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研究生:張丞賢
研究生(外文):Cheng-Hsien Chang
論文名稱:臺灣流行性病毒角結膜炎之研究
論文名稱(外文):Study on Epidemic Viral Keratoconjunctivitis in Taiwan
指導教授:陳振武 林貴香 
指導教授(外文):Chen-Wu ChenKuei-Hsiang Lin
學位類別:博士
校院名稱:高雄醫學大學
系所名稱:醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2001
畢業學年度:90
語文別:中文
論文頁數:104
中文關鍵詞:病毒角結膜炎腺病毒柯沙奇病毒A24型變異株腸病毒70型
外文關鍵詞:Viruskeratoconjunctivitisadenoviruscoxsackievirus A24variantenterovirus type 70interleukinICAM-1endothelial cells
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臺灣地處熱帶亞熱帶交接地帶,流行性角結膜炎為台灣常見之眼部流行病,主要由腺病毒(亞屬D)及腸病毒(腸病毒70型, EV70和柯沙奇病毒A24型變異型, CA24v)所引起。前者主要引起流行性角結膜炎,後者主要引起急性出血性結膜炎。回顧南臺灣流行性角結膜炎之病毒18年的變遷發現腺病毒持續一直有分離,1994年前以8型為主,而其基因型共演變成6個基因型(Ad8C-Ad8H)。1995年之後以37原型(Ad37P)和19型A型(Ad19A)為主,其基因型共有Ad7P(原)、Ad37A、Ad37B、Ad19P(原)型、Ad19A-D。隨著病毒型別及基因型之變化其臨床症狀亦隨著加重,尤其是結膜下出血和眼外症狀。結膜下出血本為腸病毒感染之特徵,但1995至1997年高醫調查結果發現腺病毒引起之結膜炎病人出血比例竟增加至45%。因此腺病毒引起之流行性角結膜炎與腸病毒引起之出血性結膜炎鑑別診斷更加困難,而迅速準確之實驗診斷相形重要。
以分子生物學方式PCR和RFLP來診斷腺病毒和分別其型態較傳統之細胞培養及血清中和方式迅速,大概可以從3週縮至3天,而其診斷率雖略高於傳統之細胞培養(41.1% vs. 34.3%),但未達統計學上有意義之差別。而PCR-RFLP放大及切割一段hexon gene無法完全分別腺病毒基因型。因此傳統之細胞培養方式仍不可或缺,它不僅提供充裕之病毒可供日後研究,亦可從中抽取核酸來做酵素切割而分出基因型。
鑑於腺病毒角結膜炎引起越來越多結膜下出血病例,為研究病毒感染之後結膜下出血之致病机轉,測試從病人分離出來的腺病毒19型、37型及柯沙奇病毒A24型對人類臍帶血管內皮細胞的相對感染力。實驗結果顯示19及37型腺病毒及柯沙奇A24變異株病毒皆會感染血管內皮細胞而產生細胞病變。在有結膜下出血與無結膜下出血病人分離出之病毒株對人類血管內皮細胞的相對感染力,兩組間無顯著差異。胞內von Willebrand factors (vWF) 經螢光染色及flow cytometry 觀察發現在病毒感染之細胞中會有減少的現象,但並非出血病人分離出來之病毒株所感染之內皮細胞vWF一定會比較少。雖然我們証明了結膜炎病毒在体外都會感染血管內皮細胞,但是此並非造成結膜炎出血之直接證據,任何其他之出血机轉都值得深入探究。
1995至1997年之腺病毒不僅引起結膜炎出血且伴隨44.7%眼外全身症狀,包括上呼吸道及消化道感染症候,因此進一步探討其感染人類表皮細胞(包括呼吸道及角膜表皮細胞)之後所引發之免疫反應,結果發現表皮細胞在腺病毒感染之後會釋出IL-1 alpha而刺激血管內皮細胞之 ICAM-1表現,這類adhesion molecules的激化,進一步之作用為吸引白血球及淋巴球穿過血管內皮細胞至病毒感染位置。以相同之實驗模式我們發現CA24v及腸病毒70型皆會在感染角膜表皮細胞後,促使其同樣釋出IL-1 alpha而刺激血管內皮細胞之 ICAM-1。同樣的以respiratory syncytial virus感染 A549細胞,發現也是同樣能釋出IL-1 alpha。因此推測IL-1 alpha從表皮細胞釋出對病毒感染後之免疫防衛機轉應佔有相當之角色。
Epidemic viral keratoconjunctivitis is a common infectious eye disease in the tropical and subtropical areas where Taiwan is located. This highly contagious disease is generally attributed to two categories of causative pathogens. The first group is adenoviruses, which cause epidemic keratoconjunctivitis (EKC) and pharyngoconjunctival fever (PCF). The second group is composed of enteroviruses, including enterovirus type 70 (EV70) and coxsackievirus A type 24 variant (CA24v), which induce acute hemorrhagic conjunctivitis (AHC). AHC is characterized by an acute course and prominent subconjunctival hemorrhage (SCH) in 50 to 100% of the infected eyes. EKC is mainly associated with adenovirus type 8, 19 and 37 (Ad8, Ad19 and Ad37), which cause SCH in 20 to 30 % of the infected patients. However, from 1995 to 1997 in southern Taiwan, about forty-five percent of Ad19 and Ad37 EKC patients presented SCH. The clinical features of this hemorrhagic EKC were not characteristic that it could be easily differenteated from AHC. A fast laboratory confirmation and differential diagnosis is required.
In contrast to a period of 3 weeks for obtaining results of viral culture, polymerase chain reaction (PCR) shortened the period of diagnosis for adenovirus to less than 2 days with slightly higher positive rates. Although the types of adenoviruses could be further identified with restriction fragment length polymorphism (RFLP) analysis of the total viral DNA in one more day, genotypes could not be detected using PCR-RFLP.
To investigate the increased incidence of SCH of EKC in the three years (1995-7) in Taiwan, we proposed the hypotheses that vascular endothelial cells are permissive to Ad19 and Ad37 and the permissiveness is different between the strains from hemorrhagic and non-hemorrhagic patients. The results from a study design using cell-culture model showed that human umbilical vein endothelial cells (HUVEC) are permissive to Ad19, Ad37 and CA24v. However, viral strains isolated from hemorrhagic and non-hemorrhagic patients are both able to infect the HUVEC without significant difference. HUVEC infected with different viruses exhibited characteristic cytopathic effects s and followed by cell death in 24 to 72 hours.
Simultaneously, we investigated if there is any vasoactive factor(s) that is released from the ocular epithelial cells upon conjunctivitis virus infection. We examined the activation of adhesion molecules on HUVEC after challenging with culture medium of infected Ad37, CA24v and EV70. Infected supernatants from ocular epithelial cells of the three viruses are found to activate intercellular adhesion molecule-1 (ICAM-1) on HUVEC by cellular ELISA test. However, vascular cell adhesion molecule-1 (VCAM-1) and E-selectin are not significantly activated. The vasoactive factor was identified as IL-1 alpha by the blocking antibody and ELISA test exhibited the IL-1 alpha concentrations are from 12 to 120 pg/ml. Further experiments were performed to test if other epithelial cells (A549) infected by Ad37 and respiratory syncitial virus (RSV) or HUVEC infected by Ad37, CA24v and EV70 follow the same immune responses. The results of experiments exhibited a similar model of immune response of viruses infecting epithelial and endothelial cells, with various magnitudes of responses and IL-1 alpha release. In conclusion, IL-1 alpha released from virus-infected cells could be an important factor in host immune response to viral infection.
