microRNA论文低温环境下microRNA对小鼠脑外伤后海马区神经干细胞增殖分化的调控作用Word格式文档下载.docx
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microRNA论文低温环境下microRNA对小鼠脑外伤后海马区神经干细胞增殖分化的调控作用Word格式文档下载.docx
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脑外伤可导致大量神经元变性坏死,进而造成神经功能缺损。
颅脑损伤后的神经功能重建一直是临床治疗中至今尚未解决的难题。
研究证实,脑外伤可诱导成体脑海马齿状回(dentategyrus,DG)亚颗粒区(subgranularzone,SGZ)和室下区(subventricularzone,SVZ)内源性神经干细胞(neuralstemcells,NSCs)增殖分化,一定程度上修复受损的神经功能。
因此,尽可能地提高脑外伤后内源性NSCs增殖分化能力,对于伤后神经功能的重建无疑具有重要意义。
但是,有关脑外伤后NSCs增殖分化调控机制目前尚不完全清楚。
微小RNA(microRNA,miRNA)为一类单链小分子非编码RNA,可通过转录后水平调控靶基因蛋白的表达,有着重要的生理功能,研究已证实其参与了多种生物学过程。
然而,哪些特异miRNA可能参与调控及其如何调控脑外伤后NSCs增殖分化目前仍所知甚少;
此外,低温环境是否会影响脑外伤后miRNA的表达进而影响NSCs增殖分化过程也不明确。
本研究通过建立闭合性脑外伤小鼠模型,4℃低温暴露4h,采用miRNA芯片技术确定脑外伤后海马区]niRNA的表达谱,筛选出差异表达且与NSCs增殖分化相关的特异miRNA,并进行real-timePCR验证。
随后挑选1-2个miRNA,进一步观察低温环境下脑外伤后不同时间点海马区特异1miRNA和其相关靶基因的表达变化以及内源性NSCs的增殖情况,以探讨miRNA对脑外伤后NSCs增殖调控作用及低温环境对该调控作用的影响。
研究内容和方法:
1.闭合性脑外伤小鼠模型的建立和实验分组采用改良的自由落体法构建闭合性脑外伤小鼠模型,于脑损伤后24h处死动物,对脑组织进行HE染色,从病理学上鉴定模型是否成功。
实验分为常温脑外伤组和低温脑外伤组,两组又分别分为假手术组、脑外伤4h、1d和3d组。
低温脑外伤组动物脑损伤后放置于~4℃低温环境中饲养4h,随后置于~25℃室温喂养。
常温脑外伤组动物脑损伤后置于~25℃室温喂养。
假手术组不做脑损伤处理。
于脑损伤后4h、1d和3d分批处死动物,收集伤侧海马组织待检。
2.脑外伤后海马区miRNA表达谱的变化取常温假手术组(对照组)、常温脑外伤4h组和低温脑外伤4h组海马标本,采用miRNA芯片技术检测低温环境下脑外伤后海马区miRNA的表达谱,筛选脑外伤后差异表达且与NSCs增殖分化相关的特异miRNA,并进行real-timePCR验证。
3.脑外伤后海马区miR-34a、Notch1信号分子的表达变化和内源性NSCs的增殖情况3.1采用real-timePCR检测脑外伤各时间点miR-34a的表达水平,明确脑外伤后miR-34a的动态表达变化。
3.2采用real-timePCR检测脑外伤后各时间Notch1mRNA的表达水平,明确Notch1基因的表达变化;
采用免疫荧光染色检测脑外伤后3d组海马DG区Notch1阳性细胞的表达,明确脑外伤后Notch1蛋白的表达变化。
3.3采用免疫荧光染色检测脑外伤后3d组海马DG区Nestin阳性细胞的表达,明确脑外伤后内源性NSCs的增殖情况。
结果:
1.成功建立闭合性脑外伤小鼠模型:
病理学观察可见脑外伤动物脑组织挫裂伤明显,细胞高度水肿,神经元坏死,数目减少,伴星形胶质细胞增生,表明本研究成功建立了闭合性脑外伤小鼠模型。
2.脑外伤后海马区miRNA表达谱的变化:
2.1芯片结果:
(1)与对照组比较,常温脑外伤组有24个miRNA上调2倍以上,7个miRNA下调2倍以上;
低温脑外伤组有20个IniRNA上调2倍以上,3个miRNA下调2倍以上,结果表明脑外伤可导致海马区多个miRNA表达发生变化。
(2)与对照组比较,常温脑外伤组和低温脑外伤组miR-200b表达分别上调(2.45±
0.21)倍和(1.11±
0.15)倍;
而miR-34a则分别下调(2.37±
0.28)倍和(1.65±
0.16)倍,表明在脑外伤后海马区1miR-200b和miR-34a的表达均发生明显变化。
2.2real-timePCR验证miR-200b和miR-34a的表达:
(1)与对照组比较,常温脑外伤组和低温脑外伤组miR-200b表达分别上调(2.95±
0.17)倍和(1.22±
0.11)倍;
而miR-34a则分别下调(2.08±
0.09)倍和(1.47±
0.03)倍,与芯片结果趋势相一致。
(2)与对照组比较,常温脑外伤组miR-200b表达显著上调(P0.05);
常温脑外伤组和低温脑外伤组miR-34a表达均显著下调(P0.05)。
结果提示,脑外伤可下调miR-34a的表达,而低温可抑制伤后miR-34a的表达下调。
3.2Notch1信号分子的表达变化:
(1)Notch1mRNA的表达变化:
real-timePCR结果显示,常温脑外伤4h、1d和3d组Notch1mRNA表达均明显高于常温假手术组(P0.05),3d组表达则明显高于低温假手术组(P0.05)。
结果提示,脑外伤可上调Notch1mRNA的表达,而低温可抑制伤后Notch1mRNA的表达上调。
(2)Notch1蛋白的表达变化:
免疫荧光染色结果显示,常温脑外伤3d组伤侧海马DG区Notch1阳性细胞数明显高于常温假手术组(18.2±
3.56比0.4±
0.55,P0.05)。
结果提示,脑外伤可诱导海马DG区Notch1蛋白表达上调,而低温可抑制伤后Notch1蛋白的表达。
3.3内源性NSCs的增殖情况:
免疫荧光染色结果显示,常温脑外伤3d组伤侧海马DG区Nestin阳性细胞数明显高于常温假手术组(21.8±
5.07比0.8±
0.45,P0.05)。
结果提示,脑外伤可诱导海马区DG区NSCs增殖,而低温可抑制脑外伤后NSCs的增殖。
结论:
1.脑外伤可导致海马区miR-200b和miR-34a等多个miRNA表达发生变化,而低温环境可影响脑外伤后海马区miRNA的表达。
2.脑外伤后海马区IniR-34a表达显著下调,靶基因Notch1显著上调,且NSCs显著增殖,提示miR-34a/Notch1信号通路可能参与了脑外伤后海马区NSCs增殖过程的调控。
3.低温环境可抑制脑外伤后海马区miR-34a/Notch1信号通路的表达,进而影响伤后NSCs增殖,提示低温环境有可能影响脑外伤的预后恢复。
【英文摘要】Backgroundand:
