基底硬度及纳米支架对胚胎干细胞（ESCs)骨分化的影响及其与Ras-MAPK-Smad通路的关系

Embryonic stem cells are a sort of important totipotent cells with the potential to differentiate into a variety of cell types.There is an evidence indicating that the microenvironment are critical for cells differentiation. It is not clear how and the underlying mechanism of matrix elasticity or three dimensional scaffold regulate ESCs differentiation. Our previous study has suggested that osteogenesis and chondrogenesis of MSCs are regulated obviously by different matrix elasticity. Hard matrix promote osteogenesis of MSCs, meanwhile, soft matrix induce chondrogeneis significantly. In addition, cytoskeleton and RhoA-Rock involved in the process of directional differentiation which was demonstrated via comparing the responsive alteration of MSCs on different elasticity matrix, the involvement of the key signaling pathway Ras-Smad1/5/8/ERK/AKT in osteogenesis differentiation was also confirmed. Based on the privious study, this project is to further study the response of ESCs to matrix elasticity and nanoscaffold in the process of osteogenic differentiation and the expression of key proteins of signaling pathway Ras-MAPK-Smad. Futhermore, we further activate or inhibit the activity of signaling pathway to regulate the osteogenic differentiation of ESCs. Elucidation of the roles of extracellular microenviroment-mechanical factor cues on ESCs differentiation will improve the control of stem cell fate to generate the desired cell types for potential therapeutic usage.

胚胎干细胞（ESCs）是一类具有全能发育潜能的细胞群，细胞所处的微环境对分化至关重要，物理因素如基底硬度和三维纳米支架如何影响分化及其作用机理尚不清楚。课题前期研究发现基底硬度对骨髓间充质干细胞（MSCs）骨分化控制明显，高硬度的基底明显促进MSCs的成骨分化而低弹性硬度的基底则促进软骨分化；通过对比MSCs在不同弹性硬度基底上的响应发现细胞骨架、RhoA-Rock参与定向分化过程，且验证硬基底促进的成骨分化中关键的信号通路Ras-Smad1/5/8/ERK/AKT参与其中。本课题拟在前期研究的基础上，进一步研究ESCs的骨分化过程对基底硬度及纳米支架三维结构的响应，观察Ras-MAPK-Smad信号通路关键蛋白的表达变化，进而通过干扰关键通路活性的方式影响或控制ESCs的骨分化。本研究旨在探讨细胞外微环境-力学因素对胚胎干细胞骨分化的影响，为ESCs在组织工程和再生医学中的应用提供依据。

聚已酸内酯（PCL）被认为是骨组织工程中一种很有前途的合成材料。因其具有独特的生物化学特性和良好的相容性，在临床应用中有望成为干细胞支持骨再生的载体。实验分别从脐带(UC)、骨髓(BM)和脂肪组织(AD)获得人骨髓间充质干细胞(MSCs)。体外实验研究了不同来源的人骨髓间充质干细胞(MSCs)对这种新型合成生物材料的成骨分化能力：首先验证人UC-，BM-和AD的MSCs呈成纤维细胞样形态，并表达特征性标记物，PCL纳米纤维支架可以支持MSC的黏附和增殖， 更重要的是，在PCL纳米纤维支架上培养，可显著提高人MSCs的成骨分化能力，其中骨髓间充质干细胞在这三种间充质干细胞之间表现出更强的分化能力。进一步研究发现PCL纳米纤维支架通过Wnt/β-catenin和Smad 3信号通路持续促进人MSCs的成骨作用。