内源性调节性T细胞在SAH后脑血管痉挛及脑损伤中的作用

内源性调节性T细胞在SAH后脑血管痉挛及脑损伤中的作用汇报人：张金卉导师：孙保亮2020/1/21研究的目的及意义SAH后脑血管痉挛、炎症反应及神经功能预后的改善是一个非常复杂的病理、生理、临床过程。虽然进行了大量研究，为止其发生机制仍未完全明了。研究小鼠体内内源性调节性T细对SAH后脑血管痉挛、炎症反应及神经功能预后的改善仍将是今后一个时期的热点。随着研究的逐渐深入，将会对内源性调节性T细胞移植SAH的临床治疗及神经功能预后带来深远的影响。2020/1/21国内外相关文献TheKineticsofLymphocyteSubsetsandMacrophagesinSubarachnoidSpaceAfterSubarachnoidHemorrhageinRatsithasbeensuggestedthathumoralimmunityplaysaroleinthepathogenesisofcerebralvasospasmaftersubarachnoidhemorrhage,therehasbeennoquantitativeassayforcellularimmunity.Westudiedthekineticsofimmunecellsinthesubarachnoidspaceaftersubarachnoidhemorrhageintherat.2020/1/21Aserialresponseofimmunoreactivecells,whichresemblesthatofthechronicallergicreactionobservedinautoimmunediseasesordelayed-typehypersensitivity,existsinthesubarachnoidspaceaftersubarachnoidhemorrhage.Thepresentresultssuggestthattheinitialresponseincellularimmunity,whichisfollowedbyhumoralimmunityandeicosanoidreactions,playsaroleinelicitingthedevelopmentofcerebralvasospasm.2020/1/21蛛网膜下腔出血后脑血管痉挛分子机制的研究进展与NO代谢有关的CVSK通道活性的改变文氧合血红蛋白的作用本磷酸二酯酶一V的作用本NO和ET一1的平衡失调2020/1/21与NO代谢无关的CVSRas蛋白的作用前列环素与血栓烷A2失衡自由基与脂质过氧化物增多血管加压素的作用ca代谢紊乱2020/1/21Statin-InducedT-LymphocyteModulationandNeuroprotectionFollowingExperimentalSubarachnoidHemorrhageStatinsinfluenceimmunesystemactivitiesthroughechanismsindependentoftheirlipidloweringproperties.Tcellscanbesubdividedbasedoncytokinesecretionpatternsintotwosubsets:T-helpercellstype1(Th1)andtype2(Th2).IndependentlaboratorystudieshaveshownstatinstobepotentinducersofaTh2switchinimmunecellresponseandbeneuroprotectiveinseveralmodelsofcentralnervoussystem(CNS)disease.Thisstudywasthefirsttoevaluatetheimmunemodulatingeffectsofstatinsinsubarachnoidhemorrhage(SAH).2020/1/21ThepresentstudyelucidatedthepotentialroleofaTh2immuneswitchinstatinprovidedneuroprotectionfollowingSAH.2020/1/21Matrixmetalloproteinase-9concentrationinthecerebralextracellularfluidofpatientsduringtheacutephaseofaneurysmalsubarachnoidhemorrhageElevatedcerebralinterstitialpro-MMP-9relatestoearlybraininjuryinaSAHpatients.Alargerprospectivestudyshouldbeperformedtoconfirmwhetherpatientswithprolongedelevationorasecondpeakofcerebralpro-MMP-9wouldbemorelikelytodevelopDCIandtoconfirmwhetherMMP-9,amediatorofneurovascularinjury,wouldbeworthtoconsiderasapredictorofvasospasm.2020/1/21Trehalosetreatmentsuppressesinflammation,oxidativestress,andvasospasminducedbyexperimentalsubarachnoidhemorrhageSubarachnoidhemorrhage(SAH)frequentlyresultsinseveralcomplications,includingcerebralvasospasm,associatedwithhighmortality.AlthoughcerebralvasospasmisamajorcauseofbraindamagesafterSAH,otherfactorssuchasinflammatoryresponsesandoxidativestressalsocontributetohighmortalityafterSAH.Trehaloseisanon-reducingdisaccharideinwhichtwoglucoseunitsarelinkedbyα,α-1,1-glycosidicbond,andhasbeenshowntoinducetolerancetoavarietyofstressorsinnumerousorganisms.Inthepresentstudy,weinvestigatedtheeffectoftrehaloseoncerebralvasospasm,inflammatoryresponses,andoxidativestressinducedbybloodinvitroandinvivo.2020/1/21trehalosehassuppressiveeffectsonseveralpathologicalventsafterSAH,includingvasospasm,inflammatoryresponses,andlipidperoxidation.TrehalosemaybeanewtherapeuticapproachfortreatmentofcomplicationsafterSAH.2020/1/21propertieswithinthecerebrospinalfluidaftersubarachnoidhemorrhageinvivoandinvitroTofunctionallycharacterizepro-inflammatoryandvasoconstrictivepropertiesofcerebrospinalfluidafteraneurysmalsubarachnoidhemorrhage(SAH)invivoandinvitro.2020/1/21Wefunctionallycharacterizedinflammatoryandvasoactivepropertiesofpatients’CSFafterSAHinvivoandinvitro.Thispro-inflammatorymilieuinthesubarachnoidspacemightplayapivotalroleinthepathophysiologyofearlyanddelayedbraininjuryaswellasvasospasmdevelopmentfollowingSAH.2020/1/21研究内容小鼠体内内源性调节性T细胞对蛛网膜下腔出血（SAH）后脑血管痉挛的影响小鼠体内内源性调节性T细胞移植SAH后神经功能预后的改善作用小鼠体内内源性调节性T细胞移植对SAH后炎症反应的影响2020/1/21每项研究内容均分4组1、Tregs删除组2、SAH模型组3、假手术组4、PBS腹腔注射组体重、窝别、喂养方法相同的小鼠随机分组每组10~15只小鼠；每只小鼠编号后按计算机随机表分4组，双盲（研究者、负责资料收集和分析的人员也不了解分组情况）较好地避免了偏倚。研究方法实验动物处理1、Tregs删除腹腔注射0.25mgCD25特异性抗体小鼠加入肝素抗凝Hank's液洗涤、ELS裂解注射后第2天取外周血加入标记的抗细胞表面特异性抗原的抗体PBS洗两遍2020/1/212、SAH模型制作感觉有阻力时再继续前进3mm,总进入长度约为10mm(已刺破动脉壁）然后迅速将线栓拔出，动脉血灌入，造成蛛网膜下腔出血将制备的线栓通过切口经颈动脉插入颈内动脉，一直送至大脑前动脉与大脑中动脉的分叉处（类似大脑中动脉阻断的方法）首先制作线栓（5-0单丝尼龙线，头端磨成子弹头状，总长度15mm，直径0.2mm）3、假手术：遇到阻力即停止继续插入，并迅速退出线栓4、PBS腹腔注射：经腹腔注射与0.25mgCD25特异性抗体等体积的PBS。2020/1/21一、内源性调节性T细胞对蛛网膜下腔出血（SAH）后脑血管痉挛的检测内容Text基底动脉形态学检测脑动脉匀浆液中一氧化氮合酶mRNA和蛋白含量；NO含量；cGMP含量局部脑血流量循环内皮细胞检测基底动脉形态学检测脑动脉匀浆液中一氧化氮合酶mRNA和蛋白含量；NO含量；cGMP含量局部脑血流量循环内皮细胞检测检测内容2020/1/21局部脑血流量（regionalcerebralbloodflow,rCBF）检测：颅骨开窗，在立体定向仪控制下，在SAH前及SAH后12小时内（或6小时）用激光多普勒血流计动态测量动物顶叶皮层rCBF。2020/1/21基底动脉形态学检测：1、立体显微镜下观察基底动脉（BA）、大脑中动脉（MCA）和大脑前动脉（ACA）并记录它们的直径2、冰冻切片上显微镜下观察基底动脉形态学变化，用图像分析仪测定基底动脉内径、外径与管腔横截面积改用10％明胶和墨水混合液灌注13.5ml／min)15s于SAH后2天、2周，经心脏灌注，10％福尔马林灌注（5.5ml／min）２min后2020/1/21循环内皮细胞检测：于SAH后3天、2周取静脉血标本，用Ⅷ因子相关抗原免疫荧光染色检测循环内皮细胞（circulatingendothelialcell,CEC），以反映血管内皮细胞的损伤。2020/1/21于SAH后3天、2周，取动物脑底Willis动脉环及其相连的动脉制备匀浆液PCR、Westernblot等检测脑动脉匀浆液中一氧化氮合酶（nitricoxidesynthase,NOS）mRNA和蛋白含量硝酸酶还原法测定NO含量放射免疫分析法测定cGMP含量2020/1/21二、内源性调节性T细胞移植SAH后神经功能预后的改善作