本文選題：H5N1 + 流感病毒��； 參考：《北京協(xié)和醫(yī)學(xué)院》2012年博士論文

【摘要】：H5N1高致病性禽流感病毒不僅會(huì)給養(yǎng)殖、畜牧業(yè)帶來災(zāi)難性的破壞,而H.人感染后死亡率高達(dá)近60%；特別是近期一些研究結(jié)果表明H5N1禽流感病毒可以通過變異、重組而能在人群中傳播,從而嚴(yán)重威脅人類的生命健康。而目前對(duì)H5N1禽流感病毒的致病機(jī)理仍然了解較少,禽流感病毒如何突破宿主細(xì)胞、免疫系統(tǒng)的防御而產(chǎn)生高病毒載量的機(jī)制還不明確,特別是具有清除外源微生物作用的溶酶體在流感病毒感染過程中的作用仍未得到解析。 本論文研究發(fā)現(xiàn)H5N1禽流感病毒感染細(xì)胞后,病毒的神經(jīng)氨酸酶(NA)能夠在細(xì)胞內(nèi)降解溶酶體膜上的糖蛋白和溶酶體內(nèi)的水解酶,從而破壞溶酶體的完整性和正常功能,完整功能的溶酶體大量喪失可能是H5N1禽流感病毒的重要致病機(jī)制之一；而達(dá)菲等NA抑制劑能在細(xì)胞內(nèi)抑制NA對(duì)溶酶體膜糖蛋白的降解,可能是其抗病毒的作用機(jī)制之一；同時(shí)也發(fā)現(xiàn),不同程度上損傷溶酶體可能是流感病毒的共同致病機(jī)理。 以上的研究發(fā)現(xiàn)提示H5N1禽流感病毒可能通過破壞溶酶體功能從而引起高病毒載量、免疫抑制等嚴(yán)重病癥,可能是H5N1禽流感病毒致病的重要原因；同時(shí)本研究結(jié)果能為將來抗流感病毒藥物的研發(fā)提供新的思路和依據(jù)。 表型和基因型關(guān)聯(lián)分析南極極端環(huán)境下科考隊(duì)員的應(yīng)激反應(yīng) 南極冰穹A (Dome A)位于南極大陸高原冰蓋最高點(diǎn),具有高海拔、低氧、酷寒、烈風(fēng)、難以到達(dá)與危險(xiǎn)性大等惡劣自然環(huán)境因素和與世隔絕的社會(huì)環(huán)境因素,被喻為地球上的“不可接近之極”之一；同時(shí)Dome A卻是科學(xué)考察的圣地,每年都有大批科考隊(duì)員前往Dome A進(jìn)行例行的考察。長(zhǎng)時(shí)間在南極生活工作,科考隊(duì)員幾乎都會(huì)出現(xiàn)不同程度的諸如沖動(dòng)、煩躁、抑郁等癥狀的“南極綜合癥”,同時(shí)高原、低氧的環(huán)境會(huì)引起肺水腫、腦水腫等致命疾病,因此研究南極Dome A復(fù)合多種極端環(huán)境對(duì)科考隊(duì)員的生理、心理影響,并在分子機(jī)制上進(jìn)行探究具有重要意義。 我們以中國(guó)第24次南極內(nèi)陸科考隊(duì)員為研究對(duì)象,分析科考隊(duì)員身處南極大陸最高點(diǎn)Dome A時(shí)的生理功能、心理情緒及外周血白細(xì)胞基因表達(dá)譜的變化。發(fā)現(xiàn)在Dome A時(shí)科考隊(duì)員表現(xiàn)出明顯的高原生理應(yīng)激反應(yīng),體內(nèi)雄性激素明顯升高,并伴隨心理情緒的異常波動(dòng),且雄性激素的升高與情緒波動(dòng)具有顯著相關(guān)性;外周血白細(xì)胞中差異表達(dá)的1,121個(gè)基因,在GO term數(shù)據(jù)庫中富集得到有關(guān)大腦神經(jīng)發(fā)育、精子生成等功能集,與表型中的情緒波動(dòng)、腦血氧含量降低、性激素上升具有顯著的相關(guān)性；基因芯片的分析結(jié)果在一定程度上與表型中的生理、心理變化相對(duì)應(yīng)。 