重复经脊髓磁刺激对Notch1通路调控小鼠脊髓神经干细胞激活效应的作用

The role of repetitive trans-spinal magnetic stimulation in regulating the activation of spinal cord derived neural stem cells through Notch1

目的:观察重复经脊髓磁刺激(repetitive trans-spinal magnetic stimulation,rTSMS)通过Notch1通路对脊髓神经干细胞(neural stem cells,NSCs)的作用。方法:孕龄16周C57BL/6小鼠处死后,迅速分离胎鼠全长脊髓,体外培养小鼠脊髓来源NSCs,通过单细胞测序及RNA速率分析检测脊髓NSCs亚群及速率变化特征。将体外培养的NSCs分为对照组、干预组及实验组。对照组将刺激探头置于培养皿上,不给予rTSMS刺激干预;干预组将刺激探头置于培养皿上,采用20Hz高频rTSMS、1500个脉冲刺激;实验组在转染Notch1 shRNA慢病毒后,进行rTSMS刺激,方案同干预组。采用Western blot检测各组Notch1表达量,免疫荧光染色和克隆球形成实验检测NSCs激活情况。结果:体外培养的脊髓NSCs可以诱导分化成为神经元、星形胶质细胞和少突胶质细胞,单细胞测序显示NSCs其可分为6个亚群并具有特殊异质性,Notch1主要表达在dNSCs和pNSCs两个亚群中。Notch1 shRNA慢病毒转染第5天后,细胞中Notch1蛋白表达量明显下降(对照组1.080±0.086 vs实验组0.520±0.041,P<0.05)。随后对照组给予假刺激,干预组及实验组给予rTSMS刺激,发现rTSMS显著提高NSCs内Notch1表达(对照组1.030±0.068 vs干预组1.480±0.087,P<0.05)。rTSMS干预后克隆球直径增大(对照组88.5±7.5 vs干预组106.2±8.8 vs实验组89.2±8.1,P<0.05)和BrdU阳性细胞率提高(对照组38.6±3.7 vs干预组50.3±4.6,P<0.05),有统计学差异。Notch1 shRNA干扰后,克隆球直径以及BrdU阳性细胞率均小于对照组,有统计学差异(P<0.05)。结论:rTSMS可在体外介导Notch1促进NSCs有效激活。

Objectives:To observe the effect of repetitive trans-spinal magnetic stimulation(rTSMS)on the activation of spinal cord derived neural stem cells(NSCs)through Notch1.Methods:The complete spinal cord of fetal C57BL/6 mice at 16 weeks of gestation was promptly extracted following euthanasia.NSCs obtained from the spinal cord were cultured in vitro.Subsets and rate changes of NSCs were analyzed using single cell sequencing and RNA profiling.The cultured NSCs were divided into control group,intervention(rTSMS)group and experiment(rTSMS+Notch1 shRNA)group.The control group was treated with sham stimulation.The rTSMS group was treated with 20Hz high frequency stimulation with 1500 pulses.The rTSMS+Notch1 shRNA group received the same rTSMS intervention after transfection with Notch1 shRNA lentivirus.The expression of Notch1 was detected by Western blot,and the activation of NSCs was detected by immunofluorescence staining and sphere formation test.Results:Spinal cord derived NSCs cultured in vitro could differentiate into neurons,astrocytes and oligodendrocytes.Single cell sequencing showed that NSCs could be divided into 6 subgroups with special heterogeneity,and Notch1 mainly expressed in the subgroups of dNSCs and pNSCs.The expression of Notch1 in the cells decreased significantly on the 5th day after transfection with Notch1 shRNA lentivirus(Control group:1.080±0.086 vs Experiment group:0.520±0.041,P<0.05).rTSMS intervention could significantly up-regulate the expression of Notch1(Control group:1.030±0.068 vs Intervention group:1.480±0.087,P<0.05).The diameter of the neurosphere(Control group:88.5±7.5 vs Intervention group:106.2±8.8 vs Experiment group:89.2±8.1,P<0.05)and the rate of BrdU positive cells were significantly increased(Control group:38.6±3.7 vs Intervention group:50.3±4.6,P<0.05)after rTSMS intervention.After Notch1 shRNA,the neurosphere diameter and BrdU positive cell rate(Intervention group:50.3±4.6 vs Experiment group:35.1±3.5,P<0.05)significantly decreased in the experiment group.Conclusions:rTSMS can promote activation of NSCs through Notch1 in vitro.