【作者】 李香丹；

【导师】 李今子；

【作者基本信息】 延边大学 ， 儿科学， 2019， 博士

【摘要】 第一部分 MiR-21对颞叶癫痫大鼠的影响目的:研究匹罗卡品诱发颞叶癫痫(TLE)的最佳剂量,并探讨miR-21在氯化锂-匹罗卡品诱导的TLE大鼠模型海马中表达的变化规律,进一步通过miR-21激动剂(agomir)及miR-21抑制剂(antagomir)对大鼠双侧海马立体定位注射,阐明不同浓度miR-21 agomir和miR-21 antagomir在体内水平改变对大鼠痫性发作及行为学改变的影响,最后,利用生物信息学方法预测miR-21靶基因,为后续研究miR-21及其靶基因在下游信号通路的作用机制奠定基础。方法:(1)选取4-6周雄性SD大鼠,体重(170±30)g,根据不同浓度的匹罗卡品随机分为25 mg/kg组、35 mg/kg组和45 mg/kg组,经腹腔给予氯化锂和不同浓度匹罗卡品,构建TLE动物模型。观察不同浓度匹罗卡品注射后大鼠行为学、诱发成功率以及死亡率的改变。(2)选取4-6周雄性SD大鼠,体重(170±30)g,随机分为正常组(n=10)与癫痫组(亚组:1d组、14d组、28d组,各组n=10),经腹腔给予氯化锂和匹罗卡品(35 mg/kg)注射,构建TLE动物模型。Digital PCR检测miR-21在正常组及不同时间点TLE大鼠海马组织中的表达变化。(3)选取4-6周雄性SD大鼠,体重(170±30)g,随机分为TLE组(10只/组),miR-21 agomir 干预组(三个浓度亚组:miRNA-21 agomir 2 nmol 组、miR-21(4)agomir 4 nmol 组和 miR-21 agomir 6 nmol 组,各组 10 只)和 miR-21 antagomir 干预组(三个浓度亚组:miRNA-21 antagomir 2 nmol 组、miR-21 antagomir 4 nmol 组和 miR-21 antagomir 6 nmol 组,每组 10 只),予氯化锂-匹罗卡品注射构建TLE模型,观察不同浓度miR-21 agomir和miR-21 antagomir干预后大鼠行为学、诱发成功率以及死亡率的改变。(5)应用TargetScan和UCSC在线工具预测mi R-21的靶基因并做分析,结合相关文献确定需要预测的靶基因。使用293T细胞,利用RT-PCR方法对m RNA进行定量分析,内参为U6。获取靶位点片段后,构建重组质粒进行质粒测序。最后使用Lipofectamine 3000转染miR-21 mimics,使用双荧光素酶基因报告验证miR-21的靶点。结果:(1)随着匹罗卡品浓度增加,发作潜伏期显著缩短(组间比较均P<0.05);与25mg/kg组相比,35 mg/kg组和45 mg/kg组Racine分级和诱发成功率显著升高(P<0.05);与25mg/kg组和35 mg/kg组相比,45 mg/kg组死亡率增加显著(P<0.01)。(2)与正常组相比,TLE大鼠海马中各时间点(亚组:1d组,14d组,28d组)的miR-21表达均增加,其中1d组和28d组表达显著(P<0.01)。(3)随着miR-21 agomir浓度增加,4nmol组与6nmol组发作潜伏期显著缩短(P<0.05);各组间Racine分级和诱发成功率无显著差异(P>0.05);6nmol组较TLE组和2 nmol组死亡率增加显著(P<0.01)。(4)随着miR-21 antagomir浓度增加,4nmol组与6nmol组发作潜伏期显著延长(P<0.05);4nmol组与6nmol组Racine分级显著降低(P<0.05);4nmol组与6nmol组诱发成功率明显降低(P<0.01);各组间死亡率无统计学差异(P>0.05)。(5)转染3’ UTR miR-21-5p mimics的双荧光素酶相对活性较对照组显著降低(P<0.01)。结论:(1)激动大鼠海马中miR-21缩短TLE大鼠发作潜伏期,增加大鼠死亡率;抑制大鼠海马中miR-21延长TLE大鼠发作潜伏期,降低Racine分级和诱发成功率,表明MiR-21参与癫痫的发生发展。(2)SOX7 3’ UTR中有明确的miR-21结合位点,受miR-21的调控。第二部分MiR-21及SOX7对TLE大鼠海马中Wnt-β-catenin通路的影响目的:使用 miR-21 agomir(4 nmol/只)及 miR-21 antagomir(4 nmol/只)干预大鼠双侧海马,利用westernblot、免疫组化、尼氏染色、Bielschowsky银染、DSI体内信号遥测系统,研究大鼠海马内相关蛋白的分子生物学指标、病理改变以及脑电图的变化,探讨TLE大鼠海马中miR-21及其靶蛋白SOX7对Wnt-β-catenin信号通路的影响,阐明miR-21在TLE大鼠海马中的作用机制。方法:(1)选取4-6周雄性SD大鼠,体重(170±30)g,随机分为正常组(n=5)、miR-21 agomir 干预组(亚组:1d 组、14d 组、28d 组,各组 n=5)和 miR-21 antagomir 干预组(亚组:1d 组、14d 组、28d 组,各组 n=5),Digital PCR检测各组大鼠海马中miR-21的表达改变。(2)选取4-6周雄性SD大鼠,体重(170±30)g,随机分为正常组(亚组:1d组、14d组、28d组,n=10)、癫痫组(亚组:1d组、14d组、28d组,各组n=10)、miR-21 agomir干预组(亚组:10d组、14d组、28d组,各组n=10)和miR-21 antagomir干预组(亚组:1d组、14d组、28d组,各组n=10),建模前使用 miR-21 agomir(4 nmol/只)及 miR-21 antagomir(4 nmol/只)干预大鼠海马,然后经腹腔予氯化锂和匹罗卡品注射,构建TLE动物模型。(3)Westernblot 和免疫组织化学法检测 SOX7、β-catenin、cyclin D1 和Wnt-3a在各组大鼠海马组织中的表达变化。(4)尼氏染色观察各组大鼠海马神经元的病理改变;Bielschowsky染色观察 28 天正常组、28 天 TLE 组、28 天 miR-21 agomir 组和 28 天 miR-21 antagomir组大鼠海马神经元轴突增殖变化。(5)选取4-6周雄性SD大鼠,体重(170±30)g,大鼠随机分为正常组、TLE 组、miR-21 agomir 组以及 miR-21 antagomir 组(n=5 只/组),建模前予miR-21 agomir(4 nmol/只)及 miR-21 antagomir(4 nmol/只)干预大鼠海马,同时植入DSI植入子后休养3天,经腹腔予氯化锂-匹罗卡品诱发癫痫,记录脑电图28天。结果:(1)经miR-21 agomir干预后,miR-21的表达在1天和14天显著增加(P<0.05);经miR-21 antagomir干预后,miR-21的表达在1天和14天显著减少(P<0.05)。(2)SOX7在TLE各组表达减少,其中14天和28天减少最显著(P<0.0001);经miR-21 agomir干预后,1天、14天及28天时SOX7表达较正常组显著减少(P<0.0001)。