Recentlyyears,moreandmoreextremelyweatherandclimateeventsfrequentlyhappenallovertheworld.Forexample,theextremelycoldandsnowyweathereventhappenedinsouthernChinainthebeginningoftheyear2008.Theextremelycoldandsnowyweathereventnotonlycanresulttrafficaccidentsandbuildingcollapse,increasingtheincidencerateoftrauma,butalsooftencangettraumapatientsexposedtolowtemperatureaperiodoftimefollowinginjurybecauseofslipperyroadandtrafficjams.Traumaticbraininjury(TBI)isaleadingcauseofdeathanddisabilitywordwidetharthreatensourlifeandhealthseriously.However,itisstillunclearthatwhatchangeswilloccuraboutthepathophisiologyofTBIfollowinglowtemperatureexposure.Hence,itisnecessarytocarryoutpathophisiologicalstudiesofTBIfollowinglowtemperatureexposureactivelyinordertomasteringitspathogenesisregularityandpreventiveandtherapeuticstrategisbetter.AlargenumberofneuralcellsnecrosisleadstopermanentlossofneurologicalfunctionafterTBI.Sofar,therearenoeffectivetreatmentsthatcouldfacilitatetherepairofthelossofneurologicalfunctional.MoreandmorestudieshavedemonstratedthatTBIcaninduceendogenousneuralstemcells(NSCs)proliferationanddifferentiationinthesubgranularzone(SGZ)ofhippocampaldentategyrus(DG)andsubventricularzone(SVZ)andreapairthelossofneurologicalfunction.Therefore,improvingtheproliferationanddifferentiationabilityofNSCsafterinjurywillbeanewtherapeuticstrategeofTBI.MicroRNAs(miRNAs)areaclassofsmallnoncodingRNAsthatactasimportantpost-transcriptionalrepressorsandhavebeenimplicatedinmanybiologicalprocesses.However,whichandhowmiRNAsareinvolvedintrauma-inducedproliferationanddifferentiationofNSCsarestilllargelyunknown;
whetherlowtemperatureexposurecanaffectmiRNAsexpressionafterTBIisstillunclearyet.Inourstudy,miceweresufferedclosedheadinjury(CHI),andthenwereexposedat4℃for4hours.WedetectedhippocampalmiRNAprofilesafterTBIbymiRNAmicroarray,screenedabnormallyexpressedmiRNAsthatareinvolvedinNSCsproliferationanddifferentiationaccordingtoreferencesandbioinformatics,andthenidentifiedtheirexpressionbyreal-timePCR.Subsequently,weinvestigatedexpressionlevelsof1-2ofthesemiRNAsandtheirtargetsandendogenousproliferationofNSCsinthehippocampusafterTBIfollowinglowtemperatureexposuretoexploretheregulationroleofmiRNAsonneuralstemcellsproliferationaftertraumaticbraininjuryfollowinglowtemperatureexposure.Methods:
1.ClosedheadinjurymicemodelandexperimentgroupsMiceweresufferedclosedheadinjuryusingamodifiedweight-dropdevice.At24hoursafterinjury,braintissueswereisolatedandstainedbyHEstainingtoassesswhetherCHImodelwassuccessfullyestablished.AnimalswererandomlydividedintonormaltemperatureexposureTBIgroup(NTBI)andlowtemperatureexposureTBIgroup(LTBI).Eachgroupwasdividedintoshamgroup,4hours,1dayand3daysgrouppost-TBI.NTBIgroupswerekeptat~25/℃.LTBIgroupswerekeptat~4℃for4hours,andthenwerekeptat~25℃.ShamgroupwasnotsufferedCHIascontrol.Animalswerekilledat4hours,1dayand3daysafterinjury.Ipsilateralhippocampustissueswereextractedforexamining.2.ExpressionchangesandvalidationofhippocampalmiRNAprofilesafterTBImiRNAmicroarrywasusedtodeterminehippocampalmiRNAprofilesat4hoursafterTBI.AbnomallyexpressivemiRNAsthatregulateNSCsproliferationanddifferentiationafterTBIwerescreenedaccordingtoreferencesandbioinformatics.Andthen,theirexpressionlevelswerevalidatedusingreal-timePCR.3.ExpressionchangesofmiR-34a,Notch1andproliferationofendogenousNSCsinhippocampusafterTBI3.1real-timePCRwasusedtoexaminemiR-34aexpressionchangesinthehippocampusatdifferenttimepointsafterTBI3.2real-timePCRwasusedtodetectNotch1mRNAexpressionchangesinthehippocampusatdifferenttimepointsafterTBI;
NotchlimmunofluorescencewasusedtoidentifyNotch1proteinexpressionchangesintheDGat3dayspostTBI.3.2ImmunofluorescencewasusedtoidentifyNSCsproliferationinDGat3daysafterTBIbyNestinstaning.Results:
1.AssessmentofCHImicemodel:
Pathologyobservationshowedthatseriouslycerebralcontusion,highlycellularedema,alargenumberofneuronsnecrosisandastrocyteproliferationafterTBI,demonstratingCHImicemodelwassuccessfullyestablished.2.ExpressionchangesofhippocampalmiRNAprofilesafterTBI2.1miRNAsmicroarrayresults:
(1)Comparedtoshamgroup,24miRNAsweremorethan2-foldup-regulated,7miRNAsweremorethan2-folddown-regulatedinNTBIgroup;
20miRNAsweremorethan2-foldup-regulated,3miRNAsweremorethan2-folddown-regulatedinLTBIgroup,reavelingthatTBIcaninducelotsofmiRNAsexpressionchangedinthehippocampus.
(2)Comparedtocontrolgroup,theexpressionlevelsofmiR-200binNTBIgroupandLTBIgroupwereup-regulated(2.45±
0.21)foldand(1.1±
0.15)fold,respectively;
theexpressionlevelsofmiR-34aweredown-regulated(2.37±
0.28)and(1.65±
0.16)fold,respectively,suggestingTBIcanchangemiR-200bandmiR-34aexpressioninthehippocampus.2.2ValidationofmiR-200bandmiR-34aexpressionlevelsbyreal-timePCR:
(1)Comparedtocontrolgroup,theexpressionlevelsofmiR-200bwerewereup-regulated(2.95±
0.17)foldand(1.22±
0.11)fold,respectively;
theexpressionlevelsofmiR-34aweredown-regulated(2.08±
0.09)and(1.47±
0.03)fold,respectively.Real-timePCRessentiallyshowed,thesamepatternofexpressionchangesofthetwomiRNAsasobservedwiththemicroarrayanalysis.
(2)TheexpressionlevelsofmiR-200binNTBIgroupwereapparentlyup-regulated(P0.05)comparedtoshamgroup;
theexpressionlevelsofmiR-34ainNTBIgroupandLTBIgroupwerebothapparentlydown-regulatedcomparedtoshamgourp(P0.05),butup-regulatedsignificantlyat3days(P<
0.05);
(3)theup-regulationlevelsofNotchlmRNAat4hours,1dayand3dayspostinjurywerelesscomparedtoNTBIrelativetimepointgroups(P<
0.05),suggestingTBIcaninduceNotchlmRNAup-regulation,whereaslowtemperaturecaninhibititsup-regulation.
(2)ExpressionchangesofNotchlprotein:
immunofluorescenceshowthat,
(1)thenumberofNotch1+cellsintheipsilateralhippocampalDGofNTBI3daysgroupwassignificantlyincreasedcomparedtoNTBIshamgroup(18.2±
3.56vs0.4±
0.55,P<
0.01);
(2)thenumberofNotchl+cellsintheipsilateralhippocampalDGofLTBI3daysgroupwasalsosignificantlyincreasedcomparedtoLTBIshamgroup(10±
2.55vs0.6±
(3)thenumberofNotchl+cellsintheipsilateralhippocamp
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- microRNA 论文 低温 环境 小鼠 脑外伤 海马 神经 干细胞 增殖 分化 调控 作用
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