纳米材料PAMAM G3及甲型H1N1流感病毒导致小鼠急性肺损伤的致病机理研究

【作者】 李承刚；

【导师】 蒋澄宇；

【作者基本信息】 北京协和医学院 ， 生物化学与分子生物学， 2010， 博士

【摘要】 纳米材料PAMAM G3引起A549细胞自噬性死亡,并导致小鼠急性肺损伤作为一项重要的和新兴的产业,纳米技术正逐步从研究领域涉及到日常生产生活。然而,直到最近,人们才开始关注纳米材料对人体及环境的安全性问题。已经有研究称：纳米材料可以造成肺部的疾病或损伤,但其所导致的肺部损伤的分子机制却仍然没有被揭示出来。本文所研究的纳米材料是一种具有高度支化、对称、呈辐射状的大分子——聚酰胺-胺树枝状大分子纳米材料(PAMAM dendrimer)。它由中心核、内层重复的分支亚单位以及表面的功能基团组成。整代的PAMAM纳米材料表面的功能基团为氨基,半代的PAMAM纳米材料表面的功能基团为羧基。由于氨基带有正电荷,整代的PAMAM常被用作载体,递送DNA,进行细胞或生物体内表达。对于这些PAMAM材料生物安全性的研究甚少。本文中,我们测定了PAMAM G1～G8以及G3.5、G4.5、G5.5和G7.5对A549细胞的影响。发现从PAMAM G3到PAMAM G8均会引起A549细胞的死亡。而表面基因为阴离子的半代PAMAM对A549细胞的活性不会产生影响。我们对PAMAM G3引起的A549细胞死亡进行了分析,发现PAMAM G3不是通过凋亡通路引起的A549细胞死亡,而是引起细胞自噬性死亡。给予自噬的抑制剂3MA或是利用siRNA技术抑制自噬基因ATG6的表达,可以有效地缓解PAMAM G3引起的A549细胞死亡。同时,自噬性细胞死亡也在PAMAM G4、G5.G6、G7和G8引起的A549细胞死亡中扮演着重要角色。同时,我们对PAMAM G3引起的自噬性细胞死亡的信号通路进行了分析。发现PAMAM G3通过Akt-TSC2-mTOR的信号通路引起A549发生自噬性细胞死亡。此外,我们发现PAMAM G3会导致小鼠死亡,引起小鼠急性肺损伤。而自噬的抑制剂3MA有效地缓解PAMAM G3引起的小鼠死亡,改善小鼠的急性肺损伤程度。说明自噬在PAMAM G3引起的小鼠死亡中发挥着重要作用。甲型H1N1流感病毒导致小鼠急性肺损伤的致病机理研究2009年3月,从美国和墨西哥流感样患者呼吸道标本中,鉴定出新的猪源性甲型H1N1流感病毒。该病毒可在人际间传播,已蔓延100多个国家、地区。人感染后的主要临床表现为急性呼吸道感染症状,部分患者病情进展迅速,继发急性呼吸窘迫综合征、肺出血、呼吸衰竭和多器官损伤,导致死亡。感染甲型H1N1流感病毒导致的急性呼吸系统损伤的致病机制尚不清楚。本研究通过建立甲型H1N1流感病毒感染的小鼠模型,揭示NF-K B信号的激活,可诱发炎症相关因子产生,形成细胞因子风暴(Cytokine storm),在小鼠感染病毒后导致的急性肺损伤中发挥重要致病作用。通过检测小鼠的死亡率、肺组织湿干比、肺组织病理、支气管肺泡灌洗液中的炎症相关因子以及肺组织中单核巨噬细胞磷酸化NF-κB及IκB-α的表达等指标,比较不同流行株的甲型H1N1流感病毒对不同周龄、不同品系小鼠的致病性。结果表明：与CA07株甲型H1N1流感病毒比较,BJ株对4周龄的C57BL/6小鼠具有更强的致肺损伤能力,其在小鼠肺组织中的复制能力也明显高于CA07株甲型H1N1流感病毒。感染甲型H1N1流感病毒BJ株的小鼠,在连续给予糖皮质激素地塞米松治疗后,其死亡率有明显降低,肺组织损伤也得到显著缓解,说明激素抑制了剧烈的炎症反应,降低了小鼠的急性肺损伤程度。上述结果的阐明,为了解甲型H1N1流感病毒导致的急性肺损伤致病机理,并临床上治疗甲型流感病毒导致的急性呼吸系统损伤提供了重要信息。更多还原

