本文關(guān)鍵詞：火雞的流感病毒唾液酸受體分布特點及其在流感病毒生態(tài)系統(tǒng)中的位置和作用　出處：《廣西醫(yī)科大學(xué)》2013年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 甲型流感病毒 跨種屬傳播 唾液酸受體 火雞

【摘要】：背景和目的：周期性的流感疫情是可以預(yù)測的,相比之下,如果大流行出現(xiàn)一種新型流感病毒亞型,則整個人群對其具有很少或缺乏免疫力,是不可預(yù)測的事件。大部分新出現(xiàn)的流感病毒亞型都是由禽流感病毒突變而來,目前研究的熱點是,流感病毒是如何從其“自然基因庫”的水禽類動物進(jìn)化為具備跨種屬障礙感染人。流感病毒外表面有兩種糖蛋白,其中一種叫血凝素(Hemagglutinin,HA),它們有助于流感病毒與宿主細(xì)胞外表面的糖鏈特異性接觸吸附、結(jié)合并進(jìn)入細(xì)胞內(nèi)的作用。大部分的禽流感病毒與唾液酸(Sialic Acid, SA) a2-3Gal受體結(jié)合,人流感病毒與SAa2-6Gal受體結(jié)合,這種差異直接導(dǎo)致了禽流感病毒很難跨種屬傳播到人群。 甲型流感病毒易發(fā)生變異,特別是膜表面的HA抗原,流感病毒發(fā)生抗原變異主要有兩種方式,一種是主要引起點突變的抗原漂移,驅(qū)使病毒發(fā)生抗原漂移的主要原因,是宿主對病毒表面抗原的免疫壓力所導(dǎo)致的適應(yīng)性突變；另一種方式主要是病毒一個或多個病毒基因片段RNA被置換的抗原轉(zhuǎn)換,導(dǎo)致新重配病毒株的產(chǎn)生�；蛑嘏鋵W(xué)說認(rèn)為流感病毒變異亞型大流行,是通過人和禽流感病毒基因重配而來。由于豬呼吸道上皮細(xì)胞表面同時存在禽類受體(SAa2-3Gal)和人流感病毒受體(SAa2-6Gal),因此,豬對禽類流感病毒和人流感病毒都具有一定的易感性,如果發(fā)生兩種病毒同時感染豬的事件,那么禽流感病毒與人流感病毒在豬體內(nèi)感染、復(fù)制的同時可能發(fā)生了基因重配,使禽流感病毒獲得與人呼吸道SAα2-6Gal受體結(jié)合的能力。但是最近幾年來,香港、東南亞等地陸續(xù)發(fā)生了多例禽流感病毒(包括HSN1,H9N2等亞型流感病毒)直接由禽類動物傳染給人的事件,隨后發(fā)現(xiàn)陸地禽類動物也同時具有兩種流感病毒受體�；痣u是一類在歐美國家常見的陸地禽類動物,1998年后在北美多次從火雞體內(nèi)分離到禽流感病毒和豬流感病毒重組的病毒,提示火雞可能是一種重要的流感病毒中間宿主,但到目前為止,對火雞體內(nèi)的唾液酸受體分布,尚未清楚.。本研究利用凝集素特異性結(jié)合SA受體的方法來確定火雞體內(nèi)SA受體的分布情況,并用禽流感病毒和人流感病毒結(jié)合實驗來驗證此結(jié)果,探討火雞在流感病毒生態(tài)中的位置及在新型流感病毒大爆發(fā)中的作用。 方法：(1)采用親和組化染色法,火雞組織切片分別滴加生物素標(biāo)記凝集素MAA-Ⅱ(特異性標(biāo)記SAa2-3Gal受體)和SNA(特異性標(biāo)記SAa2-6Gal受體),檢測火雞氣管、支氣管、次級支氣管、副支氣管以及直腸組織的SAa2-3Gal和SAa2-6Gal受體的表達(dá)。(2)利用與SAa2-3Gal受體結(jié)合的鴨源性禽流感病毒株(H9N1)和與SAa2-6Gal受體結(jié)合的人流感病毒株(H1N1),分別在雞胚和MDCK通過接種傳代法擴增病毒,純化后與熒光素Alexa488結(jié)合。然后滴加標(biāo)記了熒光素Alexa488的H9N1禽流感病毒和H1N1人流感病毒于火雞氣管、支氣管、次級支氣管、副支氣管以及直腸的組織切片上,熒光顯微鏡下觀察拍照并分析結(jié)果。 結(jié)果：(1)火雞呼吸道和消化道SAa2-3Gal的受體分布特點：禽類SAa2-3Gal受體主要分布在火雞的氣管、支氣管、次級支氣管和副支氣管四個呼吸道解剖部位中的大部分區(qū)域,親和組化分析呈強陽性分布,而在直腸內(nèi)表達(dá)較少并呈弱陽性分布,SAa2-3Gal受體在上述各部位的分布差異有統(tǒng)計學(xué)意義。(2)火雞呼吸道和消化道SAa2-6Gal受體分布特點：火雞體內(nèi)的SAα2-6Gal受體分布在呼吸道的氣管、支氣管和次級支氣管大部分區(qū)域,親和組化分析呈弱陽性分布,而在呼吸道的的終末端副支氣管分布極少,在直腸呈極少量分布,SAα2-6Gal受體在上述各部位的分布差異有統(tǒng)計學(xué)意義。(3)SAα2-3Gal受體和SAα2-6Gal受體各解剖部位分布比較：禽類SAα2-3Gal受體在火雞呼吸道的氣管、支氣管、次級支氣管以及副支氣管四個解剖部位中的分布明顯高于人SAα2-6Gal受體,而在消化道的直腸組織中火雞SAα2-6Gal受體與SAα2-3Gal受體分布無明顯差異。(4)利用熒光素Alexa488標(biāo)記的H9N1禽流感病毒以及H1N1人流感病毒與火雞呼吸道及消化道結(jié)合特點：H9N1禽流感病毒與火雞呼吸道的氣管、支氣管和次級支氣管及副支氣管的上皮細(xì)胞的大部分區(qū)域結(jié)合,而H1N1人流感病毒僅少量的與火雞呼吸道的氣管、支氣管和次級支氣管的上皮細(xì)胞結(jié)合。H9N1與H1N1流感病毒與火雞的直腸組織上皮細(xì)胞都未見熒光結(jié)合。熒光素標(biāo)記病毒結(jié)合實驗結(jié)果基本與凝集素親和組化所做出的火雞呼吸道和消化道的SAα2-3Gal受體和SAα2-6Gal受體的分布特點相一致。 結(jié)論：(1)火雞呼吸道上皮細(xì)胞均有流感病毒SAα2-3Gal受體和SAα2-6Gal受體,以SAα2-3Gal受體為主。在氣管、支氣管、次級支氣管及副支氣管四個解剖部位,SAα2-3Gal受體均呈高密度分布,火雞SAα2-6Gal受體少量分布,而且僅在氣管、支氣管、次級支氣管三個解剖部位有分布。兩種SA受體在直腸組織中均分布較少；(2)火雞呼吸道組織對H9N1流感病毒敏感,氣管、支氣管、次級支氣管及副支氣管組織均較易結(jié)合H9N1禽流感病毒,支持SAα2-3Gal受體的分布特點；(3)H1N1人流感病毒主要結(jié)合火雞上段呼吸道的氣管、支氣管、次級支氣管組織,且少量分布,與SAα2-6Gal受體的分布特點基本吻合；(4)火雞呼吸道上段均含有SAα2-3Gal受體和SAα2-6Gal受體,并且禽流感病毒和人流感病毒均能結(jié)合在呼吸道上段,提示火雞也是一種甲型流感病毒“基因混合器”,可能有助于病毒基因重配,有形成大流行株的可能。
