本文選題：血管緊張素轉(zhuǎn)換酶2　切入點：嚴(yán)重急性呼吸綜合征　出處：《中國協(xié)和醫(yī)科大學(xué)》2008年博士論文　論文類型：學(xué)位論文

【摘要】： 嚴(yán)重急性呼吸綜合征(severe acute respiratory syndrome,SARS)是由一種新的SARS冠狀病毒(coronavirus,CoV)引起的急性傳染病,在人群中傳染性極強,其病死率高達10%。彌漫性肺泡損傷(diffuse alveolar damage,DAD)是導(dǎo)致患者出現(xiàn)呼衰及死亡的主要原因之一,其發(fā)病機制至今尚未闡明。在SARS-CoV被分離后不久,人們就鑒定出了SARS受體—血管緊張素轉(zhuǎn)換酶2(angiotensin-converting enzyme 2,ACE2)。體外研究提示,SARS假病毒在小鼠和大鼠ACE2基因轉(zhuǎn)染的細胞上其病毒進入效率較人ACE2(hu-ACE2)基因轉(zhuǎn)染的細胞明顯降低,這些結(jié)果初步提示不同物種間ACE2基因的變異與SARS-CoV進入細胞的效率密切相關(guān),同時這也可能是大鼠和小鼠對SARS-CoV不易感的原因之一。體內(nèi)研究顯示SARS-CoV感染小鼠急性期可引起ACE2表達下調(diào);SARS-CoV S蛋白也可引起ACE2表達下調(diào)和急性肺損傷加重,運用ACE2底物抑制劑可明顯減輕急性肺損傷的程度。這些研究充分體現(xiàn)ACE2在SARS發(fā)病過程中扮演了重要角色。但這些研究僅局限于小鼠,尚不足以外推到人。由我們以前的研究可知,中國恒河猴被SARS-CoV感染后可出現(xiàn)輕度或重度間質(zhì)性肺炎。為了進一步明確ACE2在SARS發(fā)病中的作用,我們選擇輕度和重度SARS恒河猴為研究對象,分析恒河猴ACE2(rh-ACE2)基因變異與病毒進入之間的關(guān)系。我們希望通過對rh-ACE2基因變異與病毒進入效率之間關(guān)系的研究,從而發(fā)現(xiàn)一些與病毒進入密切相關(guān)的氨基酸位點。進而突變hu-ACE2基因的該位點殘基,觀察其對病毒進入的影響,希望找到hu-ACE2上對病毒進入有重要影響的氨基酸位點。最后我們將進一步分析rh-ACE2基因突變與SARS病變嚴(yán)重程度之間的關(guān)系。 本次研究中我們以肺部病理變化為基礎(chǔ),選取6只輕度、6只重度SARS恒河猴及2只正常對照恒河猴作為研究對象。輕度肺損傷的病理特征為肺間隔增寬伴單核巨噬細胞浸潤;重度肺損傷以彌漫性肺泡損傷為其特征,主要病理特征為彌漫性肺間隔增寬伴單核巨噬細胞浸潤、局部肺間隔融合、肺泡腔內(nèi)可見蛋白水腫液及纖維素滲出。運用RT-PCR從這些恒河猴腎組織中擴增出全長rh-ACE2基因,克隆并分析其變異規(guī)律。序列分析結(jié)果提示,在14只恒河猴中,我們共檢測到55個隨機非同義突變。以序列相對保守的8號恒河猴的8-2克隆為rh-ACE2代表,與hu-ACE2序列相比我們在8-2克隆的rh-ACE2序列中共查及38個一致性非同義突變。每只恒河猴選取2個代表性質(zhì)粒轉(zhuǎn)染HEK293T細胞后,觀察基因表達水平及病毒進入效率的變化。結(jié)果發(fā)現(xiàn)11號恒河猴11-7克隆的ACE2表達水平和病毒進入效率較11-1克隆明顯降低。序列分析結(jié)果提示,與11-1克隆相比,11-7克隆出現(xiàn)R192G、Y217N兩個位點突變。定點突變分析顯示:Y217N突變是引起rh-ACE2基因11-7克隆蛋白表達和病毒進入降低的關(guān)鍵性氨基酸位點。我們突變hu-ACE2基因獲得hu-ACE2(Y217N)突變型質(zhì)粒,轉(zhuǎn)染細胞后發(fā)現(xiàn)hu-ACE2(Y217N)基因的表達水平和病毒進入效率較hu-ACE2基因明顯下降。最后,我們分析Rh-ACE2基因變異與肺部損傷嚴(yán)重程度之間的關(guān)系。結(jié)果顯示:有1只(1/6)輕度病變的恒河猴rh-ACE2編碼區(qū)第1677位核苷酸位點存在A/G雜合性位點,有3只(3/6)重度病變的SARS恒河猴rh-ACE2編碼區(qū)第1677位核苷酸位點存在A/C雜合性位點。Fisher確切概率法檢驗提示,輕度和重度SARS恒河猴ACE2基因第1677位核苷酸位點A/G雜合率未見明顯差異。 實驗結(jié)果初步提示,中國恒河猴不同個體間rh-ACE2基因氨基酸序列存在明顯變異;Y217N突變可明顯降低rh-ACE2和hu-ACE2的蛋白表達和SARS假病毒的進入;ACE2基因變異與SARS恒洞猴肺部病變的嚴(yán)重程度無明顯相關(guān)。該研究將為設(shè)計針對ACE2的靶向藥物及進一步理解ACE2在SARS發(fā)病中的作用提供一些線索。
[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.

【學(xué)位授予單位】：中國協(xié)和醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2008
【分類號】：R373;R511.9

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本文編號：1559208