EV71脑炎血脑屏障损伤的相关性研究

课题题目：肠道病毒71(EV71)脑炎血脑屏障损伤的相关性研究实验包括：实验一、动物实验部分题目：EV71感染小鼠脑组织埘P一9、MMP一2的表达与血脑屏障损伤的相关研究关键词：肠道病毒71型；基质金属蛋白酶一9；基质金属蛋白酶2；血脑屏障；实验二、临床实验部分题目：MMP一9-1562、TNFQ-308基因多态性与EV71重症脑炎易感性研究关键词：肠道病毒71型；金属蛋白酶(MMPS)，金属蛋白酶-9(MMP一9)；肿瘤坏死因子Q(TNFQ)；多态性；综述题目：MMP一2、删P一9与中枢神经系统疾病的相关研究进展硕士研究生：辛丹丹指导教师： 陈宗波教授实验一、EV71感染小鼠脑组织MMP一9、MMP一2的表达与血脑屏障损伤的相关研究 ，中文摘要 铿燃必目的通过制作EV71感染ICRd、鼠模型，观察EV71感染后小鼠的生存情况，检测不同时间点脑干及大脑组织MMP一9和删P一2的表达水平、脑组织含水量、脑组织毛细血管形态和结构变化情况，探讨EV71感染小鼠脑组织贼P一9、麟P一2的表达与血脑屏障损伤的相关性。方法SPF级ICR小鼠腹腔注射肠道病毒7l型(EV71)制造EV71病毒性脑炎模型，于感染后l、3、5、7、lO、14天，分别取小鼠大脑及脑干组织，应用QRT-PCR检测MMP9 rnRNA和MMP2 mRNA的表达情况，ELISA方法检测MMP9和MMP-2蛋白表达情况，应用透射显微镜检测大脑及脑干脑毛细血管结构变化以及脑组织含水量了解血脑屏障损伤情况。结果1EV71感染第1、3天脑干组与同时间点EV71感染大脑组比较，MMP9mRNA表达量及其蛋白浓度显著增加(P001)。2Ev71感染后第l、3天，EV71脑干组MMP2mRNA表达量及其蛋白浓度显著高于EV71大脑组(Po05)。3EV71感染后第l、3天脑干含水量较大脑含水量显著增加(P0001)。4EV71感染后第1天电镜下观察脑干毛细血管结构损害较EV71感染大脑组明显；感染后第3、5天脑干及大脑毛细血管结构均显著破坏；EV71感染后第7天大脑及第10天脑干血脑屏障结构渐恢复。结论1EV71病毒性脑炎呈急性经过，感染极早期脑于血脑屏障较大脑的血脑屏障更易受损，且恢复较大脑组织缓慢。2EV71感染后，MMP9、MMP2的表达量增高与血脑屏障损害及脑水肿程度呈正相关，推测MMP9、MMP2参与了EV71病毒性脑炎血脑屏障损伤的病理生理过程，且MMP2可能在EV71病毒性脑炎早期的血脑屏障损害中起到更重要作用。关键词：肠道病毒71型；基质金属蛋白酶_9；基质金属蛋白酶2；血脑屏障；AbstractPart ICorrelation Research on MMP一9 and 2 Expression of EV71 InfectedMice Brain Tissue and Damages to Blood Brain BarrierObjectiveTo observe the survival of EV7 1 infected mice，detect the MMP一9 and 2 expressionlevel of brain stem and brain tissue，water content in brain tissue as well aS bloodcapillary shape and structural change of brain tissue and finally discuss the correlationbetween MMP9 and 2 expression of EV7 l infected mice brain tissue and damages toblood brain barrier through fabricating EV7 1 infection modelMethodFabricate EV7 1 viral encephalitis with SPF level ICR mice abdominal injection ofintestinal tract virus 7 1-type(EV7 1)，take out mice brain and its brain stem tissue after themice is infected for 1，3，5，7，10，and 14 days respectively；detect the expression ofMMP一9 mRNA and MMP2 mRNA through QRT-PCR,MM-9 and 2 protein expressionthrough ELISA and structural change in brain and brain stem blood capillary and watercontent in brain tissue wim transmission electron microscope tO know about the damagesto blood brain barrienResult1Through comparison of brain stem tissue group after infected witll EV71 for 1 and3 days into EV7 1 infected brain group in the same time and at same place，theMMP-9mRNA expression and its protein concentration is remarkably increased(PO01)2After infected with EV71 for l and 3 days，MMP2mRNA expression and proteinconcentration of EV7 1 brain stem group is remarkably higher than that of EV7 1 braingroup(Po05)3After infected谢m EV71 for 1 and 3 days，the water content in brain stem isremarkably increased than that in brain4After