【作者】 李杰；

【导师】 马跃文；

【作者基本信息】 中国医科大学 ， 康复医学与理疗学， 2020， 硕士

【摘要】 目的:探讨体外放散式冲击波(r ESWT)是否通过miR-124介导神经干细胞增殖。方法:神经干细胞(NE-4C)分为冲击波干预组(rESWT组)、对照组(Control组)、miR-124转染组(miR-124 mimic组)、阴性转染组(NC组)、miR-124转染冲击波干预组(miR-124 mimic+r ESWT组)和阴性转染冲击波干预组(NC+r ESWT组)。所有组别在干预后12、24、72小时进行CCK-8检测、miR-124实时荧光定量PCR检测。r ESWT组、Control组、miR-124 mimic+r ESWT组、NC+r ESWT组在干预后72小时通过Western Blot与免疫荧光检测Nestin蛋白表达情况。大鼠分组为正常组(Normal组)、模型组(I/R组)和实验组(IT组),正常组(Normal组)大鼠不做任何处理,构建大脑中动脉栓塞(MCAO)模型成功后大鼠随机分为模型组(I/R)和实验组(IT),IT组于脑梗后72小时r ESWT干预缺血侧头部,I/R组进行r ESWT假刺激,每3天1次,在MCAO后的1、3、5、7、9、11、13、15、20、29、38天处死大鼠后,检测患侧海马miR-124表达情况,IT组与I/R组选取MCAO后11天、20天、29天和38天进行神经功能评分,PCR、Western Blot与免疫荧光检测Nestin表达情况。结果:1、体外神经干细胞NE-4C在r ESWT干预后12、24和72小时CCK-8增殖曲线明显高于Control组,且与Control组相比,72小时Nestin~+/Brdu~+细胞数目及Nestin蛋白表达也明显增高(P<0.05),而miR-124在12、24小时表达则低于Control组(P<0.05)。2、与NC组相比,转染miR-124 mimic后,miR-124表达明显增高(P<0.05),而细胞增殖曲线明显下降(P<0.05)。此外,冲击波干预转染细胞后,miR-124 mimic+r ESWT组的细胞增殖曲线受到抑制(P<0.01),且72小时Nestin~+/Brdu~+细胞数和Nestin蛋白表达均低于NC+r ESWT组(P<0.05)。3、在动物实验中,脑梗大鼠患侧海马miR-124表达高于正常大鼠并在脑梗死后逐渐增加,r ESWT直接干预大鼠患侧头部后降低了miR-124的表达(P<0.05),增加了Nestin的表达,改善了大鼠神经功能评分,在MCAO后的11、20、29和38天与I/R组相比有统计学差异(P<0.05)。结论:1、过表达miR-124会抑制体外神经干细胞增殖。2、体外放散式冲击波可以通过降低miR-124表达来促进体外神经干细胞增殖。3、脑梗大鼠患侧海马miR-124表达增高,冲击波干预可降低miR-124表达,促进神经干细胞增殖,改善受损神经功能。更多还原

【Abstract】 Objective:TO investigate whether Radial extracorporeal shock wave therapy(r ESWT)regulates the proliferation of neural stem cells through miR-124.Methods:Neural stem cells(NE-4C)were divided into rESWT group,Control group,miR-124 mimic group,negative Control group(NC group),miR-124 mimic+r ESWT group,NC+r ESWT group.All groups were tested for CCK-8 and the expression of miR-124 by real-time PCR at 12,24,and 72 hours after the intervention.r ESWT group,Control group,miR-124 mimic+r ESWT group,and NC+r ESWT group were detected for Nestin protein expression by Western Blot and immunofluorescence at 72hours after the intervention.Rats were divided into normal group,model group(I/R group)and experimental group(IT group).After successfully established the model of middle cerebral artery occlusion(MCAO)model,rats were randomly divided into I/R group and IT group.The IT group were intervened by r ESWT on the ischemic head 72 hours after MCAO,once every 3 days.Rats were sacrificed on day 1,3,5,7,9,11,13,20,29,38 after MCAO,and the expression of miR-124 in the hippocampus of the affected side was measured.The modified Neurological Severity Scores in the IT group and I/R group were evaluated and the expression of Nestin was detected by PCR,Western Blot and immunofluorescence in 11 days,20 days,29 days and 38days after MCAO.Results:1.The proliferation curves of neural stem cells in vitro at12,24 and 72 hours after r ESWT intervention were significantly higher than the Control group,and72-hour Nestin~+/Brdu~+cells and Nestin protein expression also significantly increased compared with the Control group(P<0.05),while the expression of miR-124 at 12and 24 hours was lower than that of the Control group(P<0.05).2.Compared with the negative Control group,the expression of miR-124 increased significantly after transfection of miR-124 mimic(P<0.05),but the cell proliferation curve decreased significantly(P<0.05).In addition,after r ESWT intervention,the cell proliferation curve of miR-124 mimic+r ESWT group was inhibited(P<0.01),and the number of Nestin~+/Brdu~+cells and the expression of Nestin protein were lower than those in the NC+r ESWT group(P<0.05).3.In animal experiments,after cerebral infarction in rats,the expression of miR-124 in the hippocampus of the ischemic side gradually increased,and r ESWT reduced the expression of miR-124 after the intervention of the head of the ischemic side of the rat(P<0.05).r ESWT also increased the expression of Nestin and decreased m NSS score of ischemic rats,Compared with the I/R group at11,20,29 and 38 days after MCAO(P<0.05).Conclusion:1.Overexpression of miR-124 can inhibit neural stem cell proliferation in vitro.2.The r ESWT can promote the proliferation of neural stem cells in vitro by reducing the expression of miR-124.3.Compared with the normal rat,the expression of miR-124 in the hippocampus of the cerebral infarction rats was increased.r ESWT intervention could reduce the expression of miR-124 and promote the proliferation of neural stem cells.更多还原