PHLPP1调节TNF信号通路在眼镜蛇毒神经生长因子（NGF）成软骨诱导中的作用及其机制研究

Growth factors can induce chondrogenic differentiation of stem cells. The commonly used transforming growth factor (TGF-β1) falls short in meeting the needs of clinical applications because they are limited by their high cost and rapid degradation ready. Our previous study found that nerve growth factor (NGF), a natural active protein extracted from cobra venom, has specific cartilage-inducing effects and is more effective than TGF-β1. To further study its molecular biology mechanism, RNA-Seq, bioinformatics analysis and experimental verification were performed. Based on the results, we predicted that snake venom NGF may up-regulate sox9 expression by inhibiting PHLPP1, subsequently activation of TNF signaling pathway, thereby inducing chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). This project will study the role and mechanism of PHLPP1 and TNF signaling pathways in NGF-induced cartilage formation to elucidate the chondrogenic differentiation mechanism of snake venom NGF, which may provide a theoretical basis for the optimal design of cartilage-induced growth factors.

生长因子可诱导干细胞成软骨分化，但是常用的转化生长因子（Transforming growth factor β1, TGF-β1）价格昂贵、半衰期短，且容易引起功能异质性，不利于临床应用。我们的前期研究发现了从眼镜蛇毒中提取的天然活性蛋白—神经生长因子（NGF）具有特异性成软骨诱导作用，且效果相比于TGF-β1更为显著。进一步研究其分子生物学机制，基于RNA-Seq、生物信息学分析及实验验证，预测蛇毒NGF可能通过抑制PHLPP1激活TNF信号通路上调sox9表达，从而诱导骨髓间充质干细胞（BMSCs）成软骨分化。为进一步证实此假说，本项目将研究PHLPP1及TNF信号通路在NGF诱导软骨形成中的作用及机制研究，以及对软骨生成的影响，阐明蛇毒NGF成软骨分化机理，为软骨诱导生长因子的优化设计奠定理论基础。

关节软骨由于缺乏血管及淋巴管，并且软骨细胞分裂能力有限，一旦损伤，难以自我修复。骨髓间充质干细胞（BMSCs）作为关节软骨修复的潜在种子细胞，通常可通过转化生长因子（TGF-β1）诱导成软骨细胞，用于关节软骨修复。但是，由于其价格昂贵、半衰期短，且容易引起功能异质性，不利于临床推广应用。我们前期已证实眼镜蛇毒神经生长因子（NGF）具有特异性诱导干细胞成软骨分化的作用。然而，其诱导的分子生物学机制尚未清楚。本项目基于RNA-Seq、生物信息学分析及实验验证，预测PHLPP1介导TNF信号通路可能参与NGF诱导BMSCs成软骨分化过程。研究结果表明，在NGF诱导BMSCs成软骨分化过程中，敲除PHLPP1显著促进软骨特异性标记物表达及软骨再生修复，加入TNF信号通路抑制剂LEN则对成软骨分化起抑制作用；而PHLPP1过表达的BMSCs经NGF诱导后，成软骨分化作用显著降低，加入TNF信号通路激活剂PMA则成软骨分化得到恢复。表明NGF通过抑制PHLPP1介导TNF信号通路激活，从而诱导BMSCs成软骨分化。本项目阐明了对眼镜蛇毒NGF诱导软骨形成的分子机制，建立了分子与生长因子之间的联系，为解决临床关节软骨再生难题提供新思路。