基于人羊膜间充质干细胞向成骨、软骨细胞定向分化的真菌来源天然小分子诱导剂发现及调控机制

The directional differentiation of stem cells is the frontier in the field of regenerative medicine. To manipulate the fate of stem cells on the basis of the chemical genetics, small-molecule compound exerts its unique advantages with high efficiency and specificity. Human amniotic mesenchymal stem cells (hAMSCs) are recognized as the best ideal source of seed cells for bone and cartilage tissue engineering. It has shown a promising prospect for repairing defects in bone and cartilage, and their functional reconstruction. However, the routine inducers of osteogenic and chondrogenic differentiation only show poor efficiency due to low specificity, sensitivity and safety. Due to the diversity of structure and bioactivity, small-molecule compounds of fungi show a great potential to regulate the differentiation of stem cells. Our preliminary investigation showed that fifteen natural small molecules from Cordyceps species and Ganoderma lucidum could induce osteogenic differentiation of hAMSCs, but their availabilities need further improvement, and their regulatory mechanisms still remain unknown. To explore effective inducers and/or approaches involving these small molecules, this study will optimize the composition of inducer associated with osteogenic and chondrogenic differentiation of hAMSCs to obtain high efficiency inducer/approach, and the inductive efficiency is systematically evaluated at the molecular, cellular and animal level. On the basis of the above result, the molecular biology and functional genomics approaches are applied to explore the molecular mechanism of osteogenic and chondrogenic differentiation by new inducer. This work is helpful for the development of novel small molecule inducers. Also, it may provide new inductive strategy for hAMSCs osteogenic and chondrogenic differentiations. It will lay the groundwork for the hAMSCs therapy of some intractable orthopedic diseases.

基于化学遗传学调控干细胞命运的策略，小分子化合物调控展示出高效、专一的独特优势。人羊膜间充质干细胞（hAMSCs）是骨与软骨组织工程最理想的种子细胞新资源，在骨病损治疗及功能重建等方面展示了良好的前景。但常规成骨、成软骨分化诱导剂专一性差，效率低。真菌次级代谢产物具有结构和活性多样的特点，是发掘干细胞分化诱导剂的重要源泉。前期研究提示，虫草、灵芝来源的15种小分子化合物有诱导hAMSCs成骨分化的潜力。本项目拟进一步有机组合候选化合物，提高效价，获得高效调控hAMSCs成骨、成软骨分化的诱导剂，并在分子、细胞及整体水平上评价其诱导分化及修复能力。进而，采用免疫荧光、免疫印迹、分子生物学和功能基因组学相结合的方法，阐明其调控成骨、成软骨细胞分化的作用机制。这将为hAMSCs成骨、成软骨分化调控提供新的策略，为开发新型干细胞定向分化小分子诱导剂，以及临床应用hAMSCs治疗骨科疾病奠定基础。

骨及软骨缺损的修复与功能重建是生物医学领域一大难题。干细胞组织工程技术飞速发展在骨病损治疗与功能重建方面展示出良好应用前景，但仍面临体内外分化效率低、修复机制不明、疗效欠佳等制约临床应用的瓶颈问题。本项目基于化学遗传学调控干细胞命运的策略，借助天然化合物化学调控具有高效、专一的特点，以人羊膜间充质干细胞（hAMSCs）为研究对象，通过建立高通量成骨、成软骨细胞筛选模型，筛选了500余个天然化合物，发掘到GD-A、BDMC、LIM和HA等具有诱导向成骨细胞、软骨细胞分化潜力的天然化合物，并揭示了其调控分化的新机制，进而创建了 HA与hAMSCs鸡尾法治疗碘乙酸钠诱导的骨性关节炎疾病模型、BDMC联合hAMSCs移植治疗去卵巢诱导的骨质疏松症疾病模型新策略，疗效显著。因此，本研究对拓宽天然化合物新用途及创建骨病损治疗新策略奠定了良好的基础。