1天时,与TLE组相比SOX7表达明显减少(P<0.0001),14天及28天无明显差异(P>0.05);经miR-21 antagomir干预后,1天、14天及28天时SOX7表达较TLE组显著增加(P<0.0001)。与正常组相比,1天时SOX7表达明显增加(P<0.0001),14天及28天无明显差异(P>0.05);免疫组化结果示大鼠海马CA3与CA1区的SOX7表达与Westernblot一致,DG区各组间无显著差异(P>0.05)。(3)β-catenin与Cyclin D1在TLE各组表达增加,其中14天和28天增加最显著(P<0.05);经miR-21 agomir干预后,1天、14天及28天时β-catenin与Cyclinn D1表达较正常组显著增加(P<0.05)。1天时,与TLE组相比β-catenin与Cyclinn D1均表达增加(P<0.05),14天及28天无明显差异(P>0.05);经 miR-21 antagomir 干预后,1 天、14 天及 28 天时 β-catenin与Cyclinn D1表达较TLE组显著减少(P<0.05)。与正常组相比,1天时β-catenin与Cyclinn D1表达明显减少(P<0.05),14天及28天无明显差异(P>0.05);免疫组化结果示大鼠海马CA3与CA1区的β-catenin与Cyclinn D1表达与Westernblot一致,DG区各组间无显著差异(P>0.05)。(4)Wnt-3a在TLE各组表达增加,其中14天和28天增加最显著(P<0.001);经miR-21 agomir干预后,1天、14天及28天时Wnt-3a表达较正常组显著增加(P<0.0001)。1天时,与TLE组相比Wnt-3a表达明显增加(P<0.0001),14 天及 28 天无明显差异(P>0.05);经 miR-21 antagomir 干预后,1天、14天及28天时Wnt-3a表达较TLE组显著减少(P<0.0001)。与正常组相比,1天时Wnt-3a表达明显减少(P<0.01),14天及28天无明显差异(P>0.05);免疫组化结果示大鼠海马CA3、CA1与DG区的Wnt-3a表达与 Westernblot一致。(5)尼氏染色发现TLE组在14天及28天时大鼠神经元细胞结构明显改变,数目减少,尼氏小体减少,其中以CA1及CA3区神经元细胞的损伤最重;经miR-21 agomir干预发现,1天组大鼠即出现轻微神经元损伤迹象以及少量尼氏小体的减少,14天及28天时神经元损伤较TLE组更显著;经miR-21 antagomir干预发现,14天及28天时大鼠神经元尼氏小体减少不显著,神经元损伤较TLE组轻。(6)Bielschowsky染色发现TLE组可见DG、CA1和CA3区神经元纤维排列紊乱,轴突增粗,多见拖尾深染以及神经纤维缠结;经miR-21 agomir干预后,发现神经元纤维排列紊乱以及轴突增粗现象无改善,拖尾以及深染的轴突仍较多;经miR-21 antagomir干预后,发现神经元纤维排列紊乱以及轴突增粗现象减轻,拖尾以及深染的轴突减少。(7)TLE组较正常组的总棘波数、总棘波间隔时间以及平均棘波间隔时间均显著增加(P<0.0001);经miR-21 agomir干预后,总棘波数、总棘波间隔时间以及平均棘波间隔时间较正常组仍增加显著(P<0.0001);经miR-21 antagomir干预后,总棘波数、总棘波间隔时间以及平均棘波间隔时间较TLE组显著减少(P<0.05)。结论:(1)Wnt-3a在TLE大鼠海马中表达增加,miR-21调节Wnt-3a在大鼠海马中的表达。(2)MiR-21靶向SOX7增加β-catenin及Cyclin D1的表达参与癫痫发生。(3)激动miR-21促进大鼠海马神经元损伤,抑制miR-21减少大鼠海马神经元损伤。更多还原

【Abstract】 PART Ⅰ Effect of miR-21 on temporal lobe epileptic ratsObjective:To study the most suitable dose of pilocaripne-induced temporal lobe epilepsy(TLE)and to explore and discuss the change of miR-21 expression in hippocampus of lithium chloride-pilocarpine-induced TLE rat model.And further to conduct bilateral hippocampal stereotactic injection intervention by miR-21 agomir and miR-21 antagomir and to know about the effects of miR-21 agomir and miR-21 antagomir on epileptic seizures and behavioral changes in rats with different concentrations.For further research,bioinformatics methods were used to predict the target genes,which laid a foundation for the subsequent study of downstream targets in the development of TLE.Method:(1)Male Sprague-Dawley rats aged 4-6 weeks,weighing(170±30)g,were randomly divided into 25 mg/kg group,35 mg/kg group and 45 mg/kg group.The TLE rat model was constructed by intraperitoneal administration of lithium chloride and different concentrations of pilocarpine.Changes in behavior,induction success rate,and mortality of rats after injection of different concentrations of pilocarpine were observed.(2)Male Sprague-Dawley rats aged 4-6 weeks,weighing(170±30)g,were randomly divided into normal control(n=10)and epilepsy group(subgroup:1d group,14d group,28d group,n=10).The TLE animal model was constructed by intraperitoneal administration of lithium chloride and pilocarpine(35 mg/kg).The expression of miR-21 in hippocampus of TLE rats in normal control and epilepsy groups at different time points was detected by Digital PCR.