多發性硬化症(MS)是由第一型輔助型T細胞(Th1)與分泌介白素-17(IL-17)之輔助型T細胞(Th17)調控慢性自體免疫疾病，其致病原因尙不明瞭且非僅由單一基因或是環境因子造成。實驗性自體免疫腦脊髓炎小鼠(EAE)是常用於研究多發性硬化症的動物模式，由於抗原呈現細胞(APC)呈現髓鞘抗原刺激T細胞，促使T細胞釋放發炎介質與細胞激素，如:干擾素-γ(IFN-γ)與IL-17，而導致髓鞘與中樞神經系統被破壞。近來研究發現表基因體調控參與不同T細胞族群之分化，實驗室先前研究顯示，EAE小鼠前處理甲基化轉移酶抑制劑5-aza-2′-deoxycytidine (5-Aza)後，可藉由提升CD4+Foxp3+表達量抑制病症產生。除調節性輔助型T細胞(Treg)以外，其他T細胞表基因體模式尚未被詳細探討﹔因此，我們針對IFN-γ與IL-17A這兩種細胞激素，探討其DNA甲基化程度是否伴隨EAE疾病而改變。本研究第一部份我們將Th1與Th17細胞分化並處理5-Aza，接著利用亞硫酸鹽焦磷酸定序偵測IFN-γ DNA不同區域甲基化程度，包含intron1、 promoter -53CG、 CNS-6、CNS-22、CNS-54 與IL-17A 的CNS2。結果發現處理5-Aza後，Th1/Th17極化中分別於CNS-6與IL-17A之CNS2第六個與第八個CpG位置皆有顯著性差異。在蛋白質表現上，Th1極化過程處理5-Aza後發現CD4+ T細胞的IFN-γ 表現量增加﹔然而Th17極化過程中處理5-Aza後IL-17A表達量呈現抑制作用。此現象是由於5-Aza處理後，Th17有較高表現7-AAD與annexin-V，這些細胞走向細胞凋亡而導致。第二部分，我們誘導EAE老鼠並於不同疾病時期蒐集周邊血單核細胞，結果於DNA甲基化程度上發現IFN-γ intron1 於EAE疾病高峰時呈現去甲基化，年齡大(10周)的小鼠去甲基化現象較年齡小(6-8周)明顯且疾病較為嚴重。IFN-γ CNS-6 於EAE臨床疾病分數為1時呈現去甲基化﹔IL-17A CNS2第8與第10個位置於EAE小鼠發病初期與疾病到達高峰時皆呈現去甲基化現象。總結以上實驗結果顯示IFN-γ的CNS-6與IL-17A的CNS2 這些cis elements可用於預測EAE小鼠發病早期以及給予適當治療時機。

Multiple sclerosis (MS) is a Th1/Th17 CD4+ T cell-mediated chronic autoimmune disease caused by genetic and/or environmental factors. Experimental autoimmune encephalomyelitis (EAE) is the most commonly used animal model to study the MS. Because of myelin antigens presented by antigen presenting cells (APCs) and stimulated T cells that released inflammatory mediators and cytokines such as IFN-γ and IL-17 will destroy the myelin and central nerve system (CNS). In recent study, epigenetic regulation has been shown to involve in distinct T helper lineages differentiation and polarization. In our previous study, EAE mice pre treated with 5-aza-2′-deoxycytidine(5-Aza), an inhibitor of DNA nucleotide methylation transferase (DNMT), inhibited the disease through upregulated the expression of CD4+Foxp3+.In addition to the Treg cell, other T cells epigenetic pattern has not been detailed study in EAE model and MS. Therefore, we aimed to identify the cytokines of IFN-γ and IL-17A in DNA methylation pattern in vitro and in vivo EAE model. First part in this study, we carried out in vitro Th1 and Th17 polarization under 5-Aza treatment. We used bisulfite pyrosequencing to detect the regions of IFN-γ methylation pattern including intron1, promoter-53CG, CNS-6, CNS-22, CNS-54 and CNS2 of IL-17A. The CNS-6 in Th1 polarization and CNS2 site6 and site8 of IL-17A in Th17 polarization had significant difference after treated with 5-Aza. In Th1 polarization, protein level of IFN-γ was increased in CD4+ after treated with 5-Aza, however IL-17A was downregulated in Th17 polarization. The downregulation of Th17 cells were through cell apoptosis because more 7-AAD and annexin-V double positive cells was observed after treated with 5-Aza. Second part, we evaluated the methylation pattern of cytokine in different disease status in peripheral blood mononuclear cells (PBMC). In DNA level, we found the intron1 of IFN-γ was demethylated when EAE disease at peak. Older mice (more than 10 weeks) showed more demethylation and severe disease score than younger mice ( 6-8 weeks). CNS-6 of IFN-γ was demethylated when EAE score=1. Site8 and site10 in CNS2 of IL-17A was demethylated when EAE score at onset and peak. Summary, the above data show 5-Aza treatment will increase IFN-γ expression while inhibit IL-17A expression through Th17 apoptosis, and show these cis elements including CNS-6 of IFN-γ and CNS2 of IL-17A can be a tool to predict the time for EAE mice therapeutic treatment at early disease stage.