ln dayS EV71 infection,the damages to the brain stem blood capillarystructure is more obvious than that of the brain group infected witll EV7 1 when observedunder the electron microscope；after 3rd and 5tII dayS infection，the brain stem and brainblood capillary structure are remarkably damaged；the brain after infected with EV7 1 for舭7曲day and the brain stem blood brain barrier structure after infected with EV7 1 forme 1 0tIl day are gradually recoveredConelusion1EV71 viral brain encephalitis iS acute，the blood brain barrier of the brain withlarger brain stem blood brain barrier is easier to be damaged at the early stage of infection,and it is recovered more slowly than the brain tissue2After Ev7 1 infection,the increase in MMP-9 and 2 expression quantity ispositively correlated to blood brain barrier and encephaledema degreeIt is reckoned thatMMP一9 and 2 participates in pathological and physiological process of EV71 viralencephalitis blood brain barrier and lVllVlP一2 might play an important role in pathologicaland physiological process of blood brain barrier at the early stage of EV71 viralencephalitisPostgraduate student：Xin DanDan(peadiatdcs)Directed by ProL Chen ZongBoKeywords：Enterovirus 71；Matrix metailoproteinase-9(MMP-9)；Matrix metalioproteinase-2Blood brain barrier(BBB)实验二mVIP-9-1562、TNFQ-308基因多态性与EV71重症脑炎易感性研究中文摘要目的研究MMP一91562、TNF-a-308位点基因多态性与EV71脑炎病情严重程度的关系，探讨EV71重症脑炎的遗传易感因素。方法收集2010年10月至2011年10月EV71检测阳性的中枢神经系统感染患儿184例，收集同时期同地区的健康查体儿童共237例，分别提取患儿及健康儿童外周血白细胞的基因组DNA，通过序列特异性引物一聚合酶链反应(PCR-RFLP)技术检测EV71病毒性脑炎患儿及正常对照组儿童基因组TNF-a-308、MMP-9-1562位点单核苷酸多态性。结果EV71重症脑炎组TNF-Cl-308位点A等位基因频率分布明显高于轻症组(16VS64，OR 2646，95CI 12915421，P005)，EV71脑炎组与正常对照组该位点A等位基因频率分布具有统计学意义(92VS 55，OR1754，95CI10332980，P005)；EV71重症脑炎组与轻症组MMP-9-1562位点T等位基因频率分布存在明显差异(16VS 75，OR 2362，95CI 117叫768，P005)；Ev71重症脑炎组与轻症组，MMP-9-1562位点CT、TTTNFa一308位点GA、从组合基因型与MMP一91562位点CT、TTTNF-a-308位点GG组合基因型分布差异有统计学意义(467Vs 15，矛=4212，P005)。结论TNF-a一308位点A等位基因及MMP-9-1562位点T等位基因可增加EV71重症脑炎的患病风险，可能是EV71重症脑炎的遗传易感基因；TNF-Q一308位点基因型GG可能为避免感染Ev71后发展为重症脑炎的保护基因型。硕士研究生辛丹丹(研究生)指导教师 陈宗波教授关键词：肠道病毒71型；金属蛋白酶(埘Ps)，金属蛋白酶-9(MMP一9)；肿瘤坏死因子Q(TNF-a)；多态性；Part 2AbstractAssociation of Matrix Metalioproteinase一9 and Tumor Necrosisfactor-Alpha gene polymorphisms with risk of severe encephalitis inenterovirus 71 infected child renobjective：To investigate the relationship between the polymorphisms of TNFa-308 andMatrix Metalloproteinase9(MMP9、1562 with the susceptibility of enterovirus71encephalitis，and explore the risk factors of EV7 1 encephalitisMethod：Reverse transcription polymerse chain reaction(RT-PCR)was used to detect thespecific primers of EV7 1 in the faeces of patients with EV7 1 infectionGenomic DNA ofwhite blood eell were extractedJhen polymerase chain reaction-restriction fragmemlength polymorphism(PCR-RFLP)techniques were performed to determine the genotypesofn佃。