内质网应激信号在碳纳米管调控BMSC成骨分化与凋亡中的作用及机制研究

Carbon nanotubes have important application in the field of regenerative medicine, especially as the matrix for stem cell growth and differentiation. It is of great clinical significance to understand the effect on the cell differentiation and apoptosis from carbon nanotubes. So far, the mechanisms underline the effects on differentiation and apoptosis of stem cells derived from carbon nanotubes stimulation are still scarce. We know little about the signal transduction form materials to the cells and the intracellular signal transduction pathway. Our previous study suggested that endoplasmic reticulum stress occurred when the bone marrow mesenchymal stem cells co-cultured with carbon nanotubes. The endoplasmic reticulum stress(ERs) is believed to guide the bi-phasic response - differentiation pathway or the apoptotic pathway dependent on the extent of ERs. In this project, we will study how the ERs derived form carbon nanotubes regulates the bone marrow mesenchymal stem cell osteogenesis differentiation and apoptosis. Firstly, we will identify the occurrence of ERs in cells when co-cultured with carbon nanotubes and further analysis the relationship between the ERs and the time or the dose of carbon nanotubes. Secondly, in the process of cell apoptosis or cell differentiation, real-time PCR, western blot and proteomics method will be applied to determine the regulation in specific cell process from carbon nanotube and the role of the ERs. The aim of the project is to put a novel insight on the signal transduction pathway from nanomaterials to cells, in which ERs might play a key role and determine the fate of the cells. This project will provide the imformation to optimize the carbon nanotube-based tissue engineering.

碳纳米管作为干细胞生长和分化的基质在再生医学领域具有重要的应用前景，了解其对细胞分化和凋亡的影响具有重大的临床意义。目前，碳纳米管影响干细胞凋亡和分化的分子机制研究非常匮乏。申请者前期研究发现，碳纳米管刺激骨髓间充质干细胞产生了内质网应激。内质网应激具有双向介导细胞凋亡和存活的作用。特别是在成骨细胞的研究中，已证实应激能特异性促进细胞的成骨分化。本项目将围绕内质网应激，分析碳纳米管激活内质网应激信号通路调控骨髓间充质干细胞成骨分化与凋亡的作用机制。首先验证碳纳米管刺激下干细胞内质网应激的发生及其与材料剂量和作用时间之间的关系。再在诱导细胞凋亡和细胞分化的条件下，运用定量PCR,蛋白印迹和蛋白质组学方法研究碳纳米管对干细胞凋亡和分化的调控机制，分析内质网应激在调控过程中的作用，揭示纳米材料信号- - 内质网应激信号- - 细胞效应信号之间的信号传递路径，为优化碳纳米管基组织工程支架材料提供实验依据。

碳纳米管作为干细胞生长和分化的基质在再生医学领域具有重要的应用前景，了解其对细胞分化和凋亡的影响具有重大的临床意义。目前，碳纳米管影响干细胞凋亡和分化的分子机制研究非常匮乏。申请者在项目资助下，证实了碳纳米管刺激下骨髓间充质干细胞发生了内质网应激反应；并在诱导细胞凋亡和细胞分化的条件下，综合运用定量PCR、蛋白印迹、蛋白质组学等方法技术，研究碳纳米管对干细胞凋亡和分化的调控机制，分析内质网应激在碳纳米管调控干细胞凋亡和分化过程中的作用。无血清培养条件模拟了组织修复过程的缺血状态，重现了干细胞在体内经历的细胞凋亡过程。在细胞凋亡过程中，碳纳米管的存在，会通过刺激线粒体凋亡通路，细胞非外源性凋亡通路和内质网应激信号通路进一步加剧细胞的凋亡。在细胞分化过程中，碳纳米管的加入，诱发了内质网应激，包括内质网的扩张，内质网应激一些信号分子的高量表达，参与了骨分化过程中标志蛋白的合成和细胞的矿化。试验结果解释了碳纳米管在干细胞营养缺乏情况下会加剧细胞凋亡，同时在细胞分化过程中又能够促进细胞的分化。这对未来生物医学应用提出了更高的要求，既要规避碳纳米管的细胞毒性，减少细胞凋亡；又要充分利用碳纳米管促进细胞的成骨分化的功能，服务于骨组织工程的医学应用。