【作者】 吕进；

【导师】 温博海； 王希良；

【作者基本信息】 中国人民解放军军事医学科学院 ， 微生物学， 2010， 博士

【摘要】 流感是一种急性呼吸道传染病,患病率高,在老年和儿童中死亡率很高,世界范围内每年都有大量的流感死亡病例报道,流感对世界公共卫生已经构成了严重的威胁。特别是2009年世界范围内暴发的猪源性甲型流感病毒,传染性强,波及世界213个国家和地区,造成了17483例死亡病例,拉响了世界范围内防控流感的警钟,近期关于猪源性甲型流感病毒对达菲耐药的报道,使流感病毒的防控工作更为艰难。从临床学角度分析,甲型流感病毒是流感病毒三个亚型中最为重要的亚型,属正粘病毒,根据其表面血凝素(HA)及神经氨酸酶(NA)分子的抗原性不同,分别有16(HA)与9(NA)个亚型。病毒基因组的点突变或基因节段重组将会产生新的变异株,从而引起大范围的传染或暴发性流行。上个世纪共出现四次流感大流行,最为严重的是1918年的大流感,造成了世界范围内5千多万人死亡。到目前为止,每年都有一定范围内流感暴发流行的病例报道。因此,深入研究甲型流感病毒重症感染的致病机制,对于选择抗病毒治疗及控制潜在的流感暴发流行显得至关重要。流感病毒致死性感染的小鼠模型研究发现,失控的及异常激活的天然免疫应答,和感染过程中大量的巨噬细胞与中性粒细胞快速招募至肺局部是高致病禽流感致病的主要因素,此外巨噬细胞及中性粒细胞与肺部增加的细胞因子及趋化因子有关,而升高的细胞因子水平被认为是介导甲型流感病毒病症的主要原因,亦是决定高致病性禽流感病毒致死性感染症状的主要原因。然而,抑制细胞因子应答并不能保护机体免于致死性感染,感染早期清除巨噬细胞及中性粒细胞对疾病的结局亦无显著影响,这些研究提示巨噬细胞及中性粒细胞在甲型流感病毒重症感染病例中的复杂生物学效应。另外,巨噬细胞及中性粒细胞本身也是病毒感染的靶细胞,巨噬细胞及中性粒细胞被直接感染可能会导致包括抗体应答在内的获得性免疫应答不能够被有效激活。本研究主要探讨甲型流感病毒感染介导的小鼠肺免疫损伤机制,对流感病毒感染Balb/c小鼠后免疫损伤的发生、发展及转归进行动态观察,分别从细胞与分子层面揭示流感病毒感染介导的免疫损伤机制,探讨这些免疫细胞与分子在感染过程中发挥的双重作用,同时观察比较不同毒力病毒感染过程中介导免疫损伤的主要细胞与免疫分子差异。研究内容共分为三部分:1、甲型流感病毒A/PR/8/34(H1N1)感染介导的小鼠肺免疫损伤机制初步研究,分三节(1)甲型流感病毒A/PR/8/34(H1N1)感染Balb/C小鼠肺损伤动物模型的建立;(2)甲型流感病毒A/PR/8/34(H1N1)感染介导的小鼠肺免疫损伤机制初探;(3)甲型流感病毒A/PR/8/34(H1N1)感染介导的肺损伤过程中粘膜及二级淋巴器官的免疫应答分析;2、甲型流感病毒A/Beijing/501/2009(H1N1)感染介导的小鼠肺损伤机制初步研究,分三节(1)甲型流感病毒A/Beijing/501/2009(H1N1)感染Balb/C小鼠肺损伤动物模型的建立;(2)甲型流感病毒A/Beijing/501/2009(H1N1)感染介导的小鼠肺损伤机制初探;(3)甲型流感病毒A/Beijing/501/2009(H1N1)感染介导的肺损伤过程中粘膜及二级淋巴器官的免疫应答分析;3、甲型流感病毒A/Beijing/501/2009(H1N1)与A/PR/8/34(H1N1)感染介导的小鼠肺损伤机制差异分析。研究结果:1、两株病毒感染后小鼠体重变化,死亡率及肺病理变化说明,两株病毒感染介导的小鼠肺损伤模型建立成功;病毒感染后早期即可在鼻及肺中检测到病毒复制,至感染末期,病毒呈持续性复制;2、两株病毒感染造成了小鼠肺损伤的同时,介导了复杂的应答变化,无论是病毒感染早期还是晚期,均检测到了大量的巨噬细胞与中性粒细胞的累积,统计学分析结果显示,中性粒细胞与肺病毒载量、肺及血清中抗体应答密切相关,肺中抗体应答与肺中病毒载量显著相关;病毒感染后巨噬细胞及中性粒细胞的浸润,促进了肺细胞因子应答,细胞因子应答参与调节抗体应答;3、病毒感染过程中,肺部除检测到较强的抗体应答,也检测到了较强的细胞因子分泌细胞应答及抗体分泌细胞应答,这两种应答反应共同调节肺抗体应答的强度及维持的时间;4、两株病毒感染小鼠肺损伤的同时,检测到了粘膜局部及二级淋巴器官产生了T、B细胞应答反应,病变累及肺器官的同时,引起了全身免疫系统的应答反应,共同参与机体的免疫防御;5、两株病毒感染小鼠半数致死剂量,体重变化及死亡率等指标反应了两株病毒在小鼠上的毒力不同,其介导肺免疫损伤过程中诱发的各种应答反应规律不尽相同,总的来说,A/Beijing/501/2009(H1N1)病毒诱发的免疫应答反应早于A/PR/8/34(H1N1)病毒,强度亦强于后者; 6、A/Beijing/501/2009(H1N1),A/California/04/2009(H1N1)及A/PR/8/34三株病毒蛋白序列比对分析结果显示,在已知的重要功能性位点及包括受体结合区域在内的未知功能性发生了突变,这些突变与病毒的毒力直接相关。结论:甲型流感病毒感染介导的小鼠肺损伤过程是一个多因素参与的复杂过程,其中病毒持续复制是造成损伤的主要因素之一,肺中大量的巨噬细胞及中性粒细胞的累积在感染的早期是损伤的主要因素,随着疾病的进程,中性粒细胞与抗体协同作用,共同促进病毒的清除及疾病的恢复;病毒引发的肺局部损伤性应答与全身系统免疫应答的激活密不可分,肺局部的应答参与系统性应答。更多还原

【Abstract】 Influenza is an epidemic, acute respiratory disease that has a high rate of mortality, especially among the elderly and children, and a large number of deaths due to influenza are reported every year all over the world. In 2009, a novel swine-origin influenza virus capable of rapid human transmission was reported. As of 4 April 2010, worldwide more than 213 countries and overseas territories or communities have reported laboratory confirmed cases of pandemic influenza H1N1 2009, including over 17700 deaths. Surveillance network has been build worldwide since March 2009. The recent report on the drug resistance to Oseltamivir phosphate capsules made the prevention and control of pandemic influenza more difficult.Influenza A virus is the most important among the three types of the virus from a clinical point of view. Influenza A viruses