【作者】 房明丽；

【导师】 王丽颖；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2011， 博士

【摘要】 流感是一种由流感病毒感染引起的具有高度传染性的呼吸系统疾病,上世纪3次流感爆发流行造成了上千万人死亡,且大多数为青壮年。普遍认为重症流感的死亡原因与流感病毒诱导的过激性天然免疫反应以及大量细胞因子分泌造成急性肺部炎症性损伤有密切关系。因此,抑制过激性免疫应答对流感引起的急性肺损伤的防治可能有重要作用。我们在前期工作中设计并验证了一种命名为SAT05f的寡聚脱氧核苷酸(oligodeoxynucleotide, ODN),在体外及小鼠体内表现出选择性抑制TLR7/9活化的作用。为了研究SAT05f是否能够减轻流感病毒感染引起的小鼠急性肺损伤,我们选择FM1流感病毒株,以滴鼻的方式感染BALB/c小鼠建立了小鼠急性肺损伤模型。在此基础上,我们研究了SAT05f对小鼠急性肺损伤的干预作用。出乎意料的是,SAT05f只表现出轻度减轻肺病理损伤的作用,而作为对TLR7/9只在体外有微弱抑制作用的对照ODN(MS19)却明显地减轻了流感病毒引起急性肺组织病理损伤;抑制了小鼠的体重下降,减低了小鼠的死亡率。为了解释这种现象,我们对小鼠的肺组织中炎性细胞浸润、TNF-?和IL-10的表达水平进行了检测,结果显示:MS19能抑制小鼠肺组织中中性粒细胞浸润,且肺组织中TNF-?和IL-10的表达水平均降低。这些结果表明,MS19减轻肺损伤的作用与抑制肺组织炎症反应及TNF-?有关,是否其他机制也参与其中还有待进一步研究。更多还原

【Abstract】 Influenza is a highly infectious respiratory disease caused by influenza virus, posing a great global infectious disease threat. In the 20th century, three influenza pandemics emerged and killed tens of millions of people. In the patients, most deaths occurred in young people, previously healthy adults or children. Analysis showed that one of the main reasons for the death was due to acute respiratory distress syndrome (ARDS) caused by influenza virus-induced inflammatory injury.In recent years, over-activated innate immune response has been found attributable to the development of the influenza virus-induced lung inflammatory injury. Upon infection, influenza virus derived single stranded RNA and double stranded RNA, generated as intermediate molecules, are sensed by toll-like receptors (TLRs), initiating activation of the innate immune cells, such as macrophages and dentritic cells (DC). The activated cells produce type I interferon and other cytokines to combat the virus. However, if infection persists, innate immune cells could be over-activated and produce excessive pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-?), interleukin-1 (IL-1), and interleukin-6 (IL-6), undesirably. The over-produced cytokines, as found in basic and clinical studies, contribute to acute lung inflammatory injury (ALII), leading to a fatal outcome. Obviously, it is required to develop agents to curb the over-reaction and the over-produced cytokines for saving life of the patients infected with influenza virus, especially those highly pathogenic ones.Inhibitory oligodeoxynucleotides (ODNs), an artificial single-stranded DNA molecule, could regulate innate immunity activation. Recent studies demonstrate that inhibitory ODNs show a promise to develop an agent for the treatment of diseases associated with Toll-like receptor activation,including SLE and sepsis. In our previous study, SAT05f and MS19, the MS ODNs (microsatellite DNA mimicking oligonucleotides) designed with reference of the sequence of human microsatellite DNA, were shown able to rescue mice from lethal shock.In this study, to test whether the MS ODN could lessen ALII in mice infected by influenza virus, we firstly established the mouse model induced by influenza virus, a H1N1 mouse influenza virus. Then, we observe the effect of MS ODN on acute lung injury in FM1 virus infected mice and study the mechanism.1. Establishment of the mouse model of acute lung inflammatory injury induced by influenza virusIt is well known that not all influenza virus strains can cause ALII, leading to severe pneumonia by over-activating innate immune response. To study whether the MS ODN could lessen the ALII of mice, FM1 virus was used to establish a mouse model. After being inoculated intranasally with 10 LD50 of FM1 virus, the mice were observed every day for recording the morbidity and the mortality. On day 2 or 3 post infection, most of the infected mice began to exhibit clinical signs of influenza, including ruffle fur, hunched posture, neurological symptoms and weight loss. To monitor the viral infection and cytokine production in lung, the mice were sacrificed at day 0, 2, 4, 6 post infection and their lungs were removed for preparing homogenates that were used to test the FM1 virus titer by hemagglutination assay and biological activity of TNF-αby L929 cytotoxicity bioassay. The results showed that the viral titer in lung homogenate in mice began to increase on day 2, and rapidly decreased on day 4 post infection. On day 6 post infection, the virus titer in the lung homogenate was hardly detected. Interestingly, on day 6, the infected mice began to die. Within ten days, all the infected mice succumbed. Obviously, the highest virus