Notch信号系统对骨髓间充质干细胞向GABA能神经元分化的调节作用及机制研究

Evidence to date has shown that bone marrow mesenchymal stem cells (BMSCs) are capable of differentiating into gamma aminobutyric acid (GABA)ergic neurons,and our previous study indicated that Hes1 gene (which is the target of Notch signaling pathway) silencing can promote the GABAergic differentiation of BMSCs. But, the mechanism of GABAergic differentiation of BMSCs is not clear.This study was intended to establish the standard method which induced BMSCs to differentiate into GABAergic neurons firstly.Then to confirm whether Notch signals participated in the GABAergic differentiation of BMSCs or not,we examined the influence on GABAergic differentiation by inhibiting Notch signaling pathway or silencing its effector. Furthermore, we evaluated the regulatory role of Hes1 gene and its closely related upstream (RBPJ) and downstream (Mash1) gene in the Notch signals by analyzing the expression change of these genes in the process of GABAergic differentiation of BMSCs. Additionally, to further investigate the regulatory role of RBPJ, Hes1 and Mash1 gene in the GABAergic differentiation of BMSCs, we examined the influence on GABAergic differentiation with RNAi or overexpression of the RBPJ, Hes1 and Mash1 gene in BMSCs in vitro and in vivo. Upon the results of these essays, we would clarify the regulatory mechanism of Notch signals in the GABAergic differentiation of BMSCs. The current study may shed more light on the GABAergic differentiation potential of BMSCs, and provide the basis of exploring novel strategies of stem cell treatment for epilepsy.

研究证实骨髓间充质干细胞(BMSCs)可向GABA能神经元分化，且我们前期研究发现Notch信号通路靶基因Hes1沉默可促进BMSCs的这一分化潜能，但是具体机制不清。本课题拟首先通过条件诱导BMSCs向GABA能神经元分化，建立标准分化方案；然后依赖Notch信号通路阻断或效应器沉默，观察其对GABA能神经元生成的影响，确定Notch信号系统是否参与调节BMSCs向GABA能神经元分化；继而通过检测Hes1及其密切相关的上/下游基因（RBPJ/Mash1）在BMSCs分化前后的表达变化，明确其参与调节方式；而后采用RNA干扰或过表达上述基因，体内外观察其对GABA能神经元生成的影响，分析Hes1及其上/下游基因在该分化中的调节作用，最终阐明Notch信号系统对BMSCs向GABA能神经元分化的调节机制。阐明该机制将拓展BMSCs向GABA能神经元分化的潜能，为干细胞治疗癫痫开创新路径。

伽马氨基丁酸（gamma-aminobutyric acid, GABA）能神经元损失是癫痫等疾病发生的重要原因，骨髓间充质干细胞（bone marrow mesenchymal stem cells, BMSCs）向GABA能神经元分化潜能为癫痫等疾病的细胞替代治疗提供了新思路。Notch信号通路对干细胞分化的转录调节起关键作用，但在BMSCs分化中的作用机制不清。本课题组首先通过γ分泌酶抑制剂（DAPT）抑制BMSCs中的Notch信号，发现信号组件重组信号结合蛋白Jκ（recombination signal binding protein Jκ, RBPJ）/发状分裂相关增强子1（hairy and enhancer of split 1, Hes1）下调和神经母细胞特异性转移因子1（mammalian achaete-scute homologue 1, Mash1）上调并促进BMSCs向神经元（含GABA能神经元）分化，证实Notch信号通路参与BMSCs向GABA能神经元分化的转录调节。而后通过基因修饰（Hes1 shRNA, RBPJ shRNA或pGC-FU-Mash1）发现，GABA能神经元分化潜能在Hes1基因沉默（合并RBPJ下调和Mash1上调）或Mash1过表达BMSCs后均实现上升，且分化细胞具有突触后抑制的电生理功能。同时发现RBPJ siRNA处理的BMSCs（合并Hes1下调但无Mash1上调）未提高其GABA能神经元产能。体外研究综合证实Mash1依赖的Notch信号通路对BMSCs向GABA能神经元具有重要调节作用。体内实验发现Hes1抑制或Mash1过表达的BMSCs可明显抑制动物模型的癫痫发作和神经元损失，且在海马旁皮质有新生GAD67+（GABA能神经元标志物）阳性细胞产生，但在海马区域（GABA能神经元损失的关键部位）未见细胞分化。动物实验提示基因修饰可扩展BMSCs的细胞治疗潜能。