新型小分子化合物WP312诱导间充质干细胞软骨分化的机制研究

The poor self-healing ability of articular cartilage makes the traditional treatment methods of treating osteoarthritis (OA) face many challenges. In recent years, using mesenchymal stem cells (MSCs) treatment of OA has shown a broad prospect, while the poor efficiency of MSCs differentiated into chondrocytes in vivo seriously hampered the development of this method. This project intends to use small molecule compounds to improve the cartilage differentiation efficiency of MSCs, thereby enhancing the effect of MSCs in the treatment of OA. In the early stage of this project, we used human umbilical cord mesenchymal stem cells (hUCMSCs) to screen out novel compound (WP312) with higher potential for differentiation than the small ones reported in the literature, which can induce hUCMSCs to differentiate into chondrocytes. Applicants will explore the effect of WP312-induced hUCMSCs in repair of OA using an animal model of OA. At the same time, using biochemical analysis and proteomics techniques to deeply analyze the effect of WP312 regulating hUCMSCs differentiation target and molecular mechanism, combined with fluorescence labeling technology and lineage tracing method to clarify the path and mechanism of in vivo repair. This project is expected to identify a new target of stem cell differentiation induced by chondrocytes and to find novel compounds that will provide new strategies for the clinical treatment of OA and accelerate the clinical application of MSCs in the treatment of OA.

由于关节软骨自我修复能力较差，治疗骨关节炎（OA）的传统手段面临诸多挑战。近年来，利用间充质干细胞（MSCs）治疗OA呈现出广阔的前景，但MSCs在体内分化成软骨细胞的效率较低严重制约了该方法的发展。本项目拟利用小分子化合物提高MSCs软骨分化效率，从而增强MSCs治疗OA的效果。前期我们利用人脐带间充质干细胞（hUCMSCs）筛选到比文献报道的小分子KGN分化潜能更高的新型小分子化合物（WP312），能定向诱导hUCMSCs向软骨细胞分化。申请人将利用OA动物模型探究WP312诱导的hUCMSCs修复OA的效果，结合荧光标记技术和谱系追踪技术阐明体内修复的路径和机制。同时，利用生化分析和蛋白质组学等技术深入剖析WP312调控hUCMSCs分化的作用靶标和分子机理。本项目有望鉴定出诱导干细胞软骨分化的新靶标，发现新型小分子化合物，为临床治疗OA提供新的策略，加速MSCs治疗OA的临床应用。

研究背景：临界尺寸骨缺损的治疗是骨科的一大难点。成骨和血管生成是骨修复和重塑过程中的关键问题。MSCs衍生的小细胞外囊泡(sEVs)在组织再生中显示出理想的治疗前景，因为它具有令人满意的优势，包括高稳定性、易于获取和丰富的来源。然而，人脐带间充质干细胞衍生的sEVs(hUC-MSCs-sEVs)对血管化骨再生的影响及其潜在机制仍有待研究。在此，我们旨在探讨hUC-MSCs-sEVs对临界尺寸骨缺损的治疗效果和机制。本项目的顺利实施将为骨缺损的再生修复带来曙光，有望为修复骨缺损提供新的思路和理论依据。.方法：为了研究sEVs在体外潜在的成骨和血管生成作用，我们从hUC-MSCs中提取sEVs，然后将sEVs与BMSCs和HUVECs共同孵育。我们接下来通过荧光素酶报告基因检测和蛋白质印迹研究了sEVs对成骨和血管生成影响的潜在机制。随后我们设计合成了带有Gelma/纳米粘土水凝胶涂层的3D打印生物玻璃支架来加载sEV（BG-gel sEV），以确保sEV在体内的支撑效果和持续功效。最后，使用颅骨缺损模型评估复合支架的血管化骨再生能力。.结果：hUC-MSCs-sEVs促进钙沉积和内皮血管网络的形成，通过递送miR-23a-3p激活PTEN/AKT信号通路诱导成骨分化和血管生成。此外，BG-gelsEVs复合支架在体内实现了血管化骨再生。.结论：这表明hUC-MSCs-sEVs通过传递miR-23a-3p激活PTEN/AKT信号通路来促进成骨和血管生成，从而实现血管化骨再生。