探討DNA去甲基化藥劑處理之實驗性自體免疫腦脊髓炎小鼠其輔助型T細胞的調控機制

The modulatory mechanisms of DNA demethylation agent 5-Aza-2’-deoxycytidine on T helper cell function in experimental autoimmune encephalomyelitis

張家濱(Chang, Chia Bin)

指導教授 ： 吳淑芬

國立中正大學/理學院/生命科學系分子生物研究所/碩士(2012年)

摘要

調節性T細胞在生物體內是維持免疫系統恆定以及防止自體細胞遭受攻擊的重要因子，其中Forkhead box P3 (Foxp3) 是調節性T細胞發育與執行免疫抑制功能的一個重要轉錄因子。而個體內調節性T 細胞的數量或功能的異常，會導致許多自體免疫疾病的產生。在之前對自體免疫疾病-人類多發性硬化症的研究中指出，患者體內調節性T細胞功能相較正常人明顯降低。而已經有文獻報導，去氧核醣核酸的甲基化作用能夠調控Foxp3基因的表現，並且有的研究指出，利用去氧核醣核酸的去甲基化藥劑5-Aza-2’-deoxycytidine (5-Aza) 能夠加強老鼠體內調節性T細胞的功能，並抑制自體免疫糖尿病的產生。因此在我們的研究中，我們利用人類多發性硬化症的動物模式-實驗性自體免疫腦脊髓炎小鼠，在小鼠身上探討去甲基化藥劑 (5-Aza) 對於自體免疫反應的影響以及治療的可能性。本篇研究中我們發現，施打5-Aza小鼠能夠減緩及減輕實驗性自體免疫腦脊髓炎發病時間及症狀。而正常發病小鼠有嚴重的中樞神經系統髓鞘脫失現象；相對地5-Aza治療之小鼠並無看到這種情形。我們在中樞神經系統中檢測出，引起發炎反應的細胞激素IFNγ和IL-17在5-Aza治療之小鼠身上僅有少量的表現；而在正常發炎的小鼠身上會有高度的表現。另外，在調節性T 細胞功能的檢測中，我們看到5-Aza治療之小鼠會讓調節性T 細胞抑制免疫反應的作用增強。然而在更進一步的實驗當中，發現5-Aza的治療並不是讓調節性T 細胞的功能增強，而是讓引起發炎的功能性T細胞的功能變弱。
由上述實驗結果所得結論，在自體免疫腦脊髓炎小鼠的模式上，去甲基化藥劑5-Aza的處理可以防止髓鞘的脫失，並降低功能性T細胞的功能，導致攻擊中樞神經系統的免疫細胞變少，抑制疾病的產生。

關鍵字

DNA去甲基化藥劑 ； 實驗性自體免疫腦脊髓炎

並列摘要

Regulatory T (Treg) cells play a key role in maintaining self-tolerance and immunological homeostasis. Forkhead box P3 (Foxp3) is a transcription factor necessary and sufficient for Treg cells development and immunosuppressive function. Defective Treg suppressive functions were observed in many autoimmune diseases including multiple sclerosis. It had been reported that DNA methylation regulated the expression of Foxp3. In the previous report, treatment with low dose of DNA demethylation agent 5-Aza-2’-deoxycytidine (5-Aza) enhanced the Treg-mediated suppression and repressed the progression of diabetes in NOD mice. In this study, we investigated the treatment of 5-Aza in experimental autoimmune encephalomyelitis (EAE), a widely used animal model for human multiple sclerosis, to evaluate the demethylation effect in autoimmune diseases. We found that treatment of 5-Aza mice delayed the onset of EAE and only displayed the mild symptoms. Moreover, we used luxol fast blue staining to observe the demyelination in central nervous system (CNS) and found that control EAE mice developed severe demyelination but 5-Aza treated mouse did not. Next, we evaluated the inflammatory responses in CNS, there was no inflammation in 5-Aza treated mice because of the very low level of inflammatory cytokines, and on the contrary, control EAE mouse expressed very high level of IFN-γ and IL-17 in the inflamed sites. Furthermore, pretreated with 5-Aza enhanced the Treg-mediated suppression in Treg inhibitory assay. However we found that suppression of effector T cell proliferation resulted from the reduced function of effector T cells but not from the enhanced function of Treg cells.
Therefore, pretreatment of 5-Aza prevented demyelination, and reduced the effector T cells function and limited pathogenic lymphocyte into CNS, prompted the inhibition of experimental autoimmune encephalomyelitis.

並列關鍵字

DNA demethylation agent ； experimental autoimmune encephalomyelitis

參考文獻

1 Sospedra, M. & Martin, R. Immunology of multiple sclerosis. Annu Rev Immunol

23, 683-747, doi:10.1146/annurev.immunol.23.021704.115707 (2005).

2 Compston, A. & Coles, A. Multiple sclerosis. Lancet 372, 1502-1517,

doi:10.1016/S0140-6736(08)61620-7 (2008).

Blood 118, 3538-3548, doi:10.1182/blood-2010-12-328187 (2011).