臺灣博碩士論文加值系統

對於生物體而言，要如何調控免疫系統的恆定是相當重要的，過度反應的免疫系統往往會傷害宿主；而此重要調控機制可藉由調節性T細胞所負責。調節性T細胞屬於CD4輔助型T細胞之一員。調節性T細胞特定表現重要轉錄因子— Forkhead box P3 (Foxp3)；Foxp3的表現與否控制著調節性T細胞的發育以及細胞功能。個體之調節性T細胞功能若產生異變或是降低，則此個體罹患多種自體免疫疾病之機率也就大幅上升。先前針對自體免疫疾病患者的研究發現，病患體內之調節性T細胞組成比例明顯低於正常值。此外研究文獻指出，FOXP3基因的表達受到對位性基因體修飾(Epigenetic modification)的調控，且以去氧核醣核酸的甲基化為最。此外，以去氧核醣核酸之去甲基化藥劑5-Aza-2-deoxycytidine治療自體免疫糖尿病小鼠，可增加調節性T細胞抑制免疫功能以及降低糖尿病發生率。因此我們使用實驗性自體免疫腦脊髓炎做為小鼠動物模式；探討去甲基化藥劑於自體免疫疾病機制及其應用於治療之可能性。我們以轉殖FOXP3與綠螢光蛋白之基因轉殖鼠實驗發現；不論是活體外亦或活體內施予去甲基化藥劑皆能增加綠螢光蛋白的表現比率。小鼠預先施打去甲基化藥劑能減緩、延後實驗性自體免疫腦脊發病時間，同時某些實驗鼠甚至沒有實驗性自體免疫腦脊病徵。我們檢測由脊髓萃取出之核糖核酸發現；預先施予藥劑的小鼠其脊髓內與發炎反應相關的細胞激素含量明顯低於控制組。我們推論針對小鼠預先施予去甲基化藥劑能夠加強調節性T細胞抑制免疫功能並保護實驗鼠免於致病性淋巴球的攻擊；因而減緩實驗性自體免疫腦脊發病或是降低發病機率。

Regulatory T (Treg) cells play an indispensable role in immune tolerance and suppress immune responses after the infection and expressing an important transcription factor Forkhead box P3 (FOXP3). FOXP3 plays important roles for the development and function of Treg cells and has been identified as a specific marker for Treg cells. Autoimmune diseases arise from an overactive immune response against tissues normally present in the body. As previous studies shown, patients that suffered from autoimmune diseases have lower percentage of regulatory T cells including multiple scelrosis. Previous studies have indicated that epigenetic modifications were involved in the regulation of FOXP3 expression; treatment with the DNA demethylation agent enhanced the Treg-mediated suppression and decreased the occurrence of diabetes in NOD mice. In our study, we investigated the treatment of DNA demethylation agent 5-Aza (5-Aza-2- deoxycytidine) in experimental allergic encephalomyelitis (EAE), a widely used animal model for studying human multiple sclerosis, to evaluate the demethylation effect in autoimmune therapy. Using GFP knock-in FOXP3 transgenic mice to evaluate the FOXP3 expression after 5-Aza treatment, 5-Aza treatment increased the GFP expression in CD4+CD25- T cells in vitro and in vivo. Furthermore, MOG-induced mouse pretreated with 5-Aza delayed the onset of EAE at least about 20 days, and, in most cases, there is not any EAE clinical symptom. Moreover, the 5-Aza pretreated mice seem to have no inflammatory responses in central nervous system, because of the very low RNA expression level of inflammatory cytokines could be detected in central nervous system. Therefore, we hypothesized that pretreatment with 5-Aza in vivo enhanced the Treg-mediated suppression and could provide a protective effect against pathogenic lymphocyte, and as a result, decreased the occurrence of experimental allergic encephalomyelitis.

摘要 i
Abstract ii
List of Figures v
List of Tables vi
1. Introduction 1
1.1. Experimental autoimmune encephalomyelitis 1
1.1.1. Multiple sclerosis 1
1.1.2. Experimental autoimmune encephalomyelitis 2
1.2. T helper cells 4
1.2.1. Type 1 T helper cell 4
1.2.2. Type 2 T helper cell 5
1.2.3. Type 17 T helper cell 5
1.2.4. Regulatory T cell 6
1.3. Epigenetics 7
1.3.1. DNA methylation 7
1.3.2. Epigenetic modification-regulation of Regulatory T cell 8
1.3.3. Demethylating agent 5-aza-2'-deoxycytidine 8
2. Objectives of Study 10
3. Materials and Methods 11
3.1. Mice 11
3.2. EAE induction 11
3.3. Medium, buffer and reagents 12
3.3.1. T cell medium 12
3.3.2. Isolation buffer 12
3.3.3. RBC lysis buffer 12
3.3.4. Antibodies 12
3.3.5. 5-Aza-2’-deoxycytidine 13
3.4. Purification and culture of mouse T cells 13
3.4.1. In vitro 5-Aza treatment of CD4+CD25- T cells 13
3.4.2. In vitro 5-Aza treatment of CD4+GFP- T cells 14
3.4.3. Splenocytes treated with 5-aza in vitro. 14
3.4.4. In vitro Treg function assay 14
3.4.5. Isolation of CNS-infiltrating lymphocytes 15
3.5. DNA methylation analysis 15
3.5.1. Bisulphite modification of DNA 15
3.5.2. Combined Bisulphite Restriction Analysis (COBRA) 16
3.6. Analysis of mRNA expression 17
3.6.1. RNA extraction 17
3.6.2. Reverse transcription 18
3.6.3. Quantitative real-time RT PCR (qRT-PCR) 18
4. Result 20
4.1. In vitro 5-Aza treatment increased GFP expression of CD4+GFP- T cells and Foxp3 RNA level of Splenocytes 20
4.1.1. 5-Aza treatment increased Foxp3 RNA level of splenocytes 20
4.1.2. 5-Aza treatment increased GFP expression of CD4+GFP- T cells 21
4.2. 5-Aza increased GFP and RNA expression of splenocytes in vivo 22
4.3. Protection of MOG-induced EAE by 5-Aza pretreatment in B6 mice 23
4.4. Distinct differences of splenocyte population and cell number between 5-Aza and DMSO-treated EAE mice 24
4.4.1. Significant changes in cell number between 5-Aza and DMSO-treated EAE mice 24
4.4.2. Distinct differences of splenocyte population between 5-Aza and DMSO-treated EAE mice 24
4.5. Inhibited inflammatory cytokine RNA expressions in spinal cord of 5-Aza treated EAE mice 26
4.6. The expression of inflammatory cytokines increased in the CNS of 5-Aza treated mice while the disease progressed 27
4.7. Lower number of CNS-infiltrating lymphocytes in 5-Aza treated mice 28
4.8. Enhanced immune suppressive function of regulatory T cells in 5-Aza treated mice 29
5. Discussion 30
6. Figure 35
7. Supplementary Data 48
7.1. Supplementary Figure 48
7.2. Supplementary Table 55
8. References 57

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