【作者】 王革非；

【导师】 李康生；

【作者基本信息】 汕头大学 ， 免疫学， 2008， 博士

【摘要】 研究目的与意义:流感病毒感染可引起人呼吸系统疾病,同时亦可感染中枢神经系统(CNS),导致部分感染人群产生CNS病变。在流感相关性脑病与脑炎综合征病人的脑脊液中促炎症细胞因子浓度出现异常升高。神经胶质细胞,尤其是小胶质细胞和星形胶质细胞是CNS细胞因子产生的主要来源,但其在流感病毒感染CNS时的功能仍不清楚。因此,探索小胶质细胞和星形胶质细胞在流感病毒感染后所发生的病理变化及免疫反应,为明确胶质细胞在感染中的作用、理解CNS感染流感病毒后的发病机制提供必要的实验依据。此外,根据人流感病毒基因组核酸的类型、进化趋势及预实验的线索,推测流感病毒与APOBEC家族蛋白存在可能的潜在相关性。探寻APOBEC与流感病毒复制及突变的关系,为寻找对流感病毒具有潜在抑制作用的因子、理解病毒进化因素提供新的思路。实验方法:1.明确病毒遗传背景、建立反向遗传平台:选取人A/Shantou/169/2006(H1N1)和禽A/Chicken/Guangdong/1/05(H5N1)流感病毒,对病毒全基因组进行克隆测序,构建反向遗传通用载体及含上述两种病毒全基因组片段的质粒;2.探讨胶质细胞感染流感病毒后的病理变化和免疫反应:分离培养小鼠神经胶质细胞,检测流感病毒受体;使用遗传背景明确的H1N1和H5N1感染小胶质细胞及星形胶质细胞,检测病毒复制、病毒生长曲线、细胞病变、细胞因子基因转录水平及蛋白分泌变化;使用人胶质细胞,检测流感病毒感染后APOBEC家族基因转录水平变化;3.探讨APOBEC家族蛋白与流感病毒复制及突变频率的关系:克隆APOBEC家族成员并构建MDCK表达细胞系,检测流感病毒在APOBEC表达细胞上的复制效率与突变频率。实验结果:1.完成了人H1N1和禽H5N1流感病毒的纯化培养及基因组的克隆与测序;2.成功构建了CMV及Pol I双向启动子的反向遗传载体,及两株流感病毒全基因组反向遗传质粒;3.亲和荧光实验表明小鼠胶质细胞存在禽及人流感病毒受体SA-α2,3-Gal和SA-α2,6-Gal;4.小鼠小胶质细胞和星形胶质细胞均可被流感病毒感染并产生子代病毒。小胶质细胞对流感病毒的敏感性和复制效率高于星形胶质细胞,H1N1复制效率高于H5N1;5.流感病毒感染胶质细胞可产生细胞病变效应(CPE)并诱导细胞凋亡,小胶质细胞的CPE和凋亡率高于星形胶质细胞:6.流感病毒感染可诱导小胶质细胞和星形胶质细胞的促炎症细胞因子(IL-1α、IL-1β、IL-6及TNF-α)及趋化因子(CCL-2、-3、-5及CXCL-2、-9、-10)的mRNA水平上调;H5N1的感染能诱导更强烈的上调反应;7. ELISA结果表明,小胶质细胞和星形胶质细胞在流感病毒感染后,促炎症细胞因子IL-1β、IL-6和TNF-α的分泌量上调;H5N1的感染能诱导更强烈的上调反应;8.小鼠胶质细胞表达APOBEC3,人胶质细胞表达APOBEC-3B、-3C、-3F和-3G,在流感病毒感染后,其转录水平发生上调;9.克隆并表达了人APOBEC-1、-2、-3A、-3B、-3C、-3F、-3G、-3H、-4和AID,小鼠APOBEC-3和大鼠APOBEC-1,其中hA3F和hA3G构建了稳定转染的细胞系; 10.在证实hA3F和hA3G的表达不受流感病毒封闭,且具有抑制HBV复制活性的情况下,表明hA3F和hA3G对流感病毒的复制效率及基因突变频率无明显作用;其它APOBEC家族成员尚未发现具有明显抑制流感病毒复制的潜力。结论:1.成功构建了反向遗传通用载体及两株流感病毒的全基因组反向遗传质粒;2.小鼠胶质细胞表面存在人流感病毒受体SA-α2,6-Gal及禽流感病毒受体SA-α2,3-Gal,人及禽流感病毒均可感染小鼠小胶质细胞和星形胶质细胞,并进行有效复制产生具有感染性的子代病毒;3.流感病毒的感染可对小鼠胶质细胞产生以CPE和凋亡为特征的直接损伤作用,其中小胶质细胞对流感病毒更为敏感;4.流感病毒感染可诱导小鼠胶质细胞产生促炎症细胞因子反应为特征的炎症反应;其中H5N1可诱导更为强烈的促炎症因子反应;5.流感病毒可诱导胶质细胞APOBEC蛋白家族成员表达上调,但人APOBEC-3F和-3G对流感病毒的复制效率和基因突变频率无明显作用,其它APOBEC家族成员亦无明显抑制流感病毒复制的能力。更多还原

