整合素β1（ITGB1）和腱生蛋白C（TNC）在材料诱导软骨形成中的作用和机制研究

Materials that can induce chondrogenesis hold promise in the repair of cartilage defect because of their activation potential on self-regeneration of the host.Based on our previous findings that collagen hydrogels have the ability to induce chondrogenesis, we explored the underlying mechanism. The preliminary results showed that the mechanism of chondrogenesis induced by materials have common with that by growth factors, in which both materials and growth factors can regulate the downstream of TGF-β signaling pathway. But the receptors driven by materials are still unknown to us. We further analyzed the data of related gene microarrays on chondrogenic induction by the method of biomedical informatics. Based on the signaling pathway of ECM-receptor interactions, we predicted that integrin β1(ITGB1) and tenascin C (Tnc) may be involved in the materials induced chondrogenesis. Combined with reports on the two genes and the gene/protein-gene/protein interaction network, it is suggested that ITGB1 and TNC may be associated with chondrogenesis via the regulation of the downstream of TGF-β signaling pathway. In the in vitro model of chondrogenesis induced by materials, we verified that both ITGB1 and TNC were in significant relevance with the downstream of TGF-β signaling pathway and chondrogenesis. Thus, we hypothesized that materials may induce chondrogenesis by activating ITGB1 and then TNC to regulate the downstream of TGF-β signaling pathway to finally produce cartilage specific genes and proteins. To verify the presumption, the effects and underlying mechanism of ITGB1 and TNC on materials driven chondrogenesis as well as their association with materials-based cartilage repair will be explored in this study. This project is intended to establish the contact of materials with underlying molecular mechanism to conversely guide the design and optimization of materials applied in cartilage regeneration to visualize the long-term rehabilitation of cartilage defect.

软骨诱导性生物材料的提出，可望通过调动机体自身实现软骨修复。前期研究证明了胶原基水凝胶材料具有软骨诱导性，并对其分子机理进行了初步探索。结果表明，材料与生长因子均可调控TGF-β信号通路的下游基因从而诱导软骨形成，但是材料受体通路无法确定。针对这一关键性问题，我们以ECM-受体互作通路为出发点，采用生物信息学方法，通过分析软骨诱导相关芯片，预测出整合素β1（ITGB1）和腱生蛋白C（TNC）基因可能为材料诱导软骨关键基因；文献报道、基因/蛋白互作分析和进一步体外模型实验结果均表明ITGB1、TNC与TGF-β下游通路和软骨生成显著相关。因此，我们提出假说，材料可能通过激活ITGB1，途经TNC，最终调节TGF-β信号通路下游基因，从而诱导软骨形成。为证实该假说，本项目将研究ITGB1和TNC在材料诱导软骨的作用及制研究，以及对软骨修复的影响，由此建立材料与机制的联系，指导材料的优化设计。

关节软骨损伤修复是临床一大难题，目前临床上用于治疗关节软骨损伤的药物及手术均无法令人满意。软骨诱导性材料结合骨髓间充质干细胞（BMSCs）的研究有望取得突破性进展，胶原水凝胶材料已被证明具有软骨诱导性，可通过诱导干细胞成软骨细胞分化实现软骨修复。我们前期发现胶原诱导软骨与生长因子的诱导作用相近，其机制可能是胶原通过受体通路进而调控TGF-β信号通路的下游基因从而诱导软骨形成。然而，存在的主要问题是受体通路无法确定，其具体的分子生物学机制仍然不明确。本课题以ECM-受体互作通路为出发点，采用生物信息方法通过分析并结合相关文献报道及基因/蛋白互作分析，确定了关键基因整合素β1（ITGB1）和腱生蛋白C（TNC）与软骨诱导有关。本课题研究揭示，在体外实验中，ITGB1、TNC基因的敲除显著抑制了胶原诱导BMSCs的成软骨分化，而TNC的过表达显著增强了胶原诱导BMSCs向软骨细胞分化。在胶原诱导软骨缺损修复体内模型中，ITGB1、TNC基因敲除的BMSCs结合胶原水凝胶有意义地影响了软骨缺损修复的效果，而TNC基因过表达的BMSCs结合胶原水凝胶显著促进了软骨缺损修复的效果，明确了ITGB1和TNC在胶原水凝胶修复软骨缺损中的关键作用。并在机制上进一步揭示了ITGB1和TNC通过影响TGF-β信号通路下游基因SMAD2/3的磷酸化，从而诱导软骨生成。由此，本研究加深了对胶原水凝胶诱导软骨形成的分子机制的了解，建立了分子与材料之间的联系，为生物材料联合干细胞在软骨缺损修复与再生以及骨关节炎的治疗等提供重要的理论和实验依据。