Epidemiology of Respiratory Adenoviruses in Central Taiwan and Inflammatory Responses of A549 Lung Adenocarcinoma Induced by Adenovirus Infection

Abstract

呼吸道腺病毒主要感染對象為嬰幼兒，兒童及青少年，感染大多發生在冬、春兩季。腺病毒在西元1953年從兒童腺體組織中分離出來，故命名腺病毒，目前人類腺病毒有51種血清型，6個亞屬(A~F)，造成呼吸道方面感染的腺病毒主要為亞屬B的血清型3、7、11、14；亞屬C的血清型1、2、5、6及亞屬E的血清型4。 從2008年後中台灣地區便沒有腺病毒的相關研究數據，本篇研究中，我們調查了2005到2011年呼吸道病毒感染的臨床病例，回顧18,749件醫療紀錄並且比較疫情時的臨床表現。有關腺病毒造成呼吸道感染之致病機制，至今尚未完全明瞭。一般認為細胞免疫作用，扮演相當重要之角色。而細胞激素在其中所扮演之角色雖然有研究，但不夠完整。本研究在急性期採集病患喉頭檢體，首先分別做病毒培養、免疫螢光染色鑑別病毒，再藉由酵素連結免疫吸附法以及流式細胞儀在不同時間點去偵測被腺病毒感染後的A549人類肺癌細胞產生發炎反應之TNF- α、 IL-6、 IL-8濃度的變化及誘發細胞凋亡的情形。 我們收集2005年到2011年18,749 個台中榮民總醫院所提供懷疑呼吸道病毒感染的臨床特徵，其中1,479 例分離出腺病毒，平均分離率約7.89%。結果表明腺病毒發病族群主要為嬰幼兒及兒童，年齡分布為1到13歲，在1到3歲這時期為高峰期。在中台灣地區分離率最高的型別為第三型腺病毒。病毒藉由感染細胞48小時後，利用倒立式顯微鏡觀察，典型的細胞病變表現為細胞腫脹、變圓以及呈現葡萄樣聚集。此外，當A549細胞被第三型腺病毒感染24小時後，細胞因為被感染而產生許多細胞激素，甚至走向細胞凋亡。此外，被腺病毒感染後的病人血液中C-反應蛋白與平均白血球值數比健康對照組還高出許多。本篇論文對於腺病毒的流行病學提供了寶貴的信息，可幫助醫師做為中台灣地區之兒童呼吸道腺病毒臨床應用上的依據，包括評估疾病嚴重程度及臨床鑑別診斷。Respiratory adenoviruses infections mostly occur in infants, young children and adolescent in winter and spring season. Since the first isolation of adenovirus from children’s tonsils in 1953, at least 51 other human serotypes have been identified. It comprise 51 serotypes are divided into six subgroups, A to F. Respiratory infections of adenoviruses are usually caused by serotypes from subgenus B (Ad 3, Ad 7, Ad 11, Ad 14), subgenus C (Ad 1, Ad 2, Ad 5, Ad6), and subgenus E (Ad 4). There was no reported information about the prevalence of adenovirus in central Taiwan since 2008. In this study, we reviewed and compared data drawn from the medical records of 18,749 cases of adenoviral infection occurring between 2005 and 2011 to survey the clinical manifestations of the adenovirus outbreak. It is known that cellular immunity plays an important role in adenovirus infections. Therefore, the present study intends to investigate the patterns of cytokines induced by adenovirus infections. By using lung adenocarcinoma cell A549 as the respiratory cell model, production of inflammatory cytokines , including interleukin-6 (IL-6)、 IL-8 and tumor necrosis factor alpha (TNF-α), induced by adenovirus infection was determined by enzyme-linked immunosorbent assay (ELISA). Furthermore, cell apoptosis was determined by flow cytometer. To analyse the clinical features and the temporal frequency in acute adenoviral respiratory infections in hospitalized children in central Taiwan, a total of 18,749 children who were admitted to the Department of Pediatrics, Taichung Veterans General Hospital (TVGH), with clinical evidences of acute respiratory infections between 2005 to 2011 were studied. Adenoviruses were isolated from 1,479 patients with an isolation rate of 7.89 per cent. The results indicate that the incidence of adenovirus infection in infants and children has a high peak between the ages of 1 to 3 years, the age distribution ranged from 1 to 13 years, age was the most significant predictor of infection by ad 3. Ade3 has the highest separation rate , especially in central Taiwan. These data suggest that the typical CPE (cytopathic effects) characteristics of adenovirus infection were observed under an inverted microscope, where infected cells begin to swell, become rounded, and develop into grape-like clusters 48 hrs after infection. Furthermore, there is a broad array of cytokine induction in A549 during Ad3 infection, at least by 24 hrs after infection. Also, cell became apoptosis. Furthermore, the C-reactive protein (CRP) level and WBC count was much higher in adenovirus infection compared with that of other healthy control. This epidemiological study may provide valuable information that helps physicians to obtain a clinical differential diagnosis for respiratory adenoviral infection in children in central Taiwan.目錄 致謝…………………………………………………………………i 中文摘要……………………………………………………………ii 英文摘要……………………………………………………………iv 表目錄………………………………………………………………xi 圖目錄………………………………………………………………x 第一章、前言………………………………………………………1 一、腺病毒簡介……………………………………………………1 二、腺病毒特性……………………………………………………2 三、腺病毒流行病學……………………………………… ….…4 四、細胞激素之簡介………………………………………………5 五、細胞凋亡之概論………………………………………………8 第二章、研究目的…………………………………………………10 第三章、材料與方法………………………………………………12 一、疾病定義及臨床分析…………………………………………12 二、細胞培養………………………………………………………13 三、繼代培養………………………………………………………14 四、細胞計數………………………………………………………14 五、冰凍細胞………………………………………………………15 六、解凍細胞………………………………………………………15 七、腺病毒分離……………………………………………………16 八、腺病毒鑑定……………………………………………………17 九、病毒DNA的萃取……………………………………………...18 十、聚合酶連鎖反應………………………………………………19 十一、腺病毒種源建立……………………………………………20 十二、病毒定量……………………………………………………21 十三、ELISA……………………………………………….………22 十四、Annexin V/PI stain………………………………………24 十五、統計分析……………………………………………………25 第四章、實驗結果…………………………………………………26 一、臨床病例分析…………………………………………………26 二、腺病毒流行概況………………………………………………28 三、腺病毒分離與鑑定……………………………………………29 四、臨床血液分析…………………………………………………30 五、細胞激素表現…………………………………………………31 六、細胞凋亡表現…………………………………………………33 第五章、討論………………………………………………………35 第六章、結論………………………………………………………41 第七章、參考文獻…………………………………………………4