臺灣博碩士論文加值系統

人類巨細胞病毒(human cytomegalovirus; HCMV)為皰疹病毒(herpes virus)之一種，病毒本身約具二百個基因（約200kb）。 由於近年來器官移植與AIDS等免疫不全病患急速增加常導致嚴重併發症如肺炎、腸炎，腦炎、視網膜炎等。臨床上愛滋病患罹患巨細胞病毒視網膜膜炎(HCMV Retinitis)，約佔20-45%，如果不接治療大約半年內造成患者失明。在臨床的觀察中發現，巨細胞病毒視網膜炎是一種極具侵犯性及破壞性的眼部疾病，部份病患早期不會有明顯視力變化，但如果不能及時診斷並給予治療，或是中斷治療，最後皆往往造成患者失明。然而目前醫學研究對於HCMV病毒在巨細胞病毒視網膜炎的感染發展成因及HCMV病毒是如何在視網膜和眼睛內形成病毒複製循環，以及HCMV病毒又如何在視網膜傳遞 (cell to cell)，這些造成愛滋病患嚴重視力喪失-- HCMV Retinitis 之疾病重要致病機轉，至今仍不得而知。
本研究目的希望藉由對於HCMV Retinitis之病理標本之研究，配合建立HCMV Retinitis體外模型（HCMV / human retinal pigment epithelium cell; HRPE）；以進一步了解HCMV病毒在人體視網膜之感染進入路徑及病毒複製週。同時經由基礎分子生物研究，探討HCMV病毒在眼內免疫優勢區（immune privilege）中免疫變化及相互作用，以了解病毒如何規避全身免疫系統之攻擊，以及HCMV如何利用眼內特別免疫功能，而達到HCMV病毒持續感染複製及潛伏，再活化感染之HCMV Retinitis致病機制。
我們利用in situ hybridization 技術來偵測Fas ligand mRNA之表現，檢測人類視網膜組織時發現Fas Ligand在正常視網膜組織中呈現一種同質性表現分佈，主要表現在視網膜內核層(inner nuclear layer), 外核層(outer nuclear layer)，以及人類視網膜色素上皮細胞 (human retinal pigment epithelium) 。 但是對於感染CMV Retinitis的愛滋病患眼睛病灶區內之Fas ligand mRNA表現作一檢測時，我們發現在CMV感染視網膜之病灶區中Fas ligand訊號明顯增加。同時利用terminal transferase-mediated nick end labeling assay (TUNEL stain) 來分析結果發現在視網膜病灶區中有大量細胞計劃性死亡 (apoptosis) 之現象。根據以上免疫病理之發現，我們推測在愛滋病患感染HCMV Retinitis的致病機轉中HCMV病毒除了使受感染視網膜細胞壞死分解外，是不是還可經由HCMV病毒基因調控（如使Fas ligand大量表現等），而此種病理機轉也使得正常視網膜細胞以及導致來自全身性徵召 (recruitment) 之血液免疫細胞因apoptosis而不能發揮其救援功能?
為進一步驗證以上之假說，我們已將HCMV AD169病毒成功感染視網膜色素上皮細胞，以模擬HCMV病毒在眼內視網膜之致病機轉及免疫反應改變等變化。我們發現並證明HCMV immediate early gene 2 (IE2) 可以活化 Fas ligand (FasL) promotor 而造成帶有Fas receptor之免疫細胞如 T cell，因與 FasL 結合而導致apoptosis。我們實驗結果支持CMV病毒利用活化 Fas ligand表達而演化出特定之病毒免疫規避（virus evasion）效應，並且能躲避宿主免疫系統之偵測與攻擊，以調控視網膜宿主細胞存活而做出對本身病毒有利之生長環境。 其結果則導致HCMV感染越演越烈，視網膜因局部及全身性免疫自我防護之破壞，與免疫救援系統不能發揮而破壞殆盡，病人終究也喪失寶貴視覺功能。