(3)Male Sprague-Dawley rats aged 4-6 weeks,weighing(170±30)g,were randomly divided into TLE group(10 in each group),miR-21 agomir intervention group(three concentration subgroups:miR-21 agomir 2 nmol group,miR-21 agomir 4 nmol group and miR-21 agomir 6 nmol group.Ten in each group)and miR-21 antagomir intervention group(three concentration subgroups:miR-21 antagomir 2 nmol group,miR-21 antagomir 4 nmol group and miR-21 antagomir 6 nmol group.Ten in each group).They were injected with lithium chloride-pilocarpine to construct TLE model.Changes in behavior,induction success rate,and mortality were observed in rats at different concentrations of miR-21 agomir and miR-21 antagomir.(4)The Target Scan and UCSC online tools were used to predict and analyze the target genes of miR-21,and the relevant literature was used to determine the target genes to be predicted.Quantitative analysis of m RNA by RT-PCR using 293T cells with internal reference U6.After obtaining the target site fragment,a recombinant plasmid was constructed for plasmid sequencing.Finally,miR-21 mimics was transfected with Lipofectamine 3000,and the target of miR-21 was verified using a dual luciferase gene reporter assay.Results:(1)Comparison of epilepsy induced by pilocarpine with different concentrations:with the increase of concentration of the pilocarpine,seizure latency was observably shortened(P<0.05 between all groups).Compared with 25 mg/kg group,Racine classification and induction success rate in 35 mg/kg group and 45 mg/kg group increased significantly(P<0.05).Compared with 25 mg/kg group and 35 mg/kg group,mortality rate in 45 mg/kg group increased significantly(P<0.01).(2)Expression of miR-21 in the hippocampus of TLE rats:compared with the normal group,the expression of miR-21 in the hippocampus of TLE rats at different time points(subgroup:1d group,14d group,28d group)increased.And miR-21 increased most significantly in 1d group and 28d group(P<0.01).(3)Epileptic seizure and behavioral changes of TLE rats after intervention of miR-21 agomir:with the increase of miR-21 agomir concentration,seizure latency in 4nmol group and 6nmol group was observably shortened(P<0.05).There was no obvious difference in Racine classification and induction success rate among the groups(P>0.05).The mortality rate in 6nmol group was observably higher than that in TLE group and 2 nmol group(P<0.01).(4)Epileptic seizures and behavioral changes of TLE rats after intervention of miR-21 antagomir:with the increase of miR-21 antagomir concentration,seizure latency in 4nmol group and 6nmol group was significantly prolonged(P<0.05).Racine classification in 4nmol group and 6nmol group decreased observably(P<0.05).The success rate of induction in 4nmol group and 6nmol group decreased observably(P<0.01).There was no statistical difference about mortality rate among all groups(P<0.05).