目錄
中文摘要(第ⅱ頁)
英文摘要(第ⅲ頁)
縮寫對照表(第Ⅷ頁)
一、緒論(第9頁)
1.1多發性硬化症(Mutiple Sclerosis)(第9頁)
1.2實驗性自體免疫腦脊髓炎小鼠模式(Experimental autoimmune encephalomyelitis)(第10頁)
1.3輔助型T細胞(T helper cells)(第10頁)
1.4第一型輔助型T細胞(Type1 T helper cells)(第10頁)
1.5第二型輔助型T細胞(Type2 T helper cells)(第11頁)
1.6第十七型輔助型T細胞(IL-17-secreting T helper cells)(第11頁)
1.7調節性輔助型T細胞(Regulatory T helper cells)(第12頁)
1.8對位基因體學(Epigenetics)與DNA甲基化(DNA methylation)(第12頁)
1.9 DNA甲基化與免疫(第13頁)
1.10 DNA甲基化與調節性輔助型T細胞(第14頁)
1.11去甲基化試劑5-Aza-2′-deoxycytidine(5-Aza)(第15頁)
二、研究目的(第16頁)
三、材料與方法(第17頁)
3.1.小鼠(第17頁)
3.2.實驗性自體免疫腦脊髓炎小鼠模式誘導(第17頁)
3.3.培養基、溶液與試劑(第18頁)
3.4.純化與分離小鼠T 細胞(第19頁)
3.5.細胞內細胞激素染色法(Intra-cellular cytokine staining)(第21頁)
3.6.細胞凋亡染色(第23頁)
3.7. DNA甲基化分析(第23頁)
3.8.小鼠脊椎染色(第28頁)
3.9.染色分析方法(第31頁)
3.10.數據分析(第31頁)
四、實驗結果(第32頁)
4.1.第一型輔助型T細胞極化過程中處理5-Aza之IFN-γ DNA甲基化表現(第32頁)
4.2.第十七型輔助型T細胞極化過程中處理5-Aza 後IL-17A DNA甲基化表現(第37頁)
4.3.第一型輔助型T細胞極化過程中處理5-Aza 後IFN-γ蛋白質表現(第38頁)
4.4.第十七型輔助型T細胞極化過程中處理5-Aza後IL-17A與IFN-γ蛋白質表現(第38頁)
4.5.第十七型輔助型T細胞極化過程處理5-Aza後進行細胞凋亡標記之染色(第38頁)
4.6.實驗性自體免疫腦脊髓炎小鼠模式中偵測IFN-γ不同cis element DNA甲基化狀態(第39頁)
4.7.實驗性自體免疫腦脊髓炎小鼠模式中偵測IL-17A不同cis element DNA甲基化狀態(第42頁)
4.8.利用蘇木素(Hematoxylin)-伊紅(Eosin)與Luxol Fast blue於實驗性自體免疫腦脊髓炎小鼠脊椎進行組織染色(第42頁)
五、實驗討論(第43頁)
六、圖表(第47頁)
七、文獻(第86頁)

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