1、甲型H1N1流感病毒致病机理的研究2、PAMAM纳米材料毒性机理的研究

发布时间：2018-02-01 13:00

本文关键词： 凋亡呼吸猪源甲型H1N1流感纳米材料 PAMAM ACE2 急性肺损伤　出处：《北京协和医学院》2011年博士论文　论文类型：学位论文

【摘要】：2009年4月,在墨西哥首次检测并发现了一种新型的甲型流感病毒(Novel Swine-Origin Influenza A)在人群中传播,并造成感染人群的发病。这种新型的甲型H1N1流感病毒(S-OIV H1N1)迅速传播至全球许多国家和地区。2009年6月11日,世界卫生组织(WHO)发布公告,甲型流感病毒的全球警戒水平提升至6级,这也预示着全球流感大流行的爆发。尽管此次流行的甲型H1N1流感病毒感染多为散在的个体,且感染症状一般比较轻微,但对于青年人以及那些本来有基础病的人群(其中包括哮喘、糖尿病、病态肥胖症以及孕妇)而言,导致更为严重疾病的病程的情况将会大大增加。2010年8月10日,WHO甲型H1N1流感病毒大流行结束,但地区性流感爆发仍在延续。根据WHO最新统计结果,而一般情况下,因季节性流感病毒感染,每年死亡人数在250,000-500,000之间。此次甲型H1N1流感大流行共导致18,000人死亡,死亡率占4%,健康保护机构(HPA)报道,最近在英国确证因感染流感病毒导致214人死亡,其中195人确证感染了2009年的甲型H1N1流感病毒株。该机构由此提出,甲型H1N1(S-OIV H1N1)可能会卷土重来。 2009年大流行的甲型H1N1病毒可能来源于典型的猪甲型H1N1流感病毒、可能与1918年“西班牙大流感”流行的人甲型H1N1流感病毒以及禽甲型流感病毒北欧世系有同源性。研究人员对分离到的病毒进行全基因序列测定,但没有发现任何已经确定的毒力标记物。动物实验表明,造成2009年大流行的甲型H1N1病毒比人季节性甲型H1N1流感病毒具有明显的致病力。动物模型研究显示,与季节性甲型流感病毒相比,2009年的甲型H1N1病毒可以更有效地在宿主细胞内进行病毒复制,并导致更为严重的发病率和致死率。对许多生理过程,包括组织萎缩、发展以及肿瘤生物学等而言,进程性细胞死亡、或凋亡是至关重要,在众多包括疾病性感染的病理过程中,细胞凋亡发挥重要的作用。许多病毒感染均可导致宿主细胞凋亡,流感病毒可在体内和体外造成许多种类的细胞凋亡。有报道,感染H5N1-AVI患者和禽的肺泡上皮细胞或血感管内皮细胞出现细胞凋亡。还有其他一些研究报道提出,感染H5N1-AVI的人类患者,细胞发生凋亡是导致发展成为急性呼吸窘迫症(acute respiratory distress syndrome, ARDS)所必须的。通过进行细胞培养和感染小鼠的研究实验证明,导致1918-1919年爆发的“西班牙大流感”的甲型H1N1流感病毒以及流感H9N2病毒均能够导致宿主细胞凋亡。然而至今,尚未有2009年造成全球流感大流行的甲型H1N1流感病毒有关可产生细胞凋亡的报道。有研究发现,感染猪源性甲型H1N1流感死亡患者的肺部发现有肺泡的损伤,支气管坏死和组织出血。并在支气管上皮细胞以及肺泡细胞出现病变。我们在研究中,对2009年流感大流行期间在国内分离到的一些猪源性甲型H1N1流感病毒株,发现其中甲型H1N1病毒文山株(A/Wenshan/01/2009H1N1)能够导致人呼吸道上皮细胞A549株和CNE-2Z株细胞的凋亡。我们还发现,对于感染文山株病毒(A/Wenshan/01/2009H1N1),来源于上呼吸道的CNE-2Z细胞比下呼吸道的A549更为敏感,与人季节性甲型流感病毒相比,文山株显示出更高的侵入能力和病毒复制能力。我们通过研究还证实了,流感病毒是利用clathrin-依赖的和dynamin依赖的进入通路进入宿主细胞。在人类迈入二十一世纪的今天,纳米技术在医药、信息以及通讯技术产业领域里被认为是极为重要的新兴技术。纳米材料半径微小,其特殊的小尺寸效应、量子效应和巨大比表面积。树状分子材料(PAMAM)为单分散结构,且高度分枝化的纳米材料,这种结构能够设计成单分散胶体、封闭的金属簇、结合组织液、或具有生物活性的成分,并且能够在适宜的介质中溶解,并可与某些成分的表面结合。由于PAMAM的这一结构特性,因此,PAMAM可用于开发成各种功能性材料用于众多领域,包括化学物质的分离、富集、医疗影像以及DNA或药物的释放等。树状分子材料是由重复的分支组成的球型大分子,围绕核心形成三维对称结构。树状分子呈单分散对称性球型结构的结构决定了其特性。树状分子材料可分为低分子量和高分子量。前者一般指树状分支,后者包括树枝状多聚体、超枝化聚合物多聚体等。树状分枝纳米材料的特性取决于分子表面的功能基团。同时,也是的树状分子纳米材料与其他聚合材料不同,可成为水溶性材料,树状结构包裹的功能分子结构可以成为行使活性功能的场所。树状分子纳米材料可由于合成过程中重复的分支循环数量的不同分成不同的代。每新合成一代其分子量比上一代增加一倍。PAMAM G5树状分子材料是和一定数量的乙酰酐乙酰化反应的产物。高分子量的树状纳米材料具有更多的功能基团,用于商业化的树状分子纳米材料可以有更为广泛的应用。聚酰胺纳米材料(PAMAM)是目前研究比较成熟的树状分子纳米材料。PAMAM的核心为是乙二胺,和另外一个乙二胺经胺化反应形成G-0代。PAMAM纳米材料的表面功能基团被认为是链接化学,具有许多潜在的应用价值。PAMAM树枝状大分子被用广泛应用于药物研究,用于抗感染,抑制细胞、病毒、细菌、蛋白间的多价结合等。纳米材料在医学领域具有极大的应用前景。但随着纳米技术的广泛应用,纳米材料可能存在的安全性问题越来越引起广泛的关注。当前,纳米材料对机体可能会机体造成肺损伤已成为研究的热点,由此导致全世界都在呼吁在纳米材料的安全性问题彻底解决前应暂停纳米材料的使用,特别是在医药领域的呼声更为强烈。美国环境保护机构开始评价纳米技术对人类健康产生的影响,这对PAMAM纳米材料的毒性以及对环境的安全性发挥重要作用的资料。目前,关于对纳米颗粒毒性的研究的重点是导致肺部疾病。我们曾经开展过相关研究显示,血管紧张素转化酶2(angiotensin I converting enzyme2,ACE2)能够保护肺部酸吸入综合征、败血症以及感染SARS冠状病毒的小鼠避免导致严重的就急性肺损伤。由以前的研究,我们设想,是否ACE2也能够保护小鼠抵抗因纳米颗粒产生的肺损伤。目前,可供商业化提供的全代(阳离子)或半代(阴离子)的PAMAM纳米材料。由于PAMAM纳米材料正越来越广泛地在应用于医药领域,PAMAM阳离子纳米材料也即将完成临床试验,获得美国食品药品管理局(FDA)的批准。我们对阳性PAMAM纳米材料进行了深入的研究,阐明通过实验观察到的PAMAM导致肺损伤的毒性机理。我们的研究显示,给小鼠注射PAMAM,能够上调小鼠肺组织的ACE2表达,下调血管紧张素Ⅱ的产生,从而避免严重肺损伤的产生。注射ACE2基因敲除小鼠,导致动物肺损伤的发生。我们的研究解释可使纳米颗粒不能够导致肺损伤的原因,并建议为日益关注的纳米技术的安全性问题提供了可能的治疗策略。
