去分化人脐带间充质干细胞通过重编程基因表达参与缺氧缺血性脑损伤大鼠神经修复

Dedifferentiated human umbilical cord mesenchymal stem cells participate in neurological restoration in rats with hypoxic-ischemic brain damage by reprogramming gene expression

摘要:

目的：探讨人脐带间充质干细胞（human umbilical cord mesenchymal stem cells，hUC-MSCs）、神经分化hUC-MSCs（Dif）、神经去分化hUC-MSCs（De-Dif）及神经再分化hUC-MSCs（Re-Dif）4种状态的细胞移植治疗缺氧缺血性脑损伤（hypoxic-is－chemic brain damage，HIBD）大鼠治疗效果，期望找寻治疗效率更优的种子细胞。方法：体外培养hUC-MSCs，按MNM成神经诱导方法进行神经分化、去分化、再分化诱导，通过对4种状态细胞光镜下形态观察及全基因表达谱芯片分析；移植入HIBD神经损伤动物体内，进行功能学检测；应用基因和蛋白检测手段，对最优种子细胞进行可能机制探讨。结果：hUC-MSCs在体外可成功进行神经分化、去分化、再分化诱导；全基因表达谱芯片发现，诱导过程中hUC-MSCs、Dif、De-Dif及Re-Dif 4种状态细胞基因进行重编程表达，De-Dif与hUC-MSCs基因表达模式最相近；将4组细胞移植入HIBD实验大鼠体内，发现4种细胞均可促进HIBD大鼠神经功能恢复，但De-Dif较其他3组细胞发挥更大的神经修复潜能，差异具有统计学意义（P=0.000）；对hUC-MSCs与De-Dif 2组间进行GO生物学功能分析，发现De-Dif主要参与缺氧反应；De-Dif组较hUC-MSCs组高表达神经标志物MAP2、NSE、Tau、β-tubulinⅢ，干性基因C-MYC、NANOG和视黄酸核受体RARβ。结论：去分化过程可能通过重编程调控神经标志物基因MAP2、NSE、Tau、 β-tubulinⅢ，干性基因C-MYC、NANOG及RARβ基因表达，促进De-Dif参与HIBD神经修复。

Abstract:

Objective：To investigate the effect of transplantation of human umbilical cord mesenchymal stem cells（hUC-MSCs），neu－ronal-differentiation hUC-MSCs（Dif），neuronal-dedifferentiation hUC-MSCs（De-Dif），and neuronal-redifferentiation hUC-MSCs （Re-Dif） in the treatment of rats with hypoxic-ischemic brain damage（HIBD），and to find seed cells with better therapeutic efficiency. Methods：The hUC-MSCs were cultured in vitro，and neuronal differentiation，dedifferentiation，and redifferentiation were induced by the MNM neural induction method. The morphology of the cells in these four states was observed under a light microscope，and gene expression was analyzed by whole-gene expression profile microarray. The cells were transplanted into HIBD animals with nerve injury to perform functional detection. The gene and protein detection methods were used to investigate the possible mecha－nism of optimal seed cells. Results：Neuronal differentiation，dedifferentiation，and redifferentiation of hUC-MSCs were suc－cessfully performed in vitro. The whole-gene expression profile microarray showed reprogramming expression of genes in hUC-MSCs，Dif，De-Dif，and Re-Dif，and De-Dif and hUC-MSCs had similar gene expression patterns. The four groups of cells were trans－planted into the HIBD experimental rats，and the results showed that all four types of cells promoted neurological function recovery in HIBD rats，and De-Dif had a greater potential for nerve repair than the other three types of cells（P=0.000）. Gene ontology analysis was performed for hUC-MSCs and De-Dif，and the results showed that De-Dif was mainly involved in hypoxia response. Compared with the hUC-MSCs group，the De-Dif group had significantly higher expression of the nerve markers microtubule associated protein 2（MAP2），neuron-specific enolase（NSE），Tau，and β-tubulinⅢ and the stem genes C-MYC，NANOG，and RARβ. Conclusion：Ded－ifferentiation may regulate the expression of the nerve markers MAP2，NSE，Tau，and β-tubulinⅢ and the stem genes C-MYC，NANOG，and RARβ by reprogramming，which promotes the participation of De-Dif in HIBD nerve repair.