belong to the family Orthomyxoviridae. On the basis of the antigenicity of their haemagglutinin (HA) and neuraminidase (NA) molecules, they are classified into 16 HA subtypes (H1–H16) and 9 NA subtypes (N1–N9). The point mutations and reassortment events of the viral genomes contribute to the emergence of new variants or strains with epidemic or pandemic potential. Influenza A viruses have caused several pandemics during the last century, and continue to cause annual epidemics. The pandemic of 1918-1919 killed as many as 50 million people worldwide. Understanding of the mechanisms of increased pathogenicity of fatal influenza A viral infection is thus critical to optimize the antiviral treatment strategy and control the potential pandemic. The uncontrolled and aberrant activation of the innate immune system had been implicated in the mice model of fatal influenza A viral infection. A significantly rapid cell recruitment of macrophages and neutrophils into the lungs were assumed to play a role in the pathogenesis associated with H5N1 highly pathogenic avian influenza virus infection (HPAI). In addition, macrophages and neutrophils were associated with increased secretion and enrollment of some cytokine and chemokines, and the increased levels of cytokines are suggested to mediate influenza A infection signs and may play a role in the severe symptoms of fatal cases of HPAI H5N1 influenza virus infection. However, inhibition of the cytokine response was found not to protect against the lethal influenza A infection, and neutrophil or macrophage depletion initiated after infection in the early stage were also found not to have a significant effect on the disease outcome. These findings suggested complicated biological effect of macrophages and neutrophils in the fatal influenza A viral infection. In addtion, innate immune cells such as macrophages and neutrophils are the targets of influenza A viruses. The direct infection of macrophages and neutrophils may contribute to an inability to mount an adaptive immune response, such as antibody stimulation.In this study, the mechanism of immunopathology mediated by influenza A virus infection was carried out. To discover the role of these complicated factors during the infection, and evaluate the cellular and molecular discrepancy of different virulence virus, the initiation, development and outcome of the pulmonary immunopathology was investigated. Three parts were included in this study. On the first part, immunopatholgy on mice mediated by A/PR/8/34 (H1N1) influenza virus was studied. This part included: (1) The establishment and evaluation of mouse model for immunopathology on mice with influenza A/PR/8/34 (H1N1) virus infection. (2) Primary study on the mechanism of immunopathology on mice mediated by influenza A/PR/8/34 (H1N1) virus. (3) The immune response of mucosal and secondary lymphoid tissue during the pulmonary injury of mice with influenza A/PR/8/34 (H1N1) virus infection. On the second part, preliminary research on the mechanism of immunopothology on mice mediated by A/Beijing/501/2009 (H1N1) virus was studied. This part included: (1) The establishment and evaluation of mouse model