load was not directly attributed to the death, suggesting the involvement of other factors.To analyze the mechanism of acute lung injury of mice induced by influenza virus, we observe the curve of virus titer and pro-inflammation cytokines. On day 0, the mice infected 10LD50 influenza virus. On day 2, 4, and 6 post-infection, the mice were scracificed and their lungs were homogenated. The results shown that the virus titer increased gradually from day 2, reached to the maximum on day 4 and decresed on day 6 a time point when the mice began to die, indicating that the virus titer was not associated with the death. Interestingly, he TNF-α, an important pro-inflammatory factor for inducing ALII, rose rapidly in the lung on day 2 post infection and then maintained at a high level to the day 6, implying a role of TNF-αfor the death. Upon the results, the mouse infected with FM1 virus was adopted as an animal model to evaluate the effects of MS ODN on the ALII caused by FM1, possibly by modulating innate immune responses.2. The inhibitory effect of MS ODN on TLR7/9 mediated activation in innate immune response in vitroWe choose three MS ODNs, named as SAT05f, MS19 and MS33. We test the effect of MS ODNs on inhibiton of TLR9 agonist (CpG 1826) induced the proliferation of mouse spleen cells and the production of TNF-α. In addition, we also test the the effect of MS ODNs on inhibiton of TLR7 agonist (PR8-flu, inactivated FM1 virus) induced the production of TNF-αfrom RAW264.7. a) The effect of MS ODN on inhibition of CpG 1826 induced the proliferation of mouse spleen cells and the procution of TNF-αfrom RAW264.7.To observe the inhibition of MS ODN on the activiton of immune cells, we firstly isolated the mouse spleen cells. In the assay, CpG 1826 (B type CpG ODN), capable of stimulating the proliferation of mouse spleen cells, was chosen as a stimulator. Freshly isolated mouse spleen cells were incubated in the medium containing CpG 1826 alone or with SAT05f or MS19 or MS33 and then assessed for their proliferation by MTT assay. The result showed that SAT05f were capable of inhibiting CpG 1826-induced proliferation of mouse spleen cells significantly (p<0.01). MS19 and MS33 failed to inhibit the CpG 1826-induced proliferation.To determine whether MS ODNs could block the production of TNF-α, a pro-inflammatory cytokine involved in lung inflammatory injury caused by influenza virus infection (Clark, 2007), we detected the inhibitory effect of MS ODNs on blocking TNF-αproduction from RAW264.7 cells (a murine macrophage-like cell line cells) induced by CpG 1826 (B type CpG ODN) that was confirmed capable of stimulating the production of TNF-αfrom immune cells. The results showed that SAT05f could significantly inhibit the TNF-αproduction from RAW264.7 cells b) The effect of MS ODN on inhibition of TLR7 agonist induced anti-viral activity and the procution of TNF-αfrom RAW264.7To determine whether MS ODNs could block the production of TNF-αby blocking the TLR7, we detected the inhibitory effect of MS ODNs on blocking TNF-αproduction from RAW264.7 cells (a murine macrophage-like cell line cells) induced by inactivated FM1. The results showed that SAT05f could significantly inhibit the anti-VSV activity induced by PR8-flu and the TNF-αproduction from RAW264.7 cells triggered by FM1 (p<0.01), While MS19 and MS33 failed to.3. The effect of MS ODN on the morbidity, mortality, and lung inflammatory injury of mice infected with influenza virus.Upon these, a microsatellite DNA mimicking oligodeoxynucleotide (MS ODN), named as SAT05f capable of inhibiting TLR7/9-activation in vitro, was used to treat mice infected with FM1 virus. In parallel, two MS ODNs confirmed with less or no in vitro activities, named as MS19 and MS33, were used as controls. On day 0, mice were inoculated intranasally with 10 LD50 of FM1 virus. The mice (n=8) sham-inoculated with sterile PBS were used as normal control. On days 2 and 4 post infection, the model mice (n=8) were injected i.p. with PBS, SAT05f or MS19 or MS33, respectively. We observed the effect of MS ODN on the morbidity, mortality, and lung inflammatory injury of mice infected with influenza virus.a) The effect of MS ODN on weight loss and survival of mice infected with influenza virus.Unexpectively, On day 7 post infection, the model mice received PBS or SAT05f or MS19 displayed 31%, 30% and 29%,mean weight loss at day 7, respectively, while model mice