滑膜间充质干细胞亲和多肽生物学机制及在构建功能化水凝胶组织工程软骨支架中的应用研究

Articular cartilage injury, which is the most common disease of sports medicine, is very hard to be healed. Recently, after a speeding development especially in recent decades, tissue engineering (TE) technology has been a most promising way for cartilage regeneration, since cartilage can hardly regenerate by itself. However, the mobilization and transfer of MSCs to defective or damaged tissues and organs in vivo with high accuracy and efficiency has been a major problem in TE. To address this problem, in the previous study, we identified a seven amino acid peptide sequence (L7) through phage display technology that has a high specific affinity to synovium-derived mesenchymal stem cells (SMSC). In the following study, we conjugated this peptide on the surface of biomaterials for verifying its effect of specific recruitment toward SMSC in vitro, and its effect of promoting the proliferation of SMSC was also observed. However, the mechanism of this peptide is still unclear. So in the present study, we will give a further investigation into the mechanism of this peptide, including locating the target protein of this peptide and the regulation of its downstream signaling pathways. After that, based on the fully understanding of the mechanism, we will conjugate this peptide onto the hyaluronic acid hydrogel to construct an functional hydrogel scaffold for tissue engineering cartilage regeneration, which possesses the ability of the specific recruitment toward endogenous SMSC in vivo.

关节软骨损伤是运动医学领域的难治性伤病。组织工程技术的出现和不断发展，为像关节软骨这样自我修复能力差的组织再生带来了希望，而如何将间充质干细胞准确、高效地募集到组织缺损区域是目前组织工程面临的一个挑战。为此，在我们之前的研究中，成功地利用噬菌体展示技术筛选出对于滑膜间充质干细胞具有高度特异性亲和作用的多肽片段，并在体外实验中通过将该多肽片段藕连到生物材料表面，验证了其对滑膜间充质干细胞的特异性募集作用，并观察到其对于滑膜间充质干细胞的增殖具有促进作用，但其具体作用机制并不清楚。所以，在本研究中我们将对该亲和多肽具体的作用机制进行全面深入地研究，包括寻找其作用的靶蛋白以及相关下游信号通路的调控；之后，在明确该多肽的具体作用机制的基础上，利用该多肽对透明质酸水凝胶支架进行修饰，构建在体内具有特异性募集内源性滑膜间充质干细胞的功能化的水凝胶组织工程软骨支架，用于关节软骨损伤修复，提升修复效果。

本研究利用噬菌体展示技术筛选出了人来源滑膜间充质干细胞亲和多肽序列L7，并验证了其具有优良的亲和性和特异性；鉴定了滑膜间充质干细胞亲和多肽在细胞膜上作用的靶蛋白；利用蚕丝蛋白SF和透明质酸HA多次交联形成互穿网络水凝胶，并用滑膜间充质干细胞亲和多肽对支架进行了修饰，构建了模拟软骨结构并能募集关节腔内内源性干细胞的生物学功能支架；利用间充质干细胞亲和多肽对生物材料进行表面修饰的方法，对构建功能化的组织软骨再生支架， “One-Step”一步法修复关节软骨进行了多种尝试和探索：1）利用筛选出的另一种已被证明特性的骨髓间充质干细胞亲和多肽序列E7，对人脱钙骨颗粒（DBM）使用相同的藕联方式进行了表面修饰，并混合壳聚糖（CS）水凝胶制成混合支架，并对支架的性质及功能进行了评估；2）利用E7对猪腹膜脱细胞基质支架进行修饰，构建了功能化的以天然材料为主题的功能化组织工程软骨支架APM-E7，并在家兔膝关节软骨缺损模型上结合微骨折技术取得了良好的软骨修复效果；3）构建壳聚糖水凝胶-脱钙骨基质复合支架，结合同种异体软骨细胞进行“One-Step”组织工程软骨修复，取得了良好的再生效果；在软骨损伤与再生机制研究方面，发现抑制TGF-b激活酶-1（TAK1）能够有效阻止骨性关节炎中炎性相关的软骨退变发生。.本项目中的研究结果对于构建理想的组织工程软骨支架以及更好地阐明软骨损伤和再生中的具体机制都有着很深远的意义。