H5N1型禽流感病毒损伤细胞溶酶体的机制研究和南极极端环境下科考队员的应激反应研究

发布时间：2018-05-04 14:53

本文选题：H5N1 + 流感病毒　；参考：《北京协和医学院》2012年博士论文

【摘要】：H5N1高致病性禽流感病毒不仅会给养殖、畜牧业带来灾难性的破坏,而H.人感染后死亡率高达近60%；特别是近期一些研究结果表明H5N1禽流感病毒可以通过变异、重组而能在人群中传播,从而严重威胁人类的生命健康。而目前对H5N1禽流感病毒的致病机理仍然了解较少,禽流感病毒如何突破宿主细胞、免疫系统的防御而产生高病毒载量的机制还不明确,特别是具有清除外源微生物作用的溶酶体在流感病毒感染过程中的作用仍未得到解析。本论文研究发现H5N1禽流感病毒感染细胞后,病毒的神经氨酸酶(NA)能够在细胞内降解溶酶体膜上的糖蛋白和溶酶体内的水解酶,从而破坏溶酶体的完整性和正常功能,完整功能的溶酶体大量丧失可能是H5N1禽流感病毒的重要致病机制之一；而达菲等NA抑制剂能在细胞内抑制NA对溶酶体膜糖蛋白的降解,可能是其抗病毒的作用机制之一；同时也发现,不同程度上损伤溶酶体可能是流感病毒的共同致病机理。以上的研究发现提示H5N1禽流感病毒可能通过破坏溶酶体功能从而引起高病毒载量、免疫抑制等严重病症,可能是H5N1禽流感病毒致病的重要原因；同时本研究结果能为将来抗流感病毒药物的研发提供新的思路和依据。表型和基因型关联分析南极极端环境下科考队员的应激反应南极冰穹A (Dome A)位于南极大陆高原冰盖最高点,具有高海拔、低氧、酷寒、烈风、难以到达与危险性大等恶劣自然环境因素和与世隔绝的社会环境因素,被喻为地球上的“不可接近之极”之一；同时Dome A却是科学考察的圣地,每年都有大批科考队员前往Dome A进行例行的考察。长时间在南极生活工作,科考队员几乎都会出现不同程度的诸如冲动、烦躁、抑郁等症状的“南极综合症”,同时高原、低氧的环境会引起肺水肿、脑水肿等致命疾病,因此研究南极Dome A复合多种极端环境对科考队员的生理、心理影响,并在分子机制上进行探究具有重要意义。我们以中国第24次南极内陆科考队员为研究对象,分析科考队员身处南极大陆最高点Dome A时的生理功能、心理情绪及外周血白细胞基因表达谱的变化。发现在Dome A时科考队员表现出明显的高原生理应激反应,体内雄性激素明显升高,并伴随心理情绪的异常波动,且雄性激素的升高与情绪波动具有显著相关性;外周血白细胞中差异表达的1,121个基因,在GO term数据库中富集得到有关大脑神经发育、精子生成等功能集,与表型中的情绪波动、脑血氧含量降低、性激素上升具有显著的相关性；基因芯片的分析结果在一定程度上与表型中的生理、心理变化相对应。本研究全面分析了南极Dome A极端环境对人体的影响及部分表型变化的分子机制,能为将来科考队员的筛查、训练、防护和心理辅导提供重要的理论依据。HIV gp160蛋白高量表达与纯化人类免疫缺陷病毒(HIV)感染可以引起获得性免疫缺陷综合症(AIDS),感染者常常因为免疫缺陷引起继发性感染而死亡。目前全世界有超过3,000万的H1V感染者,同时每年还增加300万的新感染者,但还没有有效的疫苗和防治药物。HIV gp160蛋白构成了病毒外壳结构蛋白,介导了病毒的细胞进入以及宿主对病毒的抗体中和反应；多种病毒进入抑制剂通过干扰gp160蛋白与受体结合或者病毒包膜与细胞质膜融合而阻止病毒的进入,具有一定疗效；但目前因为未能对gp160蛋白进行晶体结构解析而阻碍了新的更有效的病毒进入抑制剂的设计和研发;2009年gp160蛋白还被Nature杂志评为最需要进行晶体结构解析的十个蛋白之一。其中表达纯化高质量的gp160蛋白是进行晶体结构解析的重要前提。本课题综合了本实验室在蛋白表达纯化技术上的优势,通过对蛋白序列进行密码子优化,使用真核高表达载体,在人源293细胞中成功表达了gp160蛋白；并结合最新的Wave大规模细胞培养技术,实现了细胞的连续灌注培养；在进一步的研究中通过在gp160蛋白末端加入T4F促三聚化末端,提高了gp160蛋白中的三聚体形式结合蛋白的比例。本研究共计纯化表达了130mg gp160蛋白,为gp160蛋白抗体制备和结晶结构解析等后续实验奠定了基础。同时,本研究内容和方法为其它蛋白的高量表达和纯化提供了一个很好的平台。
[Abstract]:H5N1 highly pathogenic avian influenza virus will not only cause catastrophic damage to breeding and animal husbandry, but the mortality of H. people is nearly 60% after infection. Especially, some recent results show that the H5N1 avian influenza virus can be transmitted through mutation and can be transmitted in the population, which is a serious threat to human life and health. At present, the H5N1 avian influenza virus can be seriously threatened. The pathogenesis of the virus is still less. The mechanism of avian influenza virus breaking through the host cells and the defense of the immune system is not clear. Especially, the role of lysosomes with the removal of exogenous microbes in the process of influenza virus infection is still not resolved.
After the study found that the H5N1 avian influenza virus infected cells, the virus's neuraminidase (NA) can degrade the glycoprotein on the lysosome membrane and the hydrolase in the lysosomal membrane, thus destroying the integrity and normal function of the lysosome, and the loss of the lysosome, the complete function of the lysosome, may be an important pathogenic mechanism of the H5N1 avian influenza virus. One of the NA inhibitors, such as Tamiflu, can inhibit the degradation of NA to lysosomal membrane glycoproteins in cells, and may be one of the mechanisms of its antiviral action. At the same time, it is also found that the damage of lysosomes to different degrees may be a common pathogenic mechanism of influenza virus.
The above findings suggest that H5N1 avian influenza virus may be an important cause of H5N1 avian influenza virus disease by destroying the function of lysosome and causing severe viral load and immunosuppression, and the results can provide new ideas and basis for the research and development of anti influenza virus drugs in the future.
Phenotypic and genotypic association analysis of stress response in the Antarctic extreme environment