[Abstract]:Background and purpose: a seasonal influenza epidemic situation can be predicted, in contrast, if the emergence of a new pandemic influenza virus subtype, the entire population has little or lack of immunity to it, is unpredictable events. Most of the new influenza virus subtype of avian influenza virus is the mutation. The focus of current research is that the flu virus is palmipeds animal evolution from their "natural gene pool" to have cross the species barrier to infect people. There are two types of influenza virus surface glycoprotein, which is called a hemagglutinin (Hemagglutinin, HA), they help sugar chain specific adsorption on influenza virus and contact the outer surface of the host cell, and combined with the role of intracellular entry. Avian influenza virus and most of the sialic acid (Sialic Acid, SA) a2-3Gal receptor binding, binding to human influenza virus and SAa2-6Gal receptor, this difference directly guide It is difficult for the avian influenza virus to spread across species to the population.
Influenza A virus to mutate, especially HA membrane surface antigen, influenza virus antigenic variation occurs mainly in two ways, one is the main starting point mutation induced antigenic drift, driven mainly due to virus antigen drift, mutation free of host adaptation pressure disease virus surface antigen caused by another; a virus is the main way of one or more virus gene fragments are converted to RNA replacement antigen, leading to new reassortant virus strains. The reassortant influenza virus subtype mutation theory that a pandemic, by human and avian influenza virus gene reassortment. Due to the surface of pig airway epithelial cells also exist in poultry receptor (SAa2-3Gal) and human influenza virus receptor (SAa2-6Gal), therefore, the pig has a certain susceptibility to avian influenza virus and human influenza virus, if two kinds of virus infected pigs incident, Then the avian influenza virus and human influenza virus infection in pigs, replication may occur at the same time the gene reassortment, the avian influenza virus acquired the ability to combine with human respiratory SA alpha 2-6Gal receptor. But in recent years, Hongkong, Southeast Asia and other places have occurred in many cases of avian influenza virus (including HSN1, H9N2 etc. influenza virus) directly infect humans events from birds to animal, then found in terrestrial poultry animal also has two kinds of influenza virus receptor. Turkey is a kind of terrestrial poultry animal common in Europe and the United States, 1998 in North America many times from Turkey isolated avian influenza virus and swine influenza virus recombinant virus, suggesting that Turkey is one of the most important intermediate host of influenza virus, but so far, the distribution of sialic acid receptors in Turkey, is not clear. This study method with the lectin binding specificity of SA receptor To identify the distribution of SA receptor in Turkey and verify the results by combining avian influenza virus with human influenza virus, we discuss the position of Turkey in the influenza virus ecology and its role in the outbreak of a new influenza virus.