for immunopathology on mice with influenza A/Beijing/501/2009 (H1N1) virus infection. (2) Primary study on the mechanism of immunopathology on mice mediated by influenza A/Beijing/501/2009 (H1N1) virus. (3) The immune response of mucosal and secondary lymphocyte tissue during the pulmonary injury of mice with influenza A/Beijing/501/2009 (H1N1) virus infection. In the third part, we compared the discrepancy pulmonary immune injury induced by A/Beijing/501/2009 (H1N1) and A/PR/8/34 (H1N1) virus infection.Results are as follows. First, we successfully established the mouse model for immunopathology on mice with two strains of influenza A virus by monitoring the weight lost, death ratio and histopathology of lung. Viral replication of two strains of influenza A virus in nasal and lung were detected in the early and late stage of infection. Second, complicated immune response was companied with the immunopathology during the infection. Large number of macrophage and neutrophils were accumulated both in the early stage and later stage of infection. Statics analysis shows that there is close correlation between the neutrophils with the viral load, serum and pulmonary antibody response. Close correlation also be found between the antibody responses with the viral load in lung. The infiltration of macrophage and neutrophils promote the cytokine response thereby implicated in the antibody response. Third, beside the robust antibody response, cytokine secreting cells (CSCs) and antibody secreting cells (ASCs) response which regulates the kinetics and magnitude of antibody response were also detected in lung of mice post infection. Fourth, cellular and humoral response in mucosal and second lymphoid organs cooperate with the immune response in lung contributes to the systemic protective response during the infection. Fifth, the different virulence of two influenza A virus strains is demonstrated by lethal dose fifty (LD50), weight lost and death ratio on mice, furthermore, the response initiated by two strains is not similar. Taken together, the response induced by A/Beijng/501/2009 (H1N1) is earlier and stronger than that induced by A/PR/8/34 (H1N1) virus. Sixth, the alignment of the protein sequency among A/Beijing/501/2009(H1N1), A/California/04/2009 and A/PR/8/34(H1N1) virus shows that there are mutations located either in known molecular determinants that confer pathogenicity and antiviral drug resistance and the glycan receptor-binding sites and antigenic loops which should be further characterized of their functional determinants.In conclusion, the process of immunopathology mediated by influenza A virus is multifaceted and highly complicated, involving different arms of the host response, all of which appear to be required for efficient virus clearance. The continuous replication of viral and accumulation of macrophage and neutophils in lung in the early stage of infection are the main reason for immune-mediated pathology. In the late stage of infection, neutrophils cooperate with the antibody response contributed to control the viral burden. Local pulmonary pathologic response is connected with the initiation of system immune response.更多还原