(5)Transfection of 3’ UTR miR-21-5p mimics and pLuc-SOX7 3’ UTR showed a significant decrease in the relative activity of the dual luciferase compared to normal control.Conclusion:(1)Agonist of miR-21 increased the Racine stage and shortened the seizure latency and increased the mortality of TLE rats;Inhibition of miR-21 reduced the Racine stage and prolonged the seizure latency;These results indicat that miR-21 is involved in the development of epilepsy.(2)SOX7 3’UTR has a miR-21 binding site and is regulated by miR-21.Part Ⅱ Effect of miR-21 and SOX7 on wnt-β-catenin pathway in hippocampus of TLE ratsObjective:By using miR-21 agomir(4 nmol each)and miR-21 antagomir(4 nmol each)to intervene bilateral hippocampus of rats and the apply of westernblot,immunohistochemistry,Nissl staining,Bielschowsky silver staining,DSI in vivo signal telemetry system,this aim is to study the molecular biological indicators,pathological changes and electroencephalogram changes of relevant proteins in the hippocampus of rats,to explore the effects of miR-21 and SOX7 in the hippocampus of TLE rats on Wnt-β-catenin signaling pathway and to clarify the action mechanism of miR-21 in the hippocampus of TLE rats.Method:(1)Male Sprague-Dawley rats aged 4-6 weeks,weighing(170±30)g,were randomly divided into normal group(n=5),miR-21 agomir intervention group(subgroup:1d group,14d group,28d group,n=5 each group)and miR-21 antagomir intervention group(subgroup:1d group,14d group,28d group,n=5 each group).Digital PCR was applied for detecting the expression changes of miR-21 in hippocampus of rats in every group.(2)Male Sprague-Dawley rats aged 4-6 weeks,weighing(170±30)g,were randomly divided into normal group(subgroup:1d,14d,28d,n=10),epilepsy group(subgroup:1d,14d,28d,n=10),miR-21 agomir intervention group(subgroup:1d,14d,28d,n=10 each group)and mir-21 antagonir intervention group(subgroup:1d group,14d group,28d group,n=10 each group).Before establishing the model,miR-21 agomir(4 nmol each)and miR-21 antagomir(4 nmol each)were used to interfere with the hippocampus of rats,and then lithium chloride and pilocarpine were injected intraperitoneally to construct TLE animal model.Lithium chloride and pilocarpine were introperitoneal inj ection to establish TLE animal model.(3)Westemblot and immunohistochemistry were used to detect the expression changes of SOX7,β-catenin,cyclin D1 and Wnt-3a in hippocampus of rats in every group.(4)Nissl staining was used to observe the pathological changes of hippocampal neurons in every group of rats.Bielschowsky staining was used to observe the axonal proliferation changes of hippocampal neurons in 28-day normal group,28-day TLE group,28-day miR-21 agomir group and 28-day miR-21 antagomir group.