【作者】 李继安；

【导师】 陈宗波；

【作者基本信息】 青岛大学 ， 儿科学， 2014， 博士

【摘要】 第一部分肠道病毒71型感染对小鼠大脑与脑干肉碱棕榈酰基转移酶2表达和作用的影响目的：肉碱棕榈酰基转移酶(Carnitine palmitoyltransferase, CPT)系统在长链脂肪酸β-氧化中起着重要作用,也是线粒体脂肪酸氧化产生ATP的关键组成部分。长链脂肪酸进入线粒体的限速步骤是在线粒体外膜CPT1的催化作用下,通过转脂作用把脂肪酰辅酶A转变成酰基肉碱,并且在CPT1的作用下进入线粒体基质；在线粒体内膜CPT2把酰基肉碱转换成酰基辅酶A,然后进入β-氧化途径。在本实验中我们研究肠道病毒71型(Entero virus71, EV71)感染对小鼠大脑和脑干肉碱棕榈酰基转移酶2表达和作用的影响,探讨EV71相关脑炎的病理生理机制。方法：1日龄ICR小鼠8窝(48-80只),每只小鼠腹腔内注射含有EV71(TCID50=107.5)的DMEM培养基O.1ml,观察小鼠感染后症状,并分别于病毒接种后5d、10d、15d各用乙醚安乐死6只感染组小鼠和6只0d(未接种病毒前)小鼠,取出大脑、脑干。应用荧光定量RT-PCR法检测小鼠脑组织中EV71病毒载量；电子显微镜检测线粒体；应用免疫组织化学(Immune histochemical, IHC)、Westblot、荧光定量RT-PCR法检测小鼠大脑、脑干CPT2的表达；检测大脑和脑干细胞的CPT2酶活性和ATP水平。结果：EV71感染小鼠后5天,在大脑检测到EV71RNA拷贝数为5.334±0.30log1ocopies/mg tissue,脑干为6.20±0.19log10copies/mg tissue;然而,随着感染时间延长,EV71RNA拷贝数逐渐降低。在感染后5天,电镜显示线粒体肿胀、线粒体嵴和线粒体膜断裂、变短或消失；CPT2表达下降,CPT2酶活性减低；ATP水平明显下降；而在感染后的10-15天,这些变化逐渐恢复到正常状态。结论：我们的研究资料显示：EV71感染损伤了脑组织的线粒体,引起CPT2的表达减少,导致CPT2酶活性降低、ATP生成减少。能量代谢障碍可能加速或加重了EV71脑炎发展,可能与脑炎的严重程度相关。本研究从能量代谢障碍方面阐述了EV71脑炎的病理生理机制。第二部分PSGL-1在肠道病毒71型感染小鼠的表达和作用目的：通过检测EV71感染小鼠不同组织中P选择素糖蛋白配体-1(Mouse P-selectin glycoprotein ligand-1, mPSGL-1)的表达,探讨P选择素糖蛋白配体-1(P-selectin glycoprotein ligand-1, PSGL-1)在EV71感染中的作用。方法：1日龄ICR小鼠11窝(66-110只)随机分为2组,实验组8窝(48-80只)每只小鼠腹腔内注射含有EV71(TCID50=107-5)的DMEM培养基0.1ml,另3窝(18-30只)为对照组,每只小鼠腹腔注射无菌DMEM培养基0.1ml。观察小鼠感染后症状,并分别于0d(接种病毒前)、病毒接种后、4d、8d、12d各用乙醚安乐死6只感染组小鼠与6只对照组小鼠,取出大脑、小脑、脑干、脊髓、心脏、肺脏。应用荧光定量RT-PCR法检测小鼠脑组织中EV71病毒载量；应用免疫组织化学、Westblot、荧光定量RT-PCR法检测小鼠大脑、小脑、脑干、脊髓、心脏、肺脏mPSGL-1的表达；应用免疫组织化学法检测CD4+、CD8+T淋巴细胞、巨噬细胞的表达；应用酶联免疫吸附法(Enzyme-linked immuno sorbent assay, ELISA)检测大脑、小脑、脑干、脊髓、心脏、肺脏的促炎细胞因子：肿瘤坏死因子-α(Tumor necrosis fector-alfa, TNF-a)、白细胞介素-6(Interleukin-6, IL-6)、白细胞介素-1β(Interleukin-lbeta, IL-1β)含量。分析EV71感染小鼠不同组织的mPSGL-1的相对表达量对CD4+、CD8+、巨噬细胞表达的影响,以及与促炎性细胞因子TNF-α、IL-6和IL-1β产生水平的关系。结果：EV71感染小鼠中枢神经系统的病毒载量较心脏和肺脏的病毒载量增多；mPSGL-1在EV71感染小鼠的组织中表达增加,在感染后一定时间内,mPSGL-1在中枢神经系统的相对表达量比在心脏或/和肺脏的相对表达量明显增多,尤其在脑干和大脑增多更为明显。在所被测组织中的CD4+、CD8+、巨噬细胞免疫反应染色显示也增多；另外,在EV71感染小鼠所被测组织中的mPSGL-1相对表达量与TNF-α、IL-6和IL-1β产生量有相关性：在感染后的4天,大脑、小脑、脑干、脊髓、心脏、肺脏的mPSGL-1相对表达量与TNF-α、IL-6和IL-1β产生量成负相关；在感染后8天,除了心脏和肺脏的mPSGL-1相对表达量与TNF-a产生量未有相关性外,大脑、小脑、脑干、脊髓mPSGL-1的相对表达量与TNF-α、IL-6和IL-1β产生量仍成负相关；在感染后12天在所有被测组织未再有相关性。结论：在EV71感染时,局部组织的mPSGL-1表达增加,可能在白细胞募集与宿主的先天性固有免疫反应中起着重要作用。第三部分SCARB2在肠道病毒71型感染小鼠的表达与促炎细胞因子的关系目的：在感染性疾病时,清道夫受体(Scavenger receptor class B, member2, SCARB2)参与早期的先天性固有免疫反应；因此,本研究探讨肠道病毒71型(Enterovirus71,EV71)感染小鼠时,小鼠清道夫受体(mouse SCARB2, mSCARB2)在不同组织的表达和作用。方法：1日龄ICR小鼠13窝(78-130只)随机分为2组,感染组10窝(60-100只)每只小鼠腹腔内注射含有EV71(TCID50=107.5)的DMEM培养基0.1ml,另3窝(18-30)只为对照组,每只小鼠腹腔注射无菌DMEM培养基0.1ml。观察小鼠感染后症状,并分别于病毒接种后2d、4d、6d、8d、12d各用乙醚安乐死6只感染组与对照组小鼠,取出大脑、小脑、脑干、脊髓、心脏、肺脏。应用荧光定量RT-PCR法检测小鼠脑组织中EV71病毒载量；应用免疫组织化学、Westblot、荧光定量RT-PCR法检测小鼠大脑、小脑、脑干、脊髓、心脏、肺脏SCARB2的表达；应用酶联免疫吸附法(Enzyme-linked immuno sorbent assay, ELISA)检测小鼠大脑、小脑、脑干、脊髓、心脏、肺脏的促炎细胞因子：肿瘤坏死因子-α(Tumor necrosis fector-alfa, TNF-α)、白细胞介素-6(Interleukin-6, IL-6)、白细胞介素-1β (Interleukin-lbeta, IL-1β)含量。分析EV71感染小鼠不同组织的SCARB2相对表达量与细胞因子TNF-α、IL-6、IL-1β含量的关系。结果：在EV71感染小鼠中枢神经系统的病毒载量较心脏和肺脏的病毒载量增多。在感染后一定时间段内,SCARB2在中枢神经系统的相对表达量较心脏或/和肺脏的相对表达量明显升高,尤其在脑干和大脑。另外,在EV71感染小鼠的不同组织的TNF-α、IL-6和IL-1p产生量与SCARB2相对表达量有相关性：在感染后4天、8天,大脑、小脑、脑干、脊髓、心脏、肺脏SCARB2的相对表达量与TNF-α、IL-6和IL-1p含量成正相关,感染后12天未再有相关性。结论：在EV71感染时,小鼠局部组织的SCARB2表达增加,可能在调节促炎细胞因子的产生中起着重要作用,尤其在中枢神经系统。更多还原

