臺灣博碩士論文加值系統

第1型糖尿病（Type 1 diabetes mellitus, T1D）是因為胰臟內產生胰島素的β細胞被白血球破壞而產生。非肥胖型糖尿病（Non-obese diabetic, NOD）小鼠是經常用於研究該疾病的動物模型。而第1型糖尿病已被認為是T細胞介導的自體免疫性疾病。其中，CD4+和CD8+ T細胞會高度破壞郎格罕氏胰島內的β細胞。
在先前的研究已經知道，調節性T細胞（Regulatory T cells, Treg）會受到抗原刺激，而在免疫系統的動態平衡和耐受性中擔任調節免疫系統的關鍵角色，從而防止自體免疫的發生。最近的研究已經發現，在體內使用組蛋白去乙醯酶（Histone deacetylase, HDAC）抑製劑（HDACi）能夠增加調節性T細胞的關鍵轉錄因子（Forkhead box P3, FOXP3）的表達，以及增加調節性T細胞的產生和抑制功能。
丙戊酸（Valproic Acid, VPA），一種短支鏈脂肪酸，被廣泛用作抗癲癇藥和情緒穩定劑。在最近，VPA已被證明可以用作HDAC抑製劑並具有增加調節性T細胞的功能。可降低膠原誘導關節炎（Collagen - induced arthritis, CIA）和實驗性自體免疫腦脊髓炎（Experimental autoimmune encephalomyelitis, EAE）的發生率和嚴重性。根據以往的研究，我們評估了VPA對移植排斥反應和NOD小鼠胰島移植後復發的自體免疫抑制機制。
我們通過使用免疫組織化學和HE染色觀察胰島移植物的存活和胰島素生成。我們也通過西方墨點法和流式細胞儀來研究其保護機制。本研究的結果可以提供NOD小鼠在進行VPA治療後其脾細胞中Treg的比例增加的證據。

Type 1 diabetes mellitus (T1D) results from the destruction of insulin-producing β cells in the islet of the pancreas by leukocytes. Non-obese diabetic (NOD) mouse is an animal model frequently used for this disease. It has been considered that T1D is a T cell-mediated autoimmune disease. CD4+ and CD8+ T cells are highly responsible for the destruction of β cells within the pancreatic islets of Langerhans. Previous studies have revealed that regulatory T cells (Tregs) play a key role in immune system homeostasis and tolerance to antigens, thereby preventing autoimmunity. Recent studies have found that HDAC inhibitor (HDACi) treatment in vivo increases the expression of forkhead box P3 (FOXP3), a critical transcription factor for Tregs, as well as the production and suppressive function of Tregs. Valproic acid (VPA), a branched short-chain fatty acid, is widely used as an antiepileptic drug and a mood stabilizer. Recently, VPA has been demonstrated to acts as a HDAC inhibitor and VPA treatment increases Tregs function and decreases the incidence and severity of collagen-induced arthritis and experimental autoimmune neuritis. Based on previous studies, we evaluate the potential and the mechanism of VPA treatment on the suppression of allograft rejection and autoimmune recurrence in islet transplantation of NOD mice. We observed the survival and insulin production in islet grafts by using immunohistochemistry and H-E stain. We also investigated the protective mechanism by western blot and flow cytometry. The results of this study can provide evidence for an increased proportion of Tregs in the splenocytes in VPA-treated NOD mice, which will also contribute to their subsequent study of T-cell differentiation after VPA administration.

本文目錄..................................................I
圖目錄....................................................V
摘要.....................................................VI
ABSTRACT...............................................VIII
第一章 緒論................................................1
第一節 糖尿病..............................................1
壹、病因...................................................1
貳、併發症.................................................2
參、分類...................................................3
肆、診斷...................................................4
伍、治療...................................................5
陸、預防...................................................6
第二節 T細胞...............................................7
壹、效應型T細胞.............................................7
貳、調節型T細胞.............................................7
參、免疫細胞攻擊β細胞........................................8
第三節 非肥胖型糖尿病小鼠....................................9
第四節 丙戊酸..............................................10
壹、臨床應用...............................................10
貳、組蛋白去乙醯酶抑制劑....................................11
第二章 實驗目的............................................12
第三章 材料與方法..........................................13
第一節 材料................................................13
壹、實驗動物...............................................13
貳、胰島移植用器械..........................................14
參、丙戊酸.................................................15
肆、實驗試劑...............................................15
伍、組織包埋 ..........................................16
陸、HE stain..............................................17
柒、IHC stain.............................................17
捌、手動磁珠分離CD4+ T 細胞與CD4+CD25+ T 細胞................18
玖、調節型T細胞之FACS分析...................................19
壹拾、Treg proliferation之FACS分析.........................20
壹拾壹、Treg apoptosis之FACS分析............................21
壹拾貳、Treg differentiation之western blot分析..............21
第二節 方法................................................24
壹、移植前後施打VPA.........................................24
貳、小鼠胰島的分離..........................................24
參、胰島移植手術............................................25
肆、腎臟石蠟包埋............................................26
伍、切片及HE染色............................................27
陸、IHC染色................................................28
柒、體外脾臟細胞懸浮液製備...................................29
捌、CD4 T細胞純化...........................................30
玖、誘導naïve T細胞分化及添加VPA.............................31
壹拾、流式細胞儀分析Treg的population.........................32
壹拾壹、分析Treg的proliferation.............................33
壹拾貳、分析Treg的apoptosis.................................34
壹拾參、分析Treg的differentiation signaling pathway.........35
壹拾肆、體內施予VPA後之Treg分析..............................38
第四章 實驗結果.............................................39
壹、施打丙戊酸後，胰島的型態完整且功能正常.....................39
貳、在體外Treg分布明顯；但在體內目前效果不明顯.................39
參、體外施加VPA後，Treg的proliferation增加...................40
肆、體內給予VPA後，Treg的proliferation同樣會增加..............40
伍、體外施加VPA後，Treg的apoptosis增加.......................41
陸、體內給予VPA後，Treg的apoptosis同樣會增加..................41
柒、體外施加VPA後，Treg的differentiation有些微增加............41
第五章 討論.................................................43
第六章 結論.................................................49
第七章 參考文獻.............................................50
第八章 結果圖表.............................................53