9 gene and a polymerase chain reaction with sequence-specific primers(PCRssP)system were used to determine genotype for the AG polymorphism at308position of 1冬一a-308 gene in encephalitis group and in control groupResults：The TNFa308 A allele was observed with sigificantly greater frequency inpatients with serious EV71 encephalitis compared with slight EV71 encephalitispatients(16VS 64，OR 2646，95CI 1291-5421，P005)；In EV7l encephalitisgroup，A allele of n虾仅308 were more common than that in control group(92VS55OR 175495CI 10332980，P005)Similarly,the MMP一9一1562 T allele wasobserved with significantly greater frequency in patients with serious EV7 1encephalitis(1 6)compared with slight EV7 1 encephalitis patients(75)(OR 2362，95C111 70-4768，P005)；People whose genotypes cornbination wasMMP91 562(C1r，1mMGt308(GAAA)were more easily to suffer with serious EV71e n c e P h a li t i S t h a n t h O S e w h O S e e n o t y P e s c o m b i n at i O n w a sMMP91 562(CTTT)TNFix308(GG)(467VS 1 5，)Cz=42 1 2，P005)Conclusion：The polymorphism of 1小限一a-308 and MN皿-9-1 562 single nucleotide maycorrelate wim EV7 1 serious encephalitismaybe 11、IF-a一308 GG is the protectivegenotype which protect children keep away from EV71 serious encephalitisthat there is aclose relationship between genetic polymorphism 308AG on TNFa gene and geneticpolymorphism 1 562C厂r on M卟僵P-9 gene with the severity of EV7 1 infected childrenPostgraduate student：Xin DanDa(peadiatrics)Directed by ProfChen Zong-BoKeywords：Enterovirus 71；Matrix metalloproteinase；Matrix metalloproteinase-9(MMP-9)；Tumor necrosis factor-G(TNF-帕O；Polymorphisms,single nueIeofid目录一、EV71感染小鼠脑组织MMP9、MMP-2的表达与血脑屏障损伤的相关研究引言1第一章材料与方法211材料。212方法。3第二章结果621小鼠生存情况622小鼠脑组织EV71表达情况。623 QRT-PCR检测脑组织MMP-9、MMP-2mRNA表达情况624 Elisa检测脑组织MMP一9、MMP-2表达水平825小鼠脑组织血脑屏障结构改变情况。1l26脑组织含水量变化情况j13第三章讨论：14第四章结论16参考文献1 7二、MMP91562、TNF仅308基因多态性与EV71重症脑炎易感性研究引言：1 8第一章材料与方法-1911材料1 912方法。20第二章结果2321一般情况：2322 TNFa一308及MMP一91562位点基因多态性分析23第三章讨论27第四章结论29参考文献30综述32综述参考文献36攻读学位期间的研究成果39致谢40学位论文独创性声明、学位论文知识产权权属声明41英文缩写EV71h心但NEMP9N附2ECMBBBTNFSNP几CNS缩略词索引表英文全称Enterovirus 7 1Matrix metalloproteinaseMatrix metalloproteinase-9Matrix metalloproteinase-2Extracellular matrixBlood Brain BarrierTumor Necrosis FactorSingle Nucleotide PolymorphismInterleukinCentral nervous system中文译名肠道病毒71型基质金属蛋白酶基质金属蛋白酶9基质金属蛋白酶2细胞外基质血脑屏障肿瘤坏死因子单核苷酸多态性白介素中枢神经系统引言实验一、EV7 1感染小鼠脑组织MMP一9、MMP-2的表达与血脑屏障损伤的相关研究引言肠道病毒71型(Enterovirus 71)是单链小RNA病毒由7408个核苷酸组成，EV71感染在全球范围内已有多次爆发与流行，近年来EV71在我国的流行呈显著上升的趋势n喇。EV71感染通常主要累及5岁以下的儿童，重症致死者多见于3岁以下的d,JL，一般儿童感染EV71后临床表现主要以手足口疱疹为特征，