第一章 引言與介紹....................1
1.1流行性結膜炎..................... 1
 1.1.1 腺病毒的介紹...................1
 1.1.2腺病毒之構造及成份................ 2
 1.1.3 腸病毒的介紹...................2
 1.1.4 腸病毒的介紹...................3
1.2 結膜炎病毒的診斷...................3
 1.2.1 臨床診斷.....................3
 1.2.2實驗診斷..................... 4
1.3 病毒性結膜炎的傳染與流行...............5
1.4 病毒感染與宿主的防衛.................5
1.5 流行性病毒角結膜炎之防疫與治療............7
第二章 南臺灣傳染性病毒角結膜炎十八年之流行病學探討...9
2.1 摘要.........................9
2.2 前言.........................10
2.3 前人的研究......................11
2.4 材料與方法......................11
 2.4.1 檢體收集與資料記錄................12
 2.4.2 實驗診斷.....................12
2.5 結果.........................12
 2.5.1 病原分析.....................12
 2.5.2 臨床症狀分析...................13
2.6 討論.........................15
第三章 出血性的腺病毒結膜炎之臨床資料分析........20
3.1 摘要.........................20
3.2 前言.........................20
3.3 前人研究.......................21
3.4 材料與方法......................21
3.5 結果.........................21
 3.5.1 腺病毒感染的臨床症狀分析.............21
 3.5.2 出血型態與就診時間................22
 3.5.3 結膜下出血與結膜發炎之關係............22
 3.5.4 感染途徑之分析..................23
3.6 討論.........................23
第四章 比較分子生物診斷及細胞培養方法診斷結膜炎腺病毒..25
4.1 摘要.........................25
4.2 前言.........................25
4.3 前人研究.......................26
4.4 材料與方法......................27
 4.4.1 病人與樣本收集..................27
 4.4.2 病毒之細胞培養與鑑定...............27
 4.4.3 病毒之分子生物學診斷...............27
4.5 結果.........................29
 4.5.1 病例收集.....................29
 4.5.2 以RT-PCR及病毒培養方式偵測腸病毒.........30
 4.5.3 以PCR及病毒培養方式偵測腺病毒.......... 30
 4.5.4 病毒累積陽性率與採檢日的關係...........30
 4.5.5 腺病毒的血清型及基因型..............31
4.6 討論.........................31
第五章 表皮細胞被結膜炎病毒感染後之免疫反應.......33
5.1 摘要.........................33
5.2 前言.........................33
5.3 前人研究.......................34
 5.3.1 病毒感染表皮細胞之機制..............34
 5.3.2 表皮細胞在病毒感染後所釋出之發炎因子.......35
 5.3.3 血管內皮細胞上adhesion molecules的激發......35
 5.3.4 表皮細胞被感染病毒後之adhesion molecules表現...36
 5.3.5 腺病毒與apoptosis................ 36
5.4 材料與方法......................37
 5.4.1 試劑與材料....................37
 5.4.2 細胞、病毒、和免疫血清..............37
 5.4.3 血管內皮細胞分離.................38
 5.4.4 病毒之製備....................39
 5.4.5 病毒蛋白之免疫沉澱................39
 5.4.6 以ELISA偵測細胞ICAM-1表現及溶液中之IL-1濃度... 40
 5.4.7 DNA fragmentation for Apoptosis......... 41
5.5結果......................... 41
 5.5.1 腺病毒感染細胞後之表現..............42
 5.5.2 腺病毒感染後細胞之ICAM-1表現...........42
 5.5.3 IL-1為腺病毒感染表皮細胞後所釋出的
    血管內皮細胞刺激因子...............43
 5.5.4 病毒感染引發感染表皮細胞分泌IL-1α的主因..... 44
 5.5.5 IL-1對ICMA-1,VCAM-1和E-selectin刺激的差異.... 44
 5.5.6 腺病毒蛋白質不會激發血管內皮細胞ICAM-1的表現...45
 5.5.7 病毒感染表皮細胞後產生之血管內皮細胞
    刺激因子之免疫反應與比較.............45
 5.5.8 病毒感染液中IL-1的濃度..............46
 5.5.9 表皮細胞被感染病毒後之adhesion molecule的表現.. 46
 5.5.10 腺病毒感染A549細胞並不會引起apoptos...... 47
5.6 討論.........................47
第六章 出血性病毒結膜炎之病理機轉探討..........51
6.1 摘要.........................51
6.2 前言.........................51
6.3 前人研究.......................53
 6.3.1 感染血管內皮細胞的病毒..............53
 6.3.2 血管內皮細胞被感染後其adhesion molecules表現或
    cytokine/chemokine之釋放.............54
 6.3.3 血管內皮細胞中的vWF及其在病毒感染後之變化.... 54
6.4 材料與方法......................55
 6.4.1 血管內皮細胞之分離及其病毒感染..........55
 6.4.2 螢光抗體染色內皮細胞中的von Willebrand factor
    (vWF)VIII.................... 56
6.4.3 以FACS分析病毒感染之血管內皮細胞vWF之
    表現及定量....................56
 6.4.4 以ELISA測ICAM-1在血管內皮細胞之表現....... 57
6.5 結果.........................57
 6.5.1 腺病毒、柯沙奇病毒A24變異型和腸病毒70型皆會
    感染人類血管內皮細胞...............57
 6.5.2 出血病毒株未有較高之內皮細胞感染力........58
 6.5.3 內皮細胞中之vWF在病毒感染後減少......... 58
 6.5.4 以腺病毒、柯沙奇病毒A24變異型和腸病毒70型
    感染血管內皮細胞後其adhesion molecules之
   表現及其影響...................59
6.6討論......................... 59
第七章  總結與展望................... 62
附圖...........................65
附表...........................92
參考文獻.........................104
附錄和已發表之論文....................131
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