Methods: (1) using affinity histochemical staining, Turkey tissue sections were dropping biotinylated lectin II (MAA- specific marker SAa2-3Gal receptor) and SNA (a specific marker for SAa2-6Gal receptor), detection of Turkey trachea, bronchus, secondary bronchi, the expression of SAa2-3Gal and SAa2-6Gal receptor in rectal tissue and bronchial side. (2) the use of duck derived avian influenza virus binds to the SAa2-3Gal receptor (H9N1) and human flu virus binds to the SAa2-6Gal receptor (H1N1), respectively in the chick embryo and MDCK by inoculating passage amplification of virus, purified with fluorescein Alexa488. Then add the marker of H9N1 avian influenza virus and H1N1 the human influenza virus Alexa488 in Turkey fluorescein trachea, bronchus, secondary bronchi, bronchial and rectal side on tissue sections were observed under fluorescence microscope and photographed the analysis results.
Results: (1) receptor distribution characteristics of Turkey respiratory and digestive tract SAa2-3Gal: avian SAa2-3Gal receptor mainly located in Turkey's trachea, bronchus, bronchus and four secondary side bronchial airway anatomy in most area, affinity group analysis showed strong positive expression in the rectum and distribution, and less positive distribution. There were statistically significant differences in the distribution of the various parts of the SAa2-3Gal receptor. (2) Turkey respiratory and digestive tract SAa2-6Gal receptor distribution in Turkey: SA alpha 2-6Gal receptor distribution in respiratory trachea, tracheal and bronchial branches in most regions of the secondary, affinity group analysis showed weak positive distribution, and in the end side of respiratory tract bronchial distribution rarely, is a very small amount of the distribution in the rectum, there were statistically significant differences in the distribution of the various parts of the SA alpha receptor. 2-6Gal (3) SA and SA receptor alpha 2-3Gal alpha 2-6Gal The receptor anatomic site distribution: trachea, avian SA alpha 2-3Gal receptor in Turkey respiratory bronchi, bronchial and secondary distribution parabronchi four anatomic sites are significantly higher than that in human SA alpha 2-6Gal receptor, and SA receptor SA and Turkey alpha 2-6Gal alpha 2-3Gal receptor distribution had no obvious difference in rectal tissue of digestive tract (. 4) using fluorescein labeled Alexa488 H9N1 avian influenza virus and human influenza virus H1N1 and Turkey respiratory and digestive tract: H9N1 combined with the characteristics of avian influenza virus and Turkey respiratory trachea, bronchi and secondary bronchi in most regions and side bronchial epithelial cells with H1N1, and only a small amount of human influenza virus and respiratory tract in Turkey there was no fluorescence with the trachea, bronchi and secondary bronchi epithelial cells with.H9N1 and H1N1 influenza virus and Turkey rectal tissue epithelial cells were labeled. The results of virus binding test were basically consistent with the distribution characteristics of SA alpha 2-3Gal receptor and SA alpha 2-6Gal receptor in Turkey respiratory tract and alimentary tract made by lectin affinity histochemistry.
Conclusion: (1) Turkey had respiratory epithelial cells of influenza virus SA alpha 2-3Gal receptor and SA receptor alpha 2-6Gal, alpha SA to 2-3Gal receptor. In the trachea, bronchus, bronchus and four secondary side bronchial anatomy, SA alpha 2-3Gal receptor showed a high density distribution, Turkey SA alpha 2-6Gal receptor and only a small amount of the distribution. In the trachea, bronchus, secondary bronchus three anatomic site distribution. Two SA receptors are distributed less in rectal tissues; (2) Turkey respiratory tissue sensitivity to H9N1 influenza virus, trachea, bronchus, bronchus and trachea tissue secondary collateral branch were more easily with the H9N1 avian influenza virus, the distribution characteristics of SA alpha 2-3Gal support receptor; (3) H1N1 human influenza virus with upper respiratory tract mainly Turkey trachea, bronchus, secondary bronchial tissue, and a small amount of the distribution, basically consistent with the distribution of SA alpha 2-6Gal receptor; (4) the upper respiratory tract of Turkey Both of them contain SA alpha 2-3Gal receptor and SA alpha 2-6Gal receptor, and avian influenza virus and human influenza virus can bind to the upper respiratory tract, suggesting Turkey is also a "gene mixer" of influenza A virus, which may help virus gene redistribution and have the possibility of forming a pandemic strain.

【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：S852.65;R373.13

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