经颅磁诱导3D微凝胶包载的骨髓源神经祖细胞向神经元分化技术基础研究

How to reduce the heterogeneity of bone marrow mesenchymal stem cells, improve the survival rate of transplanted stem cells, and promote their directional differentiation into neural cells, are the key technical problems that need to be solved in the field of stem cell therapy for nervous system injury diseases. Our previous studies showed that 50Hz/5mT electromagnetic fields can promote bone marrow derived neural progenitor cells (BM-NPCs) differentiation into functional neurons. In this study, single-cell cloning technique is proposed for BM-NPCs purification with suspension culture, the method of whole protein spectrum is used to analyze the specific markers of cell membrane, as well as the method of enriching BM-NPCs by flow cytometry will be established. 3D microgel with emulsion polymerization is used to encapsulate stem cells. The mechanism of calcium related signal transduction pathway will be explored during the process that low intensity transcranial magnetic fields induce BM-NPCs encapsulated with microgel and co-cultured with cortical neurons to differentiate into neurons. By establishing rat models of traumatic brain injury，using techniques of single neuron electrophysiological recording and single cell gene analysis, and methods of cortical somatosensory evoked potential and animal behavior evaluation, to investigate the effects of low intensity transcranial magnetic fields on BM-NPCs encapsulated with microgel for nerve regeneration of brain injury rats and functional recovery, in order to provide a new technique and experimental basis for the treatment of nervous system injury diseases.

如何减少骨髓间充质干细胞异质性的影响，提高移植干细胞的存活率，促进其向神经细胞定向分化，是干细胞移植治疗神经损伤疾病的关键技术问题。我们前期研究显示，50Hz/5mT电磁场可促进骨髓源神经祖细胞(BM-NPCs)向功能性神经元分化。本研究拟采用单细胞克隆技术对BM-NPCs进行悬浮纯化培养，通过蛋白质全谱分析细胞膜特异性标志物，建立流式细胞仪富集BM-NPCs方法，选用乳液聚合法制备3D微凝胶，在与皮层神经元共培养环境中，探讨低强度经颅磁诱导微凝胶包载的BM-NPCs向神经元分化过程中，钙离子相关信号转导通路的作用机制；建立创伤性脑损伤大鼠模型，利用脑片单个神经元电生理记录与单细胞基因分析技术，和皮层体感诱发电位检测与动物行为学评估等方法，探讨低强度经颅磁诱导微凝胶原位包载的BM-NPCs定向分化技术，对脑损伤神经再生和功能恢复的作用，为治疗中枢神经损伤疾病提供新技术和实验依据。

本研究对BM-NPCs进行了悬浮纯化培养，建立了富集BM-NPCs方法，减少干细胞异质性的影响；观察与皮层神经元共培养环境中，探讨了低强度经颅磁诱导微凝胶包载的BM-NPCs向神经元分化过程中的作用机制 ；建立了创伤性脑损伤大鼠模型，探讨了低强度经颅磁诱导微凝胶原位包载的BM-N PCs定向分化技术，对脑损伤神经再生和功能恢复的作用，为治疗中枢神经损伤疾病提供新技术和实验依据。通过两年来课题组努力工作，我们建立了BM-NPCs进行悬浮纯化培养，富集BM-NPCs的方法，对其细胞学特性进行了鉴定，体外进行了与与皮层神经元共培养环境，体内移植到脑损伤大鼠动物模型，观察到了其向神经细胞分化，促进神经功能恢复的作用。同时，研究中我们体外研究BM-NPC会影响小胶质细胞自噬，其携带外泌体miR‑32‑3p，可能参与调控免疫微环境，减少神经炎症反应，为下一步研究做了准备。同时，我们观察到低强度经颅磁具有促进BM-NPC向神经细胞诱导分化，提高脑损伤神经修复作用。