ACE2基因变异与SARS冠状病毒进入及肺部病变严重程度之间的关系研究

发布时间：2018-03-03 02:35

本文选题：血管紧张素转换酶2　切入点：严重急性呼吸综合征　出处：《中国协和医科大学》2008年博士论文　论文类型：学位论文

【摘要】： 严重急性呼吸综合征(severe acute respiratory syndrome,SARS)是由一种新的SARS冠状病毒(coronavirus,CoV)引起的急性传染病,在人群中传染性极强,其病死率高达10%。弥漫性肺泡损伤(diffuse alveolar damage,DAD)是导致患者出现呼衰及死亡的主要原因之一,其发病机制至今尚未阐明。在SARS-CoV被分离后不久,人们就鉴定出了SARS受体—血管紧张素转换酶2(angiotensin-converting enzyme 2,ACE2)。体外研究提示,SARS假病毒在小鼠和大鼠ACE2基因转染的细胞上其病毒进入效率较人ACE2(hu-ACE2)基因转染的细胞明显降低,这些结果初步提示不同物种间ACE2基因的变异与SARS-CoV进入细胞的效率密切相关,同时这也可能是大鼠和小鼠对SARS-CoV不易感的原因之一。体内研究显示SARS-CoV感染小鼠急性期可引起ACE2表达下调;SARS-CoV S蛋白也可引起ACE2表达下调和急性肺损伤加重,运用ACE2底物抑制剂可明显减轻急性肺损伤的程度。这些研究充分体现ACE2在SARS发病过程中扮演了重要角色。但这些研究仅局限于小鼠,尚不足以外推到人。由我们以前的研究可知,中国恒河猴被SARS-CoV感染后可出现轻度或重度间质性肺炎。为了进一步明确ACE2在SARS发病中的作用,我们选择轻度和重度SARS恒河猴为研究对象,分析恒河猴ACE2(rh-ACE2)基因变异与病毒进入之间的关系。我们希望通过对rh-ACE2基因变异与病毒进入效率之间关系的研究,从而发现一些与病毒进入密切相关的氨基酸位点。进而突变hu-ACE2基因的该位点残基,观察其对病毒进入的影响,希望找到hu-ACE2上对病毒进入有重要影响的氨基酸位点。最后我们将进一步分析rh-ACE2基因突变与SARS病变严重程度之间的关系。本次研究中我们以肺部病理变化为基础,选取6只轻度、6只重度SARS恒河猴及2只正常对照恒河猴作为研究对象。轻度肺损伤的病理特征为肺间隔增宽伴单核巨噬细胞浸润;重度肺损伤以弥漫性肺泡损伤为其特征,主要病理特征为弥漫性肺间隔增宽伴单核巨噬细胞浸润、局部肺间隔融合、肺泡腔内可见蛋白水肿液及纤维素渗出。运用RT-PCR从这些恒河猴肾组织中扩增出全长rh-ACE2基因,克隆并分析其变异规律。序列分析结果提示,在14只恒河猴中,我们共检测到55个随机非同义突变。以序列相对保守的8号恒河猴的8-2克隆为rh-ACE2代表,与hu-ACE2序列相比我们在8-2克隆的rh-ACE2序列中共查及38个一致性非同义突变。每只恒河猴选取2个代表性质粒转染HEK293T细胞后,观察基因表达水平及病毒进入效率的变化。结果发现11号恒河猴11-7克隆的ACE2表达水平和病毒进入效率较11-1克隆明显降低。序列分析结果提示,与11-1克隆相比,11-7克隆出现R192G、Y217N两个位点突变。定点突变分析显示:Y217N突变是引起rh-ACE2基因11-7克隆蛋白表达和病毒进入降低的关键性氨基酸位点。我们突变hu-ACE2基因获得hu-ACE2(Y217N)突变型质粒,转染细胞后发现hu-ACE2(Y217N)基因的表达水平和病毒进入效率较hu-ACE2基因明显下降。最后,我们分析Rh-ACE2基因变异与肺部损伤严重程度之间的关系。结果显示:有1只(1/6)轻度病变的恒河猴rh-ACE2编码区第1677位核苷酸位点存在A/G杂合性位点,有3只(3/6)重度病变的SARS恒河猴rh-ACE2编码区第1677位核苷酸位点存在A/C杂合性位点。Fisher确切概率法检验提示,轻度和重度SARS恒河猴ACE2基因第1677位核苷酸位点A/G杂合率未见明显差异。实验结果初步提示,中国恒河猴不同个体间rh-ACE2基因氨基酸序列存在明显变异;Y217N突变可明显降低rh-ACE2和hu-ACE2的蛋白表达和SARS假病毒的进入;ACE2基因变异与SARS恒洞猴肺部病变的严重程度无明显相关。该研究将为设计针对ACE2的靶向药物及进一步理解ACE2在SARS发病中的作用提供一些线索。
[Abstract]:Severe acute respiratory syndrome (severe acute respiratory syndrome, SARS) is a new kind of SARS coronavirus (coronavirus, CoV) of acute infectious disease caused in the crowd is highly contagious, the mortality rate of up to 10%. diffuse alveolar damage (diffuse alveolar damage, DAD) is one of the main and lead to patients with respiratory failure the cause of death, its pathogenesis has not been elucidated. Shortly after the separation of SARS-CoV, people have identified SARS receptor angiotensin converting enzyme 2 (angiotensin-converting enzyme 2, ACE2). In vitro studies suggest that SARS pseudotyped virus in mouse and rat ACE2 gene transfected cells on the virus entry efficiency is ACE2 (hu-ACE2) gene transfected cells decreased significantly, these results preliminarily suggested the efficiency variation and SARS-CoV among different species of ACE2 gene into cells is closely related, it also may be the rat and One of the reasons for not susceptible SARS-CoV mice. In vivo studies revealed that acute SARS-CoV infection in mice can cause the down-regulation of ACE2 expression; SARS-CoV S protein also induced ACE2 down-regulation and enhance acute lung injury, the use of ACE2 substrate inhibitor can significantly reduce the degree of acute lung injury. These studies show that ACE2 play a important role in the pathogenesis of SARS. In the process. But these studies are only limited to mice. The lack of outside to push people. Our recent studies indicated that Chinese Ganges RIver monkey infected by SARS-CoV can occur after mild or severe interstitial pneumonia. In order to further clarify the role of ACE2 in the pathogenesis of SARS, we choose Ganges RIver for mild and severe SARS monkey