[Abstract]:In April 2009, the first detection and discovery of a novel influenza A virus in Mexico (Novel Swine-Origin Influenza A) spread in the population, the incidence of infection and cause. The new H1N1 influenza virus (S-OIV H1N1) quickly spread to the world in many countries and regions in.2009 June 11th, WHO (WHO) announced influenza A virus, the global alert level raised to 6, which also indicates that the global influenza pandemic outbreak.
Although the H1N1 influenza virus infection of the epidemic is more scattered in the individual, and the symptoms of infection is generally mild, but for young people and people who have underlying diseases (including asthma, diabetes, morbid obesity and pregnant women), which is a serious disease course situation will greatly increase.2010 in August 10th WHO, H1N1 influenza virus pandemic is over, but regional flu outbreak continues. According to the results of the latest statistics WHO, under normal circumstances, due to seasonal influenza virus infection, the annual death toll in 250000-500000. The H1N1 flu pandemic caused a total of 18000 deaths, mortality accounted for 4%, the Health Protection Agency (HPA) reported recently in the UK was due to a flu virus that killed 214 people, including 195 people were infected with influenza a H1N1 influenza virus strain in 2009. The agency which provided A H1N1 (S-OIV H1N1) may come back again.
The 2009 pandemic influenza H1N1 virus may be derived from classical swine H1N1 influenza virus may have homology with the 1918 "Spanish flu" epidemic H1N1 human influenza virus and avian influenza virus. The researchers determined the Nordic descent from the virus genome sequence has been determined, but found no toxicity marker. Animal experiments show that caused the 2009 pandemic influenza a H1N1 virus has obvious pathogenicity than human seasonal influenza a H1N1 influenza virus. Animal model studies show that compared with the seasonal influenza virus, 2009 H1N1 influenza virus can be more effective for virus replication in host cells, and lead to more morbidity and the fatality rate of serious.
In many physiological processes, including tissue atrophy, development and tumor biology, the process of cell death, or apoptosis is critical in many pathological processes, including disease infection, cell apoptosis play an important role in host cells. Apoptosis can be many viral infections, influenza viruses can cause apoptosis in many types of in vivo and in vitro.