本研究全面分析了南極Dome A極端環(huán)境對(duì)人體的影響及部分表型變化的分子機(jī)制,能為將來科考隊(duì)員的篩查、訓(xùn)練、防護(hù)和心理輔導(dǎo)提供重要的理論依據(jù)。HIV gp160蛋白高量表達(dá)與純化 人類免疫缺陷病毒(HIV)感染可以引起獲得性免疫缺陷綜合癥(AIDS),感染者常常因?yàn)槊庖呷毕菀鹄^發(fā)性感染而死亡。目前全世界有超過3,000萬的H1V感染者,同時(shí)每年還增加300萬的新感染者,但還沒有有效的疫苗和防治藥物。HIV gp160蛋白構(gòu)成了病毒外殼結(jié)構(gòu)蛋白,介導(dǎo)了病毒的細(xì)胞進(jìn)入以及宿主對(duì)病毒的抗體中和反應(yīng)；多種病毒進(jìn)入抑制劑通過干擾gp160蛋白與受體結(jié)合或者病毒包膜與細(xì)胞質(zhì)膜融合而阻止病毒的進(jìn)入,具有一定療效；但目前因?yàn)槲茨軐?duì)gp160蛋白進(jìn)行晶體結(jié)構(gòu)解析而阻礙了新的更有效的病毒進(jìn)入抑制劑的設(shè)計(jì)和研發(fā);2009年gp160蛋白還被Nature雜志評(píng)為最需要進(jìn)行晶體結(jié)構(gòu)解析的十個(gè)蛋白之一。其中表達(dá)純化高質(zhì)量的gp160蛋白是進(jìn)行晶體結(jié)構(gòu)解析的重要前提。 本課題綜合了本實(shí)驗(yàn)室在蛋白表達(dá)純化技術(shù)上的優(yōu)勢(shì),通過對(duì)蛋白序列進(jìn)行密碼子優(yōu)化,使用真核高表達(dá)載體,在人源293細(xì)胞中成功表達(dá)了gp160蛋白；并結(jié)合最新的Wave大規(guī)模細(xì)胞培養(yǎng)技術(shù),實(shí)現(xiàn)了細(xì)胞的連續(xù)灌注培養(yǎng)；在進(jìn)一步的研究中通過在gp160蛋白末端加入T4F促三聚化末端,提高了gp160蛋白中的三聚體形式結(jié)合蛋白的比例。本研究共計(jì)純化表達(dá)了130mg gp160蛋白,為gp160蛋白抗體制備和結(jié)晶結(jié)構(gòu)解析等后續(xù)實(shí)驗(yàn)奠定了基礎(chǔ)。同時(shí),本研究?jī)?nèi)容和方法為其它蛋白的高量表達(dá)和純化提供了一個(gè)很好的平臺(tái)。
[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.

【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R373.1

【參考文獻(xiàn)】

相關(guān)期刊論文 前4條

1 張?jiān)轿?;中國(guó)南極考察的醫(yī)學(xué)保障[J];中國(guó)臨床保健雜志;2007年02期

2 王興春;楊致榮;王敏;李瑋;李生才;;高通量測(cè)序技術(shù)及其應(yīng)用[J];中國(guó)生物工程雜志;2012年01期

3 閆鞏固;葉芊;;極地環(huán)境中的心理學(xué)研究[J];心理科學(xué)進(jìn)展;2009年01期

4 高麗;潘霄;嚴(yán)進(jìn);劉偉志;唐云翔;;極端環(huán)境下個(gè)體績(jī)效的負(fù)性表現(xiàn)[J];心理科學(xué)進(jìn)展;2010年06期

相關(guān)博士學(xué)位論文 前1條

1 王洪亮;SARS冠狀病毒與H5N1型禽流感病毒致病機(jī)理的研究[D];中國(guó)協(xié)和醫(yī)科大學(xué);2009年

，

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