(5)Male Sprague-Dawley rats aged 4-6 weeks,weighing(170±30)g,were randomly divided into normal group,TLE group,miR-21 agomir group and miR-21 antomir group(n=5 each group).Before establishing the model,miR-21 agomir(4 nmol each)and miR-21 antomir(4 nmol each)were given to interfere with the hippocampus of the rats.At the same time,DSI implants were implanted and rest for 7 days,lithium chloride-pilocarpine was given through introperitoneal injection to induce epilepsy.And electroencephalogram was recorded for 28 days.Result:(1)After the intervention of miR-21 agomir,the expression of miR-21 increased significantly on the first and fourteenth days(P<0.05);After the intervention of miR-21 antagomir,the expression of miR-21 decreased observably on the first and fourteenth days(P<0.05).(2)The expression of SOX7 in all TLE groups reduced.Among them,there was the most obvious decrease on the fourteenth and twenty-eighth days(P<0.0001);After the intervention of miR-21 agomir,compared with the normal group,the expression of SOX7 was significantly reduced on the first,fourteenth and twenty-eighth days(P<0.0001).On the first day,compared with TLE group,the expression of SOX7 was significantly reduced(P<0.0001).But there was no significant difference on the fourteenth and twenty-eighth days(P>0.05).After the intervention of miR-21 antagomir,compared with TLE group,the expression of SOX7 was significantly increased on the first,fourteenth and twenty-eighth days(P<0.0001).Compared with the normal group,the expression of SOX7 was significantly increased on the first day(P<0.0001).But there was no significant difference on fourteenth and twenty-eighth days(P>0.05).Immunohistochemical results showed that the expression of SOX7 in CA3 and CA1 regions about the hippocampus of rats was consistent with Westernblot.There was no significant difference among groups in DG region(P>0.05).(3)Expression of β-catenin and Cyclin D1 increased in all TLE groups.Among them,there was the most obvious increase on the fourteenth and twenty-eighth days(P<0.05).After the intervention of miR-21 agomir,compared with the normal group,the expression of β-catenin and Cyclin D1 was significantly increased on the first,fourteenth and twenty-eighth days(P<0.05).On the first day,compared with TLE group,the expression of β-catenin and Cyclin D1 was significantly increased(P<0.05).But there was no significant difference on the fourteenth and twenty-eighth days(P>0.05).After intervention of miR-21 antagomir,compared with TLE group,the expression of β-catenin and Cyclin D1 was significantly decreased on the first,fourteenth and twenty-eighth days(P<0.05).Compared with the normal group,the expression of β-catenin and Cyclin D1 was significantly decreased on the first day(P<0.01).But there was no significant difference on the fourteenth and twenty-eighth days(P>0.05).Immunohistochemical results showed that the expression of β-catenin and Cyclin D1 in CA3 and CA1 regions about the hippocampus of rats was consistent with Westernblot.There was no significant difference among groups in DG region(P>0.05).