Human cytomegalovirus (HCMV), a member of the beta subfamily of herpes viruses, contains a double-stranded DNA genome of 229,354 base pairs with a potential to encode more than 200 proteins. HCMV infection usually develops asymptomatic lifelong infection in healthy individuals, but can cause severe clinical complications such as HCMV retinitis when reactivated in immunocompromised patients. Although the detailed mechanisms of HCMV latency and reactivation are not yet well understood, accumulating evidences suggest that the virus can use a panel of viral proteins to escape from cellular immune control and thus, successfully survive and replicate in host cells.
Cellular immune reactions and the associated inflammatory responses can be harmful to nearby tissues. Since minor inflammation can result in impaired vision or even blindness, the eye is naturally designed as an immune privileged site where infections usually do not lead to destructive immune reactions. The underlying mechanism has been hypothesized to involve Fas ligand (FasL)-mediated programmed cell death (also called apoptosis) of Fas (CD95)-expressing T cells when attracted to the infection sites. In this case, activated T cells are eliminated through ligation of Fas by FasL and no serious immune reactions are induced. Thus, the damage to the eye is minimized. However, HCMV infection of human eyes is shown to cause large-scaled cell death and tremendous visual dysfunction. Whether HCMV takes the advantage of the FasL-dependent immune evasion to exert its destructive effects remains an important issue.
Here we report that viral immediate early gene 2 (IE2), but not IE1, upregulates the Fas ligand (FasL) expression in HCMV-infected human retinal pigment epithelium (HRPE) cells. Increased secretion of FasL from virally infected cells into cultured medium was observed upon HCMV infection. The capability of such cell-free medium to induce apoptosis of Fas (CD95)-expressing Jurkat cells further implies that Fas-FasL interaction might mediate cell death in the lesion of HCMV retinitis. To support this idea, we observed augmented soluble FasL (sFasL) levels in vitreous from AIDS patients with HCMV retinitis as compared to that from AIDS patients without HCMV infection. In addition, by in situ hybridization and immunohistochemistry, we detected enhanced signals of FasL, the existence of viral IE antigens and apoptotic cells at the same sites in the lesion of HCMV-infected retina. These results strongly suggest that IE2 induction of FasL expression in human retina might be an important event that takes place in the early stage of infection and finally leads to visual loss in individuals affiliated with HCMV retinitis.

目 錄
頁數
目 錄.…………………………………………………………………….. 3
中文摘要………………………………………………………………….. 5
英文摘要………………………………………………………………….. 7
第一章 緒論……………………………………………………………… 9
( 一 ) 巨細胞病毒感染與臨床疾病之相關性………………. 10
( 二 ) 巨細胞病毒視網膜炎之臨床病程表徵………………. 11
( 三 ) 巨細胞病毒視網膜炎之輔助實驗診斷………………. 13
( 四 ) 眼免疫特區與病毒感染之互動………………………. 15
第二章 材料與方法……………………………………………………… 21
第三章 細胞計劃性死亡在巨細胞病毒視網膜炎中所扮演之角色…… 28
（一） 巨細胞病毒視網膜炎病患之臨床病理研究及細胞
計劃性死亡之現象…………………………………… 29
（二） 巨細胞病毒視網膜炎體外模型之建立………………. 32
（三） 巨細胞病毒體外感染導致視網膜色素上皮細胞執行
細胞計劃性死亡……………………………………… 34
第四章 巨細胞病毒基因對於人類視網膜Fas / Fas Ligand系統之作用
及影響……………………………………………………………. 45
（一） 巨細胞病毒感染視網膜色素上皮細胞引發Fas Ligand
之表現…………………………………………………. 46
（二） 巨細胞病毒IE2基因與Fas Ligand作用及影響……… 47
（三） 受巨細胞病毒感染之HRPE細胞釋放出大量游離型
Fas Ligand並造成Jurkat細胞進行細胞計劃性死亡.. 48
（四） Fas / Fas Ligand系統所引發的細胞計劃性死亡在巨細
胞病毒視網膜炎中所扮演之角色…………………… 49
第五章 問題討論與未來研究方向………………………………………. 57
第六章 總 結…………………………………………………………….. 65
參考文獻………………………………………………………………….. 68
附錄……………………………………………………………………….. 77

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