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研究生:陳意芃
研究生(外文):Yi-Peng Chen
論文名稱:以NOD基因轉殖小鼠模式探討Ptpn22基因對於 CD8+ T細胞之免疫功能影響
論文名稱(外文):To investigate the modulatory role of Ptpn22 in effector functions of CD8+ T cells in nonobese diabetic mice: approaches by transgenic models
指導教授:司徒惠康司徒惠康引用關係
指導教授(外文):Huey-Kang Sytwu
口試委員:繆希椿莊依萍
口試委員(外文):Shi-Chuen MiawYi-Ping Chuang
口試日期:2014-05-20
學位類別:碩士
校院名稱:國防醫學院
系所名稱:微生物及免疫學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2014
畢業學年度:102
語文別:中文
論文頁數:89
中文關鍵詞:Ptpn22基因CD8 T淋巴細胞NOD小鼠NOD 8.3小鼠
外文關鍵詞:Ptpn22CD8+ T cellNOD miceNOD 8.3 mice
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Ptpn22 基因可轉錄轉譯為富含脯氨酸、谷氨酸、絲氨酸與蘇氨酸片段之酪氨酸磷酸酶 (PEST domain-enriched tyrosine phosphatase, Pep),並透過對T細胞接收器 (T cell receptor, TCR) 下游的遠端訊息進行負向調控維持作用型/記憶型T細胞 (effector/memory T cell) 在體內的細胞衡定。PTPN22基因中帶有一個C1858T的single nucleotide polymorphism (SNP),導致蛋白的第620個胺基酸由arginine (R) 變成tryptophan (W),而這個SNP與多種自體免疫疾病具有高度相關性。先前研究中指出,Pep在先天與後天免疫中可調控不同免疫細胞的發育與功能。然而Pep在CD8+ T淋巴細胞中所扮演的角色與其影響的免疫生理仍有許多未知之處。在我們實驗室先前研究中,葉禮慈博士製造了專一性在T細胞增加Pep表現的基因轉殖非肥胖型糖尿病小鼠 (non-obese diabetic mice, NOD),並探討Pep對第一型糖尿病的影響。研究結果指出增加Pep會對小鼠的作用型CD4+ T細胞與調節型T細胞 (Regulatory T cells, Treg) 造成不同調控,導致第一型糖尿病的發病減緩。在本篇研究,我們同樣利用這隻基因轉殖小鼠,透過探討Pep對CD8+ T淋巴細胞功能以及致糖尿病的影響,研究Pep蛋白在CD8+ T淋巴細胞的角色。
我們的研究結果顯示,CD8+IFN-gamma+、CD8+granzyme B+、CD8+TNF-alpha+和CD8+FasL+細胞的比例在12到14週大的基因轉殖小鼠與非基因轉殖小鼠的脾臟中不具有統計上差異,唯有在基因轉殖小鼠胰臟淋巴結的CD8+TNF-alpha+ T淋巴細胞比例有減少。當基因轉殖CD8+ T淋巴細胞受到TCR刺激時,細胞增生有下降的現象。為了探討CD8+ T淋巴細胞的致病能力,我們將不同基因轉殖小鼠的CD8+ T淋巴細胞加上非基因轉殖小鼠的CD4+CD25- T淋巴細胞,移殖到免疫缺陷NOD/SCID小鼠後監測尿糖濃度。實驗結果顯示增加Pep削弱了CD8+ T淋巴細胞轉移自體免疫糖尿病的能力,而且這樣的削弱程度與Pep的表現量成正比。為了進一步探討Ptpn22轉殖基因是否影響CD8+ T淋巴細胞對自體抗原的反應,我們生產了Ptpn22/NOD8.3雙基因轉殖小鼠。令人驚訝的是,基因轉殖Pep對Ptpn22/NOD8.3小鼠第一型糖尿病發病、細胞增生與細胞胞殺能力並未造成任何影響。我們推測這樣的結果可能是因為在NOD8.3 TCR基因轉殖小鼠中,基因轉殖Pep的功能在CD8+ T淋巴細胞被選擇性的遮蔽或影響。因此在未來的實驗中,我們會深入探討造成基因轉殖Pep在NOD小鼠與NOD8.3 TCR轉殖小鼠之間差異的作用機制。
Ptpn22 encodes PEST domain-enriched tyrosine phosphatase (Pep), a negative regulator of TCR proximal signaling molecules, to maintain effector/memory T cell homeostasis. It has been demonstrated that a single nucleotide polymorphism (SNP) in PTPN22 (C1858T) leads to an amino acid (R620W) substitution in humans and has been identified as a critical factor for a variety of autoimmune diseases. Previous studies demonstrated that Pep regulates the development and/or functions of various immune cells in innate and adaptive immunity. However, the role of Pep in CD8+ T cells is largely unknown and its immunophysiological effects are still controversial. We previously generated a transgenic nonobese diabetes (NOD) mouse model overexpressing Pep in T cells and demonstrated that a different modulation of this molecule