(4)Expression of Wnt-3a increased in all TLE groups.Among them,there was the most obvious increase on the fourteenth and twenty-eighth days(P<0.0001).After intervention of miR-21 agomir,compared with the normal group,expression of Wnt-3a was significantly increased on the first,fourteenth and twenty-eighth days(P<0.0001).The expression of Wnt-3a in TLE group was significantly increased on the first day(P<0.0001).But there was no significant difference on the fourteenth and twenty-eighth days(P>0.05).After the intervention of miR-21 antagomir,compared with TLE group,expression of Wnt-3a was significantly decreased on the first,fourteenth and twenty-eighth days(P<0.0001).Compared with the normal group,the expression of Wnt-3a was significantly decreased on the first day(P<0.01).But there was no significant difference on the fourteenth and twenty-eighth days(P>0.05).Immunohistochemical results showed that expression of Wnt-3a in CA3,CA1 and DG regions of in hippocampus of rats was consistent with Westernblot.(5)Nissl staining:The structure of neuron cells in TLE group changed obviously on the fourteenth and twenty-eighth days.The number of neuron cells decreased and Nissl corpuscles decreased.Among them,neurons in CA1 and CA3 regions had the most severe damage.After intervention of miR-21 agomir,it was found that the rats in the 1d group appeared signs of slight neuronal damage and a small quantity of Nissl corpuscles reduced.And the neuronal damage was more serious than that in the TLE group in the 14d and 28d groups.After the intervention of miR-21 antagomir,it was found that Nissl corpuscles in neurons of rats did not decrease significantly on the fourteenth and twenty-eighth days.And neuronal damage was lower than that in TLE group.(6)Bielschowsky staining:In TLE group,there is a disordered arrangement about neuronal fibers in DG,CA1 and CA3 regions.Axons are thickened.Tail deep staining and neurofibrillary tangles were more universal.After intervention of miR-21 agomir,it is found that the disorder of neuronal fiber arrangement and axon thickening did not be improved.There were still more trailing and deeply stained axons.After intervention of miR-21 antagomir,it is found that the disorder of neuronal fiber arrangement and axon thickening phenomenon were reduced and the number of trailing and deeply stained axons were decreased.(7)Compared with the normal group,Total Number of Spike Trains,Total Spike Train Duraiton and Average Spike Train Duration in TLE group increased significantly(P<0.0001).After intervention of miR-21 agomir,compared with the normal group,Total Number of Spike Trains,Total Spike Train Duraiton and Average Spike Train Duration in TLE group still increased significantly(P<0.0001).After intervention of miR-21 antagomir,compared with the TLE group,Total Number of Spike Trains,Total Spike Train Duraiton and Average Spike Train Duration decreased observably(P<0.05).Conclusion:(1)The expression of Wnt-3a increased in the hippocampus of TLE rats,and the expression of Wnt-3a was regulated by miR-21.(2)MiR-21 targeted SOX7 to increase the expression of β-catenin and Cyclin D1,thus participating in the development of TLE.(3)Agonist of miR-21 promotes hippocampal neuron injury of TLE rat and inhibition of miR-21 reduces hippocampal neuron injury of TLE rat.更多还原