treated with MS19 only displayed 22 % mean weight loss, indicated that MS19 significantly inhibit the weight loss of mice infected influenza virus. While SAT05f displayed a weaker role. On day 16, the mortality rates of the mice treated with PBS and MS33 successively increased to 87.5% and 100%, whereas the mortality rates of the mice treated with SAT05f or MS19 decreased to 62.5% or 37.5%, respectively.b) The effect of MS ODN on lung inflammatory injury of mice infected with influenza virus.On day 17, the survived mice in each group and normal control mice were sacrificed and their lungs and spleens were removed for gross observation, histopathological examination and body weight measurement. The whole lungs of the mice treated with PBS and MS33 displayed edema, consolidation and profuse hemorrhage. Comparably, in the mice treated with SAT05f, the consolidated change and profuse hemorrhage only occurred on 25% areas of the lung. Unexpectedly, MS19, an ODN unable to inhibit TLR7/9 activation in vitro, could almost completely prohibit the development of the consolidation and hemorrhage in lung of the mice. Histopathologically, mice treated PBS showed thickened and congested alveolar walls, intra-alveolar edema, and numerous infiltrated leukocytes. In contrast, the lung of the mice treated with SAT05f, the areas with thickened alveolar walls, intra-alveolar edema and infiltrated leukocytes were fewer. The lung of the mice treated with MS19 manifested thinner alveolar walls, lacking intra-alveolar edema fluid and infiltrated leukocytes. Furthermore, the pathological score of the lung tissue of MS19-treated mice was lower than that in the mice treated with PBS and SAT05f group. The data indicated that MS19 could lessen the ALII of mice infected with influenza virus.c) The effect of MS ODN on lung index and spleen index of mice infected with influenza virus.Based on the weights of the lungs and spleens of the sacrificed mice, lung index (lung weight/body weight) and spleen index (spleen weight/body weight) were determined. The lung index of the mice infected with FM1 virus increased significantly, compared to that in normal mice, indicating that a strong inflammatory reaction was induced by FM1 virus in lung of the model mice. The lung index of the mice in MS19 group was lower than that in the PBS group, implying that MS19 could significantly inhibit the FM1 virus-induced ALII. The inhibition was correlated with the inhibitory effect of MS19 on immune reactions, manifested by that the spleen index of the mice received MS19 was 6.29±1.32; in contrast, that in the mice treated with PBS was 11.79. The spleen index of the mice in MS19 group was as low as that in the normal control group.d) The effect of MS ODN on the infliltration of immune cells and production of inflammatory cytokines in lung tissue of mice infected with influenza virus. To find how MS ODN inhibited the development of ALII caused by FM1 virus, we detected the the infliltration of immune cells and production of inflammatory cytokines in lung tissues of the mice infected with FM1 virus. The results showed that MS19 significantly inhibit the infiltrated immune cells. In addition, we test the mRNA levels of TNF-αand IL-10 in the lung tissues of mice. The results showed that MS19 significantly inhibit the expression of TNF-αand IL-10 in the lung tissues. In addition, MS19 could inhibit TNF-αproduction induced by FM1 virus.e) The effect of MS ODN on the virus titer in lung tissue of mice infected with influenza virus.Upon that the FM1 virus titer reached its peak in lungs of the model mice, as determined above, we detected the FM1 virus titers in the homogenates collected on day 4 after infection. FM1 virus titers in lung homogenates of the mice treated with MS19 was within the same range as that of the homogenates from the mice treated with PBS, suggesting that MS19 mediated inhibition on the development of ALII induced by FM1 virus was unrelated to the direct action of MS19 on replication of FM1 virus.In present study, we found that MS19 significantly inhibited the weight loss and displayed dramatic effect on lessening the ALII by reducing consolidation, hemorrhage, intra-alveolar edema and leukocyte infiltration in lungs of the mice. Meanwhile, MS19 could prolong the survival of the mice. The data suggest that MS19 might be developed a candidate drug for the treatment of acute lung injury induced by other virus.更多还原