in effector and regulatory CD4+ T cells leads to attenuation of autoimmune diabetes in these mice. In this study we take advantage of this transgenic model to investigate the potential role of Pep on effector functions of CD8+ T cells and evaluate the contribution of transgenic CD8+ T cells in the protection of autoimmune diabetes. Our data revealed that the percentages of CD8+IFN-gamma+, CD8+Granzyme B+, CD8+TNF-alpha+ and CD8+FasL+ T cells in spleen of Ptpn22 transgenic mice are comparable with that of non-transgenic mice at 12–14 weeks of age. However, the percentage of CD8+TNF-alpha+ T cells was decreased in pancreatic lymph nodes of Ptpn22 transgenic mice, compared to non-transgenic controls. Transgenic CD8+ T cells showed decreased TCR-mediated effector responses such as proliferation and expression of effector molecules. To investigate the diabetogenic property of transgenic CD8+ T cells, we adoptively transferred these cells combined with non-transgenic CD4+CD25- T cells into NOD/SCID mice and monitored the development of diabetes. Our data revealed that transgenic Pep attenuates the ability of CD8+ T cells to transfer diabetes in a dosage-dependent manner. To further examine whether transgenic Ptpn22 modulates the pathogenic features of transgenic CD8+ T cells when encounter islet antigens in vivo, we generated Ptpn22/NOD8.3 doubly transgenic mice. Unexpectedly, the regulatory effects of transgenic Pep on the development of spontaneous diabetes and effector functions of cytotoxic CD8+ T cells were not observed in these doubly transgenic mice, suggesting that the regulatory potential of transgenic Pep on CD8+ T cells may be affected and/or masked by a selection bias of 8.3 TCR transgenic T cells. We will further investigate the underlying mechanisms involved in this discrepancy of transgenic Pep between Pep transgenic and Pep/NOD8.3 doubly transgenic mice.
致謝 i
目錄 ii
表目錄 iv
圖目錄 v
中文摘要 vi
英文摘要 viii
第一章 緒論 1
第一節 第22型非受體型蛋白酪氨酸磷酸酶基因 (Protein tyrosine phosphatase, non-receptor type 22, Ptpn22) 1
第二節 第一型糖尿病 (Type 1 Diabetes) 4
第三節 CD8+ T淋巴細胞與自體免疫疾病 10
第四節 研究策略與目的 12
第二章 材料與方法 13
第一節 小鼠來源與繁殖 13
第二節 基因轉殖小鼠之篩選 14
第三節 偵測Pep蛋白在CD8+ T淋巴細胞中表現 16
第四節 細胞表面抗原與胞內蛋白質染色分析 19
第五節 T淋巴細胞刺激增殖 (proliferation) 23
第六節 小鼠免疫細胞移植 (adoptive transfer) 23
第七節 CD8+ T淋巴細胞胞殺能力分析 23
第三章 實驗結果 26
第一節 Ptpn22基因轉殖非肥胖型糖尿病小鼠轉殖基因分析 26
第二節Pep蛋白質在Ptpn22基因轉殖非肥胖型糖尿病小鼠CD8+ T淋巴細胞中表現量確認 26
第三節 探討Ptpn22基因轉殖非肥胖型糖尿病小鼠之Ptpn22轉殖基因是否影響CD8+ T淋巴細胞發育 28
第四節 探討Ptpn22轉殖基因是否影響CD8+ T淋巴細胞之免疫功能 29
第五節 探討Ptpn22轉殖基因是否影響CD8+ T淋巴細胞致病能力 31
第六節 探討Ptpn22轉殖基因是否影響AutoAg-specific CD8+ T淋巴細胞之胞殺能力 33
第四章 討論 377
第五章 參考文獻 43
第六章 表附錄 48
第七章 圖附錄 49

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