1.Shoback, Greenspan's basic & clinical endocrinology. 9th ed. 2011, New York: McGraw-Hill Medical.
2.Kitabchi, A.E., et al., Hyperglycemic crises in adult patients with diabetes. Diabetes Care, 2009. 32(7): p. 1335-43.
3.Stephen MS, J.R., JR KP, RKP, Diabetes. 8 edition ed. 2006, United States: LWW: Textbook of Therapeutics: Drug and Disease Management. 1045.
4.Power, C. and C. Thomas, Changes in BMI, duration of overweight and obesity, and glucose metabolism: 45 years of follow-up of a birth cohort. Diabetes Care, 2011. 34(9): p. 1986-91.
5.Shi, Y. and F.B. Hu, The global implications of diabetes and cancer. Lancet, 2014. 383(9933): p. 1947-8.
6.Kim, J.J., et al., Analysis of compensatory beta-cell response in mice with combined mutations of Insr and Irs2. Am J Physiol Endocrinol Metab, 2007. 292(6): p. E1694-701.
7.Butler, A.E., et al., Beta-cell deficit and increased beta-cell apoptosis in humans with type 2 diabetes. Diabetes, 2003. 52(1): p. 102-10.
8.Ferrannini, E., The stunned beta cell: a brief history. Cell Metab, 2010. 11(5): p. 349-52.
9.Gepts, W., Pathologic anatomy of the pancreas in juvenile diabetes mellitus. Diabetes, 1965. 14(10): p. 619-33.
10.Butler, A.E., et al., Modestly increased beta cell apoptosis but no increased beta cell replication in recent-onset type 1 diabetic patients who died of diabetic ketoacidosis. Diabetologia, 2007. 50(11): p. 2323-31.
11.Dustin, M.L., Coordination of T cell activation and migration through formation of the immunological synapse. Ann N Y Acad Sci, 2003. 987: p. 51-9.
12.Mosmann, T.R., et al., Differentiation and functions of T cell subsets. Ciba Found Symp, 1997. 204: p. 148-54; discussion 154-8.
13.Saouaf, S.J., et al., Deacetylase inhibition increases regulatory T cell function and decreases incidence and severity of collagen-induced arthritis. Exp Mol Pathol, 2009. 87(2): p. 99-104.
14.Heath, W.R. and F.R. Carbone, Dendritic cell subsets in primary and secondary T cell responses at body surfaces. Nat Immunol, 2009. 10(12): p. 1237-44.
15.Lieberman, S.M. and T.P. DiLorenzo, A comprehensive guide to antibody and T-cell responses in type 1 diabetes. Tissue Antigens, 2003. 62(5): p. 359-77.
16.Delovitch, T.L. and B. Singh, The nonobese diabetic mouse as a model of autoimmune diabetes: immune dysregulation gets the NOD. Immunity, 1997. 7(6): p. 727-38.
17.Inoue, Y., et al., Activating Fc gamma receptors participate in the development of autoimmune diabetes in NOD mice. J Immunol, 2007. 179(2): p. 764-74.
18.Koarada, S., et al., Increased nonobese diabetic Th1:Th2 (IFN-gamma:IL-4) ratio is CD4+ T cell intrinsic and independent of APC genetic background. J Immunol, 2002. 169(11): p. 6580-7.
19.ES, E.L.D., et al., Pharmacokinetic modelling of valproic acid from routine clinical data in Egyptian epileptic patients. Eur J Clin Pharmacol, 2004. 59(11): p. 783-90.
20.Thelen, P., et al., Expressional changes after histone deacetylase inhibition by valproic acid in LNCaP human prostate cancer cells. Int J Oncol, 2004. 24(1): p. 25-31.
21.Lackmann, G.M., Valproic-acid-induced thrombocytopenia and hepatotoxicity: discontinuation of treatment? Pharmacology, 2004. 70(2): p. 57-8.
22.Lv, J., et al., The antiepileptic drug valproic acid restores T cell homeostasis and ameliorates pathogenesis of experimental autoimmune encephalomyelitis. J Biol Chem, 2012. 287(34): p. 28656-65.
23.Mantel, P.Y., et al., Molecular mechanisms underlying FOXP3 induction in human T cells. J Immunol, 2006. 176(6): p. 3593-602.
24.Ngalamika, O., et al., Epigenetics, autoimmunity and hematologic malignancies: a comprehensive review. J Autoimmun, 2012. 39(4): p. 451-65.
25.Wu, S.Y., et al., Valproic acid attenuates acute lung injury induced by ischemia-reperfusion in rats. Anesthesiology, 2015. 122(6): p. 1327-37.