治疗效果，具有十分重要的学术和应用价值。

在骨组织工程中，间充质干细胞（MSCs）等具有多向分化潜能的细胞常包埋于骨材料中，辅以成骨诱导因子，以促进MSCs成骨分化，显著提高成骨、骨缺损修复的效率。蚕丝因其低免疫原性、血液相容性、良好的物理性能等特性，成为极具潜力的骨组织工程候选材料。通过转基因技术可将蚕丝作为生物反应器，制备表达外源活性蛋白的功能性生物材料。课题组利用前期获取的表达人重组蛋白PDGF-BB的功能化蚕丝，通过改进Na2CO3溶解-透析法，制备成丝胶水凝胶材料以诱导MSCs成骨分化。实验结果表明，PDGF-BB蛋白活性的丝胶水凝胶细胞毒性极小，MSCs能够正常增殖和分化。碱性磷酸酶及茜素红染色实验结果表明PDGF-BB水凝胶不能独立诱导MSCs成骨分化，但可增强BMP9诱导的MSCs成骨分化，且随着蚕丝水凝胶中PDGF-BB浓度增加，MSCs成骨分化能力增强。PCR及Western blot检测发现MSCs中成骨分化标记基因Runx2、OPN蛋白表达量显著增加。micro-CT检测表明，PDGF-BB水凝胶可协同BMP9诱导MSCs在裸鼠皮下异位成骨。Western blot检测进一步发现蚕丝水凝胶中PDGF-BB可能通过BMP/Smad磷酸化信号通路调控BMP9对MSCs成骨分化的诱导作用。此外，课题组发现PDGF-BB能显著抑制BMP9诱导MSCs成脂分化，抑制BMP9对C/EBPa及PPARg表达的促进作用。CoIP及ChIP实验结果表明，PDGF-BB可能通过激活Stat3信号，且抑制PPARg表达，抑制BMP9诱导的MSCs成脂分化，进而进一步促进MSCs成骨分化。基于上述研究成果，我们推测PDGF-BB丝胶水凝胶是一种有效的骨组织工程材料，有一定的医学应用价值和前景。