The Antarctic ice dome A (Dome A), which is located at the highest point of the ice cover on the Antarctic continental plateau, has high altitude, low oxygen, cold, strong wind, difficult to reach and dangerous natural environmental factors and isolated social environmental factors. It is regarded as one of the "unapproachable poles" on the earth. At the same time, Dome A is the holy place of scientific investigation, and every year there is a large scale. The members of the team went to Dome A for a routine investigation. For a long time in the Antarctic life, the team members almost all appeared in different degrees of "Antarctic syndrome" such as impulse, irritability, depression and other symptoms. At the same time, the plateau, the hypoxic environment could cause pulmonary edema, brain edema and other fatal diseases, so the Antarctic Dome A complex was studied in a variety of extremes. It is of great significance to explore the physiological and psychological effects of the environment on the team members and explore the molecular mechanism.
Taking the twenty-fourth Chinese Antarctic expedition members as the research object, we analyzed the physiological functions, psychological emotions and the changes of the gene expression profiles in the peripheral blood leucocytes at the highest point Dome A in the Antarctic continent, and found that the athletes showed obvious plateau physiological stress response at the time of Dome A, and the male hormone increased significantly in the body. With the abnormal fluctuation of psychological emotion, the increase of the male hormone is significantly correlated with the mood fluctuation; 1121 genes expressed differently in the peripheral blood leukocytes are enriched in the GO term database to get the function set of the brain nerve development, spermatogenesis and so on, and the mood fluctuation in the phenotype, the cerebral blood oxygen content decrease, and the sex hormone rise. There is a significant correlation between the results of gene chip analysis and physiological and psychological changes in phenotype.
This study comprehensively analyzes the influence of the extreme environment of Antarctic Dome A on the human body and the molecular mechanism of some phenotypic changes, which can provide an important theoretical basis for the high expression and purification of.HIV gp160 protein for the screening, training, protection and psychological counseling of the team members in the future.
Human immunodeficiency virus (HIV) infection can cause acquired immunodeficiency syndrome (AIDS). Infected people often die from secondary infection caused by immunodeficiency. There are more than 30 million H1V infected people all over the world, and 3 million of the new infected people are added each year, but there is no effective vaccine and control drug.HIV gp160 protein. The virus shell structure protein is formed, which mediates the cell entry of the virus and the antibody neutralization reaction of the host to the virus; many virus entry inhibitors have a certain effect to prevent the virus from entering the virus by interfering with the gp160 protein with the receptor or the fusion of the virus envelope with the cell membrane. The analysis of crystal structure has hindered the design and development of a new and more effective virus entry inhibitor. In 2009, gp160 protein was also one of the ten proteins that need to be analyzed by Nature. The expression and purification of high quality gp160 protein is an important prerequisite for crystal structure analysis.
This project combines the advantages of the protein expression and purification technology in our laboratory. Through the optimization of the codon of protein sequences and the use of eukaryotic high expression vector, gp160 protein is successfully expressed in human 293 cells, and continuous perfusion and culture of cells is realized with the latest Wave large-scale cell culture technology. In this study, the proportion of trimer binding proteins in gp160 protein was increased by adding T4F to the terminus of gp160 protein at the end of the protein. This study has purified and expressed 130mg gp160 protein, which lays the foundation for subsequent experiments of preparation of gp160 protein antibody and crystallization structure analysis. Meanwhile, the content and methods of this study are other proteins. High expression and purification provide a good platform.

【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】：2012
【分类号】：R373.1

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