神经营养因子3-壳聚糖载体诱导神经干细胞分化为神经元的亚型及其电生理特性

Neurotrophic factor 3-chitosan carrier induces neural stem cells to differentiate into neuronal subtypes and their electrophysiological properties

背景:前期研究已证实神经营养因子3-壳聚糖载体可支持神经干细胞的存活和增殖,同时可高效诱导神经干细胞向神经元方向分化。目的:观察神经营养因子3-壳聚糖载体对神经元发育进程、发育各阶段电生理特性及发育成熟神经元亚型的影响。方法:取第3代新生大鼠脊髓神经干细胞,分4组培养:空白对照组加入神经干细胞培养基,壳聚糖组加入含壳聚糖的神经干细胞培养基,NT3组加入含神经营养因子3的神经干细胞培养基,NT3-壳聚糖组加入含神经营养因子3-壳聚糖载体的神经干细胞培养基。利用免疫荧光染色观察神经干细胞发育各阶段标志物表达情况,借助全细胞膜片钳技术评价神经干细胞发育过程中电生理特性的变化情况,利用免疫荧光染色观察神经干细胞分化21 d后中间神经元的亚型。结果与结论:①Nestin、DCX、Tuj1及MAP2免疫荧光染色显示,神经营养因子3-壳聚糖载体维持了神经干细胞池的稳态,并且通过加速神经母细胞的发育进程来促进神经元发育成熟;②全细胞膜片钳记录发育过程中的细胞发现,营养因子神经营养因子3和神经营养因子3-壳聚糖在发育早期对神经干细胞膜功能以及细胞膜上离子通道的发育成熟具有一定的促进作用,但是仅有神经营养因子3-壳聚糖可将这一优势维持到发育中后期,即分化后7-14 d;③免疫荧光染色显示,神经干细胞分化21 d后,NT3-壳聚糖组成熟神经元可表达运动神经元特异性标记物HB9、V1类型中间神经元FOXP1、V2类型中间神经元特异性标记物LHX3,以及调控机械性痛觉感觉中间神经元的特异性标记物VGLUT3;④结果显示,神经营养因子3-壳聚糖载体促进了神经干细胞向神经母细胞的发育,在发育早期对细胞膜功能及细胞膜上的离子通道发育成熟具有一定的促进作用,可诱导发育成熟的神经元亚型多样化。

BACKGROUND:Previous studies have confirmed that the neurotrophic factor 3-chitosan carrier can support the survival and proliferation of neural stem cells,and can efficiently induce neural stem cells to differentiate into neurons.OBJECTIVE:To observe the effects of neurotrophic factor 3-chitosan carrier on neuronal development and its electrophysiological properties at various stages of development and the subtypes of mature neurons.METHODS:Passage 3 spinal cord neural stem cells of neonatal rats were collected and cultured in four groups.The blank control group was added with neural stem cell culture medium.Chitosan group was added with neural stem cell culture medium containing chitosan.Neurotrophic factor 3 group was added with neural stem cell medium containing neurotrophic factor 3.Neurotrophic factor 3-chitosan group was added with neural stem cell medium containing neurotrophic factor 3-chitosan carrier.Immunofluorescence staining was performed to observe the development of neurons at various stages.The changes in electrophysiological properties during neural stem cell development were evaluated by whole-cell patch-clamp technology.Interneuron subtypes were visualized by immunofluorescence staining 21 days after neural stem cell differentiation.RESULTS AND CONCLUSION:(1)Immunofluorescence staining for Nestin,DCX,Tuj1 and MAP2 showed that the neurotrophic factor 3-chitosan carrier maintained the homeostasis of the neural stem cell pool and promoted neuronal maturation by accelerating the developmental process of neuroblasts.(2)Whole-cell patch-clamp technology results demonstrated that the neurotrophic factor 3 and neurotrophic factor 3-chitosan promoted cell membrane function and the maturation of ion channels on the cell membrane early in development.However,only neurotrophic factor 3-chitosan can maintain this advantage to the middle and late stages of development,that is,7-14 days after differentiation.(3)Immunofluorescence staining exhibited that after 21 days of neural stem cell differentiation,mature neurons in the neurotrophic factor 3-chitosan group could express the motor neuron specific marker HB9,V1 type interneuron FOXP1,V2 type interneuron specific marker LHX3,and regulate a specific marker VGLUT3 that regulates mechanical pain sensory interneurons.(4)These results have verified that the neurotrophic factor 3-chitosan carrier can promote the development of neural stem cells to neuroblasts,and the function of the cell membrane and the maturation of ion channels on the cell membrane in the early stage of development to a certain degree,and can induce the diversification of mature neuronal subtypes.