MAPK/ERK通路对eMSCs/fMSCs干细胞亚群能量代谢的差异性调控在ENS神经再生优化中的应用

Injury or neurodegenerative disorders of the enteric nervous system (ENS) cause gastrointestinal dysfunctions for which there is no effective therapy so far. Our previous study showed that bone marrow mesenchymal stem cells (MSCs) are considered as the hope of compensatory ENS nerve regeneration. However, the maturity, network structure and functional repair of regenerative intestinal neurons are still insufficient and are needed to be optimized. Our previous data have originally investigated that MSCs present as two different phenotypes in vitro: epithelial-like MSCs (eMSCs) and fibroblast-like MSCs (fMSCs) showing different characteristics of stem cell biology. However, the effect and mechanism of eMSCs/fMSCs on neuron regeneration remains unknown. The neuron injury could lead to the typical high glutamate microenvironment. Notably, the glutamate metabolism in tricarboxylic acid cycle (TCA cycle), which mainly regulated by MAPK/ERK signal, has been recently demonstrated to be involved in the regulation of methylation status of stem cells and further effects on stem cell biology. And most interesting, MAPK/ERK signal was differentially expressed in eMSCs/fMSCs. Therefore, we address the hypothesis that the differentially expressed MAPK/ERK signal in eMSCs/fMSCs could differentially progress to neuroregeneration through differential regulation of the tricarboxylic acid cycle metabolism, methylation status and "gliocyte-like" characteristics of eMSCs/fMSCs. The project aims to emphasis the differential regulation of metabolism on eMSCs/fMSCs in neuroregeneration under the typical high glutamate microenviroment of neuron injury. To understand the real pathophysiology regulation of microenviroment, metasbolism and phenotypes diversity of MSCs (eMSCs/fMSCs) in starting neuroregeneration holds the insightful therapeutic potential and optimizing clinical application for patients with gastrointestinal dismotility. The MSCs is a typical cell type with high plasticity. To emphasis the effects of disease-specific microenvironment on cell determination of MSCs helps to overall reveal the nature of biological characteristics of MSCs, and evaluate the benefits and risks of its application in different clinical diseases.

肠神经（ENS）损伤目前尚缺乏有效治疗手段。我们前期报道显示骨髓基质干细胞（MSCs）是代偿性ENS神经再生的希望，然而再生肠神经元的分化程度、网络结构和功能修复仍明显不足。本课题基于“MSCs表型多态性”这一原创发现，充分利用“神经损伤高谷氨酸微环境与干细胞能量代谢”、“ eMSCs/fMSCs与MAPK/ERK差异性表达”这两个重要原创契合点，提出在神经损伤高谷氨酸特征性微环境下，MAPK/ERK通路可能通过差异性调控MSCs两亚群三羧酸循环能量代谢，改变细胞甲基化状态，从而差异性刺激两亚群的“类胶质细胞”特性，差异性诱导神经再生的观点。首次探讨eMSCs/fMSCs通过能量代谢在启动神经再生方面作用机制及效能的异同，为更优化地诱导ENS再生提供理论基础。MSCs是高度可塑的干细胞，强调微环境对MSCs生物学走向的影响，对整体认识MSCs在多种疾病中的临床价值及风险有重要提示意义。

肠神经（ENS）损伤目前尚缺乏有效治疗手段。我们前期报道显示骨髓基质干细胞（MSCs）是代偿性ENS神经再生的希望，然而再生肠神经元的分化程度、网络结构和功能修复仍明显不足。研究发现神经损伤可导致高谷氨酸微环境，细胞能量代谢是维持干细胞生物学特性的重要因素，但细胞能量代谢如何调节 ENS 损伤微环境中 MSCs 的命运尚不清楚。第一部分，我们用BMP-7干预MSCs构建经典的EMT转化模型，使以纤维样细胞为主的fMSCs 转化为以上皮样细胞为主的eMSCs，观察两细胞亚型增殖迁移分化等生物学特性的改变。第二部分，我们测定了在ENS损伤的高谷氨酸微环境中，MSCs的生物学特性、能量代谢和组蛋白甲基化水平的变化。第三部分，谷氨酸脱氢酶(Glud1)催化谷氨酸氧化脱氨为α-酮戊二酸(α-KG)。我们检测了高表达Glud1的MSCs生物学特性、能量代谢和组蛋白甲基化水平的变化。第四部分，我们将高表达Glud1的MSCs定向移植到ENS损伤小鼠的神经损伤部位，以探究它们对肠神经再生的影响。体外实验结果显示：fMSCs的增殖分化能力显著高于eMSCs。在ENS损伤的高谷氨酸微环境中，MSCs的MAPK/ERK信号通路被激活，上调MSCs胶质细胞特征性蛋白的表达。此外，在高谷氨酸微环境中，MSCs细胞内的α-酮戊二酸（α-KG）与琥珀酸的比例、组蛋白去甲基化水平和神经胶质细胞特征性蛋白的表达显着增加（P<0.05）。外源性α-KG干预上调 MSCs H3K9和 H3K27的单甲基化水平，降低其三甲基化水平（P<0.05），且显著上调了神经胶质细胞特征性蛋白的表达（P<0.01）。过表达Glud1的MSCs H3K9和H3K27的单甲基化水平增加，三甲基化水平降低（P<0.05），神经胶质细胞特征性蛋白表达上调（P<0.01）。在体内，过表达Glud1的MSCs通过上调H3K9和H3K27的去甲基化，上调神经胶质细胞特征性蛋白的表达从而促进肠神经的再生修复。MSCs是高度可塑的干细胞，强调微环境对MSCs生物学走向的影响，对整体认识MSCs在多种疾病中的临床价值及风险有重要提示意义。