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研究生:簡含書
研究生(外文):Han-Shu Jian
論文名稱:Lyn基因缺陷的調節性樹突細胞功能之研究
論文名稱(外文):The Function of Lyn-Deficient Regulatory Dendritic Cells in vitro
指導教授:朱清良
指導教授(外文):Ching-Liang Chu
口試委員:繆希椿廖南詩
口試委員(外文):Shi-Chuen MiawNan-Shih Liao
口試日期:2015-07-09
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:免疫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:英文
論文頁數:65
中文關鍵詞:Lyn酪氨酸激酶調節性樹突細胞腸道CD103+樹突細胞第二型視黃酸合成酶調節性T細胞
外文關鍵詞:Lynregulatory dendritic cellCD103+ DCRALDH1a2regulatory T cell
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Lyn 酪氨酸激酶是Src酪氨酸激酶家族中的一員,它表現在很多的白血球上,但只有T細胞不表現Lyn。Lyn具有獨特的調節特性,因為它不僅會誘發激活信號,也具有調節抑制信號的能力,進而促使免疫耐受性。在Lyn基因剔除 (lyn-/-)的老鼠身上會自發性發展成一種類似人類紅斑性狼瘡的自體免疫疾病,這表示說Lyn在抑制信號上扮演重要的角色。之前有文獻指出lyn-/-樹突細胞 (dendritic cell, DC)會增強誘發免疫反應的能力,但實際上DC具有很多不同的分類去調控不同的免疫反應,就像腸道有一群很特別的CD103+ DCs,它會促使口服耐受性,進而維持腸道的恆定。之前有文獻指出老鼠腸道固有層 (lamina propria, LP)和腸系膜淋巴結 (mesenteric lymph nodes, MLN)的CD103+ DCs會表現第二型視黃酸合成酶 (RALDH1a2),RALDH1a2可以將維生素A代謝成視黃酸 (retinoic acid, RA),故腸道的CD103+ DCs會產生高量的RA。在有RA的情況下可促使腸道的CD103+ DCs誘導T細胞表現腸道歸巢接受器 (gut-homing receptor) α4β7和CCR9,而且會促使T細胞分化成Foxp3+調節性T細胞 (regulatory T cell, Treg cell),故RA在維持腸道的恆定性也是扮演重要的角色。迄今為止,Lyn在調節性樹突細胞 (regulatory DC, reg-DC)上的角色尚未被研究,因此在我的研究論文中會探討Lyn是否會影響reg-DCs的功能。首先,我先建立體外的reg-DCs培養方法和比較野生型(wild type, WT) reg-DCs和lyn-/- reg-DCs在功能上的差異。接續我會探討lyn-/-老鼠體內的reg-DCs與WT老鼠的差異。實驗結果顯示體外培養的lyn-/- reg-DCs其RALDH1a2酵素活性會降低,而且lyn-/- reg-DCs誘發T細胞分化成Foxp3+ Treg cells能力也較弱,此外體外培養的lyn-/- reg-DCs相較於WT reg-DCs顯得比較不成熟。另外lyn-/- MLN的CD103+ reg-DCs其RALDH1a2酵素活性也較低,這與體外的實驗結果相吻合。這些實驗結果意味著lyn-/- reg-DCs的功能有缺損,進而導致嚴重的自體免疫疾病,因此我推測Lyn在reg-DCs中是扮演正向調控的角色。然而Lyn是經由何種機轉去調控reg-DCs的功能仍需再進一步的研究。