【Abstract】 Impact of enterovirus71infection on the expression and role of carnitine palmitoyltransferase II in mice brain and brain stemBackground:The carnitine palmitoyltransferase (CPT) system has crucial roles in the β-oxidation of long-chain fatty acids, and is a pivotal component of ATP generation through mitochondrial fatty acid oxidation. The rate-limiting step in the importation of long-chain fatty acids into the mitochondria is the transesterification of acyl-coenzyme A (CoA) to acylcarnitine by CPT I, while CPT II changes the imported acylcarnitine back to acyl-CoA. Here we investigated the impact of enterovirus71(EV71) infection on the expression and role of CPT II in mice brain and brain stem to explore the pathophysiology of EV71-associated encephalitis.Methods:48-80one day old ICR mice. ICR mice were inoculated intraperitoneally (i.p.) with EV71, and were sacrificed by aether anesthesia at days5,10and15post infection (p.i.), and day0(before EV71infection), their brain and brain stem were dissected out for determining the number of copies of viral RNA by quantitative real-time PCR (qRT-PCR). Mitochondria detected by electron microscopy. Detection of expression of CPT II by immunohistochemistry, qRT-PCR and western blot. CPT II activity and ATP level were also measured in brain and brain stem cells.Results:In EV71-infected mice, the number of copies of EV71RNA detected at day5p.i. were brain (5.33±0.30log10copies/mg tissue), brain stem (6.20±0.19log10copies/mg tissue). However, the virus was gradually eliminated in later days. Mitochondria undergone a markedly amplitude swelling in brain and brain stem at day5p.i., and shown some recovery from swollen in later. At day5p.i., expression of CPT II decreased, CPT II activity reduced and ATP levels lowed more obviously, and these changes gradually restored to basic normal state at10-15days p.i..Conclusion:Our data suggests that EV71infection impaired mitochondria, reduced the CPT Ⅱ expression and activity, and decreased local ATP level, which might be important factors to trigger the pathomechanism of acute encephalitis in EV71infection, which may be causally related to the severity of disease. The expression and role of P-selectin glycoprotein ligand-1in different tissues of enterovirus71-infected miceBackground:Aim was to elucidate the role of P-selectin glycoprotein ligand-1(PSGL-1) in Enterovirus71(EV71) infection by evaluating the expression of mouse PSGL-1(mPSGL-1) in EV71-infected mice.Methods:66-110one day old ICR mice were divided into infected group (48-80) and control group (18-30). ICR mice were inoculated intraperitoneally (i.p.) with EV71, and were sacrificed by aether anesthesia at days4,8and12post inoculation (p.i.), and day0(before