【Abstract】 Background and aimsInfluenza virus infection can cause human respiratory illnesses and occasional central nervous system (CNS) disorders. Influenza virus can infect the CNS. Besides, pro-inflammatory cytokines are reportedly increased in the cerebrospinal fluids and plasma of patients with influenza encephalopathy and encephalitis. Microglia and astrocytes are capable of producing cytokines in the CNS. Nevertheless, the role(s) of microglia and astrocytes in acute encephalopathy and encephalitis caused by influenza virus infection remains unexplored. Therefore, to understand the immune responses mediated by microglia and astrocytes after influenza virus infection and the mechanism underlying the associated encephalopathy and encephalitis, mouse microglia and astrocytes were isolated and infected with human H1N1 and avian H5N1 influenza viruses, then their viral susceptibility and productivity, and cytopathic, apoptotic, and pro-inflammatory cytokine responses were examined.A steady increase of uracil (U) and adenine (A) along with the decrease in cytosine (C) and guanine (G) was detected in human influenza virus genomes but not in the avian influenza virus genomes. One of those speculations is that humans have a native defense against RNA viruses similar to APOBEC proteins. Based on other reports and our previous results, we speculated that APOBEC might be involved in the inhibition or hypermutation of influenza virus. There is no report on whether APOBEC enzymes can inhibit or hypermutate human IAV until now. It will be beneficial to explore and understand the novel potential inhibitors and hyper-mutagenesis factors among cytidine deaminase or intercellular immunity mediators might be found.Methods1. To clarity the genetics background and construct the reverse genetics system, the universal reverse genetics vector was constructed. All the eight genome-fragments of human H1N1 and avian H5N1 influenza viruses were cloned using RT-PCR and sequenced. Then the fragments were inserted into the universal vectors.2. To investigate the cytopathy and immune reactions of glial cells infected influenza viruses, mouse microglia and astrocytes were isolated and infected by human H1N1 and avian H5N1 influenza viruses. Then their viral receptors distribution, infection, apoptosis, cytopathy, and immune responses were examined using affinity fluorescence, immuno- fluorescence, semi-quantitative RT-PCR, ELISA and other assays. The mRNA levels of APOBEC members in infected human glial cells were determined by semi-quantitative RT-PCR.3. To clarify the role of APOBEC3 members in cell defenses against influenza A virus, the effects of APOBEC members on influenza A virus replication and hypermutation were investigated. The APOBEC members were cloned and expressed in transfected cells and confirmed by Western blotting and FCM. The viral replication in APOBEC expressed cells were examined by TCID50 and PFU assay. The viral mutations were checked by sequencing.Results1. The genome fragments of H1N1 and H5N1 strains were cloned and sequenced successfully;2. The universal reverse genetics vector was constructed and confirmed by sequencing;3. Viral receptors, sialic acid (SA)-α2,3-Galactose(Gal) and SA-α2,6-Gal, were both observed to be homogeneously distributed on microglia and astrocytes;4. Both H1N1 and H5N1 viruses were replicative and productive in microglia and astrocytes;5. Virus-induced apoptosis and cytopathy in infected cells were observed at 24 h post-infection (p.i.);6. The mRNA expression of pro-inflammatory cytokines (IL-1α, IL-1β, IL-6 and TNF-α) and chemokines (CCL-2, -3, -5, and CXCL-2, -9, -10) examined at 6 h and 24 h p.i. was up-regulated, and their expression levels were considerably higher in H5N1 infection. The protein levels of secreted pro-inflammatory IL-1β, IL-6 and TNF-αat 6 h and 24 h p.i. were also induced, with greater induction by H5N1 infection;7. Mouse APOBEC3 and human APOBEC-3B, -3C, -3F, and -3G were detected in mouse and human glial cells, respectively. Their mRNA levels were up-regulated after influenza virus infection;8. The APOBEC members, including human APOBEC-1, -2, -3A, -3B, -3C, -3F, -3G, -3H, -4, AID, mouse APOBEC-3, and rat APOBEC-1, were cloned and confirmed by sequencing, and expressed in transfected cells.9. The results of viral titers assay showed that there were no significant differences in replication of influenza virus between APOBEC proteins expressed cells and the control cells. It indicated that APOBEC3F and 3G could inhibit the replication of HBV in HepG2.2.15 cells. Western blotting indicated that APOBEC3F and 3G proteins were both over-expressed during the infection of influenza virus. DNA sequencing showed that there was no hypermutation in influenza virus genome induced by APOBEC3F or 3G.Conclusions1. The genome fragments of H1N1 and H5N1 strains were cloned and sequenced successfully; The universal reverse genetics vector was constructed and confirmed by sequencing;2. This study firstly demonstrated that both human H1N1 and avian H5N1 influenza viruses could infect and induce apoptosis, cytopathy, and pro-inflammatory cytokine production in mouse microglia and astrocytes in vitro. The results suggest that direct cellular damage and the consequences of immunopathological injuries in the CNS contribute to the influenza viral pathogenesis.3. APOBEC3F and 3G could effectively inhibit HBV replication, but could not inhibit human influenza A virus replication or induce its hypermutation. And other APOBEC members, including human AID, APOBEC1, APOBEC3A, 3B, 3C, 3H and rat APOBEC1, have no inhibition effect on the replication of influenza A virus, too.更多还原