Reported that patients infected with H5N1-AVI and avian alveolar epithelial cells or blood vascular endothelial cells appeared apoptosis sense. Some research reports suggested that H5N1-AVI infection in human patients, cell apoptosis is lead to the development of acute respiratory distress syndrome (acute respiratory distress syndrome, ARDS) must be.
Through the study of cell culture and experimental infection of mice that led to 1918-1919 years of the outbreak of the "Spanish flu" influenza a H1N1 influenza virus and influenza H9N2 virus can cause the host cell apoptosis. However, so far, has not yet been reported in 2009 caused a global influenza pandemic influenza a H1N1 virus can produce cell apoptosis.
It has been found that alveolar injury, bronchial necrosis and tissue bleeding are found in the lungs of patients infected with swine influenza a H1N1 influenza, and there are lesions in bronchial epithelial cells and alveolar cells.
In our research, we detected some swine influenza A virus strains isolated from China during the 2009 influenza pandemic. We found that the H1N1 strain of Wenshan virus (A/Wenshan/01/2009H1N1) can induce apoptosis of A549 and CNE-2Z cells of human respiratory epithelial cells, H1N1.
We also found that for infected strains of Wenshan virus (A/Wenshan/01/2009H1N1), CNE-2Z cells from the upper respiratory tract were more sensitive than A549 in the lower respiratory tract. Compared with the seasonal influenza A virus, Wenshan strains showed higher invasion ability and virus replication ability.
We have also demonstrated that influenza viruses are entering the host cells using clathrin- dependent and dynamin dependent access pathways.
As mankind enters the twenty-first Century, nanotechnology is regarded as a very important new technology in the fields of medicine, information and communication technology industry.
Nano materials and its special radius of small, small size effect, quantum effect and large specific surface area. Dendrimer material (PAMAM) for monodisperse and highly branched structure, nano materials of this structure can be designed into monodisperse colloidal metal clusters, closed, combined with tissue fluid, or biologically active ingredients. And can be dissolved in a suitable medium, and can be combined with certain surface components. Due to the structural characteristics of PAMAM, therefore, PAMAM can be used to develop into various functional materials used in many fields, including separation, chemical enrichment, medical imaging and DNA or drug release.
Dendrimer material is spherical macromolecules composed of repeated branches, around the core to form a three-dimensional symmetric structure. A monodisperse dendrimer structure symmetry spherical structure determines its characteristics. Dendrimer materials can be divided into low and high molecular weight. The former refers to the tree branch, the latter including dendritic poly dimers, super branched polymer polymer. Functional groups dependent on dendritic nano materials on the molecular surface. At the same time, but also the dendrimers of different nano materials and other polymeric materials, can be water soluble material, tree structure package molecular structure can become active functional exercise in place. The tree molecular nano materials may be due to the synthesis process of repeated cycles of different number of branches into different generations. Each new generation of synthetic molecular weight than the previous generation of double.PAMAM G5 dendrimer material The products of acetylation reaction with a certain number of acetyl anhydride. High molecular weight dendrimers have more functional groups. The commercialized dendrimer nano materials can be widely applied.
Polyamide nano material (PAMAM) is the core of the dendrimer nanoparticles.PAMAM mature as ethylenediamine, and another ethylenediamine by amination of functional groups formed on the surface of G-0 generation of.PAMAM nano material is considered to be the link to chemistry, with many potential applications of.PAMAM dendrimers are widely used in medicine study for anti infection, inhibiting cell, virus, bacteria, protein between multivalent binding. Nano material has great application prospect in the field of medicine.
But with the wide application of nanotechnology, safety of nanomaterials may exist more and more attention. At present, nano materials may cause body lung injury has become a research hotspot in the body, resulting in the world calls for the problem of nano materials should be used to solve an before suspension of nano materials especially in the field of medicine, more and more applause. The U.S. Environmental Protection Agency began to impact assessment of nanotechnology to human health, the toxicity of PAMAM nano materials and environmental security play an important role.
At present, the focus on research of nanoparticle toxicity is the cause of lung disease. We have carried out relevant studies show that angiotensin converting enzyme 2 (angiotensin I converting enzyme2, ACE2) can protect the lung acid aspiration syndrome, sepsis and infection of SARS coronavirus in mice to avoid severity of acute lung injury. From previous studies, we assume that whether ACE2 can protect mice from nanoparticles due to lung injury.
At present, for commercial whole generation (cationic) or half generation (anionic) PAMAM nano materials. Because PAMAM nano materials are becoming more and more widely applied in the field of medicine, PAMAM cationic nano materials will also be completed clinical trials, the U.S. Food and Drug Administration (FDA) to obtain approval. We are a further study on the positive PAMAM nano materials, clarify the experimentally observed PAMAM induced toxicity mechanism of lung injury.
Our study shows that mice injected with PAMAM can increase the ACE2 expression in lung tissue of mice, decreased the production of angiotensin II, so as to avoid serious lung injury. Injection of ACE2 gene knockout mice, resulting in animal lung injury. Our study can make the interpretation of nanoparticles can cause lung injury the security problem and suggest more attention nanotechnology provides possible therapeutic strategies.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R373.13;R318.08

【共引文献】