【Abstract】 PAMAM dendrimer (polyamidoamine dendrimer) is a polymer with dentritic branching and radial symmetry. It consists of three parts:a central core, an interior dendritic structure (the branches) and an exterior surface (the end group).PAMAM dendrimers can be divided in to the whole (cationic) or half (anionic) generation polymers. The surface of the whole generation is functionalized with primary amino surface groups and the half generation is with carboxylate surface groups. Due to the cationic surface, the whole generation PAMAM could be used for gene delivery for expression in cells or in animals. Therefore, the biosafety of these PAMAMs need to be evaluated.In this dissertation, we determine the cells viability after PAMAM G1to G8and G3.5, G4.5, G5.5and G7.5treatment. We find that treatment of A549cells by cationic PAMAMs, from G3to G8could cause cells death, while the anionic PAMAMs have no effect on cells viability. Furthermore, we find that PAMAM G3causes A549cells death through autophagy, but not through apoptosis. Treating cells with inhibitor of autophagy,3-methyladenine (3MA), or by knockdown the autophagy related gene, beclin, could improve the cells viability treated by PAMAM G3. In the meanwhile, autophagic cell death happens in the PAMAM G4, G5, G6, G7and G8treated A549cells.In the following step, we analyze the signal transduction pathways in the PAMAM G3induced autophagic cell death. We find that PAMAM G3induced A549autophagic cell death through Akt-TSC2-mTOR pathway.Furthermore, we discover that PAMAM G3could induce acute lung injury and cause death in mice, while the autophagy inhibitor3MA could improve the situation. It is indicated that autophagy plays an important role in PAMAM G3induced mice acute lung injury. In March2009, in the United States and Mexico, a new porcine influenza A H1N1virus was identified from specimens of patients with influenza-like symptom. The virus can spread from person to person has been spreading to more than100countries and regions.Main clinical manifestations of human infected with influenza A H1N1virus was acute respiratory infection symptom. And rapid progression in some patients, sudden high fever, severe pneumonia or secondary acute respiratory distress syndrome, pulmonary hemorrhage, respiratory failure and multiple organ injury were observed. Mechanism of acute respiratory infection related injury caused by pathogenic is not clear yet.In this study, we established animal model of Influenza A H1N1virus infection in mice, found that:the activation of NF-κB signaling can induce production of inflammation-related factors (cytokines, chemokines), then the formation of cytokine storm, which plays an important role in the process of acute lung injury in mice after Influenza A H1N1virus infection.By detecting the mortality of mice, the lung wet to dry ratio, lung pathology, the level of the inflammation-related factors (cytokines, chemokines) in broncho-alveolar lavage fluid and phosphorylation of NF-K B and IK B-a in lung mononuclear macrophages as indicators, we compare the virulences of different strains of influenza virus after infection. Influenza A H1N1virus BJ strain on the4-week-old C57BL/6mice was found with a stronger ability of lung injury, in comparison to CA07strain. And the viral load in lung tissue of mice was significantly higher than CA07strain.Infected with influenza A H1N1virus strain BJ, mice given continuous steroid Dexamethasone, which significantly reduced mortality and markedly respited the lung injury.These findings expand our understanding of influenza A H1N1virus induced lung injury and also provide valuable information for the anti-viral research.更多还原