Lyn, a Src family tyrosine kinase, is expressed in all leukocytes except T cells. Lyn has unique regulatory properties, because it not only triggers activation signals but also regulates inhibitory signals for tolerance. Lyn-deficient mice spontaneously develop an autoimmune disease that resembles human systemic lupus erythematosus (SLE), suggesting that Lyn plays a more important role in mediating inhibitory function. It has recently been reported that lyn-/- dendritic cells (DCs) enhance the capacity to induce immune responses. Actually, DCs have distinct subsets for regulating different immune responses. A special intestinal subset of DCs with characteristic of CD103 expression (CD103+ DCs) was reported involving in oral tolerance. Mouse CD103+ DCs isolated from intestinal lamina propria (LP) and mesenteric lymph nodes (MLN) show a high level of retinoic acid (RA) production. RA, a metabolite of vitamin A, can be induced by retinal aldehyde dehydrogenase family 1, subfamily A2 (RALDH1a2), an enzyme for RA synthesis in DCs. RA plays a critical role in maintaining intestinal immune homeostasis. Thus, intestinal CD103+ DCs can induce gut-homing receptors α4β7 and CCR9 on T cells and drive the differentiation of transforming growth factor (TGF)-β-mediated Foxp3+ regulatory T (Treg) cells. Up to date, the role of Lyn tyrosine kinase in reg-DCs has not been studied. Hence, my thesis is to study whether Lyn tyrosine kinase affect the function of reg-DCs. First, I established bone marrow-derived reg-DCs in vitro culture and compared the function between wild type (WT) and lyn-/- reg-DCs. Second, I examined the phenotype of reg-DCs in lyn-/- mice. The results showed that Lyn deficiency led the lower RALDH1a2 activity in reg-DCs in vitro. Also, lyn-/- reg-DCs impaired the function of inducing CD4+ Foxp3+ Treg cells in vitro. In addition, lyn-/- reg-DCs impaired maturation after innate immune stimulation. Consistent with in vitro data, reg-DCs in lyn-/- MLN showed lower RALDH1a2 activity. These results suggested that lyn-/- mice may have weak reg-DC function compared to WT mice and lead to autoimmune disease. Also, these results suggested that Lyn may positively regulate the function of reg-DCs. However, the mechanism of how Lyn participates in reg-DCs function needs to be further explored.

Contents
誌謝 i
中文摘要 ii
Abstract iii
Abbreviations iv
List of Figures viii
Chapter 1 Introduction 1
1.1 Background 2
1.1.1 SFKs 3
1.1.2 Lyn 4
1.1.3 The role of Lyn in myeloid cells 5
1.1.4 Reg-DCs 6
1.1.5 Strategies to induce reg-DCs 8
1.2 Rationale 10
1.3 Specific aims 11
Chapter 2 Materials and Methods 12
2.1 Mice 13
2.2 Bone marrow-derived DCs culture in vitro 13
2.3 Bone marrow-derived reg-DCs culture in vitro 14
2.4 Aldehyde dehydrogenase activity assay 14
2.5 Reg-DCs maturation 14
2.6 Isolation of BMDCs, reg-DCs and OT-II T cells 15
2.7 T cell priming and differentiation in vitro 15
2.8 Analysis of spleen, MLN and peripheral LN DC subsets and lymphocytes 16
2.9 Isolation of SI-LP DC subsets and lymphocytes 16
2.10 Real-time PCR (qPCR) analysis 18
2.11 Assessment of cytokine production by ELISA 19
2.12 Statistical analysis 19
Chapter 3 Results 20
3.1 The induction of aldh1a2 expression and RALDH activity by RA and IL-4 in BMDC cultures 21
3.2 Reg-DCs in vitro culture system 22
3.3 Reg-DCs augmented il-10 gene expression in response to RA and IL-4 co-treatment 23
3.4 Reg-DCs promoted Foxp3+ Treg cells differentiation 23
3.5 Lyn-deficient reg-DCs had lower aldh1a2 expression and RALDH activtiy compared to WT reg-DCs in vitro 24
3.6 Lyn-deficient reg-DCs impaired maturation after LPS stimulation 25
3.7 Lyn-deficient reg-DCs impaired the function for inducing Foxp3+ Treg cells in vitro 26
3.8 Lyn deficiency led the lower RALDH activity of CD103+ reg-DCs in MLN 26
Chapter 4 Discussion 28
4.1 The induction of aldh1a2 expression and RALDH activity by RA and IL-4 in BMDC cultures 29
4.2 Reg-DCs promoted Foxp3+ Treg cells differentiation 30
4.3 The role of Lyn in RA or IL-4 signaling in reg-DCs 32
4.4 Lyn activity protected mice from DSS-induced colitis 33
Chapter 5 Conclusions 34
Figures 36
References 56



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