the object of study, analysis of Ganges RIver monkey ACE2 (rh-ACE2) the relationship between gene mutation and viral entry. We hope that through the research on the variation and virus rh-ACE2 gene into the relationship between efficiency, In order to find some virus into the amino acid sites are closely related. Thus the mutation site residues of hu-ACE2 gene and to observe its effect on virus entry, hoping to find hu-ACE2 on the virus into amino acid sites have important influence. Finally, we will further analyze the relationship between rh-ACE2 gene mutation and SARS severity.
We are based on the pathological changes in this study, a total of 6 mild, 6 severe SARS and 2 normal control Ganges RIver monkey monkey in Ganges RIver as the research object. Pathological features of mild lung injury for pulmonary septal thickening and infiltration of mononuclear macrophages; severe lung injury with diffuse alveolar damage is characterized mainly. Pathological features of diffuse pulmonary septal thickening and infiltration of mononuclear macrophages, local pulmonary septal fusion, alveolar edema fluid and fibrin protein. Using RT-PCR amplified the full-length rh-ACE2 gene from the Ganges RIver monkey kidney tissues, cloning and analysis of its variation. Sequence analysis indicated that, in the 14 Ganges RIver monkey. We detected a total of 55 random non synonymous mutations. In a relatively conservative sequence No. 8 Ganges RIver monkey 8-2 clones as the representative of rh-ACE2, and the hu-ACE2 sequence in the rh-ACE2 sequence we compared 8-2 clones of the check and 38 A The consistency of non synonymous mutations. Each selected 2 representative of Ganges RIver monkey plasmid were transfected into HEK293T cells, changes in the level and efficiency of virus gene expression were observed. Results showed that 11 monkeys in Ganges RIver 11-7 cloning ACE2 expression and virus entry efficiency is significantly reduced. 11-1 cloning sequence analysis results indicated that, compared with the 11-1 clones 11-7 clones R192G, Y217N, two. Mutation analysis showed point mutations: Y217N mutation is caused by cloning rh-ACE2 gene 11-7 protein expression and virus into the key amino acid sites decreased. We obtained the hu-ACE2 mutation in the hu-ACE2 gene (Y217N) mutant plasmid transfected cells after hu-ACE2 (Y217N) expression and viral gene enter the efficiency than hu-ACE2 gene decreased significantly. Finally, we analyze the relationship between the severity of the Rh-ACE2 gene mutation and pulmonary injury. The results showed: there were 1 mild lesions (1 /6) The Ganges RIver monkey 1677th rh-ACE2 encoding region of nucleotide sites in A/G heterozygous loci, 3 (3/6) of severe lesions of the SARS Ganges RIver monkey 1677th rh-ACE2 encoding region nucleotide site A/C heterozygosity locus.Fisher exact probability test indicated that mild and severe SARS Ganges RIver monkey ACE2 gene 1677th nucleotide A/G heterozygous there was no significant difference.
The experimental results indicated that significant variation between different individuals Chinese Ganges RIver monkey rh-ACE2 gene amino acid sequence; Y217N mutation can significantly reduce the access to the rh-ACE2 and the protein expression of hu-ACE2 and SARS pseudovirus; was not related to severity of ACE2 gene mutation and SARS constant hole monkey pulmonary lesions. The study for design of targeting ACE2 drugs and further understanding of the role of ACE2 in the pathogenesis of SARS provide some clues.

【学位授予单位】：中国协和医科大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R373;R511.9

【参考文献】