EV71infection), their lung, heart, brain, brainstem, spinal cord and cerebellum were dissected out for determining the number of copies of viral RNA by quantitative real-time PCR (qRT-PCR), detection of expression of mPSGL-1by immunohistochemistry, qRT-PCR and Western blot. Detection of expression of CD4+. CD8+T lymphocyt, macrophagocyt, natural killer cell of tissues by immunohistochemistry. Cytokines quantification by ELISA.Results:Viral loads in central nervous system (CNS) were higher than in lung or/and heart. Expression of mPSGL-1increased more obviously in CNS than in lung or/and heart within a certain period of time, particularly in brain stem and brain. In the tisssues, the expression of mPSGL-1increased, mean while, immunohistochemistry shown that the expression of CD4+、CD8+T lymphocyte, macrophagocyte, natural killer cell also increased. Local TNF-α, IL-6and IL-1β production were consistent with expression of mPSGL-1. Surprisingly, it presented a negative correlation between relative mPSGL-1mRNA level and TNF-α, IL-6and IL-1β levels in local tissues at day4p.i. and at day8p.i.(excluded TNF-α in lung and heart).Conclusion:The elevated local mPSGL-1may be important in the leukocyte recruitment and in host innate immune response against EV71infection. Association of the expression of SCARB2in EV71-infected mice with inflammatory cytokinesBackground:Scavenger receptor class B, member2(SCARB2) participates in early innate immune responses to infection, so our aim was to explore the expression and role of mouse SCARB2(mSCARB2) in different tissues in EV71-infected mice.Methods:78-130one day old ICR mice were divided into infected group (60-100) and control group (18-30). ICR mice were inoculated intraperitoneally (i.p.) with EV710.1ml107.5TCID50/ml. The control mice were injected i.p. with the same volume RD cell lysate. Mice were sacrificed by aether anesthesia at day4,8and12post infection (p.i.), their brain, brainstem, spinal cord, cerebellum, lung and heart were dissected out for determining the number of copies of viral RNA by quantitative real-time PCR (qRT-PCR), detection of expression of mSCARB2by immunohistochemistry, qRT-PCR and Western-blotting. Cytokines quantification by ELISA.Results:The viral loads in central nervous system (CNS) were higher than in lung or/and heart. The expression of mSCARB2increased in tissues of EV71-infected mice, however, the levels of mSCARB2increased in CNS were higher than in lung or/and heart within a certain period of time, particularly in brain stem and brain. In addition, local TNF-a, IL-6and IL-1β levels of production were consistent with mSCARB2levels of expression in tissues of EV71-infected mice. However, it presented a positive correlation between relative mSCARB2mRNA level and TNF-a, IL-6and IL-1β levels in local tissues at day4and8p.i..Conclusion:Our data revealed that the elevated local mSCARB2may modulate pro-inflammatory cytokines induction in local tissues, particularly, in CNS of EV71-infected mice.更多还原