臺灣博碩士論文加值系統

糖尿病 (diabetes mellitus, DM) 世界上是最常見的代謝性疾病。糖尿病病患的先天免疫反應比正常人差，容易受到疾病感染。在先天免疫系統中Toll-like receptors (TLRs)，負責偵測病原並調控血球細胞激素(cytokine)分泌。近來研究發現glycogen synthase kinase 3 (GSK3)可以負向調控TLR誘發的激素反應。糖尿病患的先天免疫缺陷可能的調控有關，然而目前對其調控機制並不明瞭。為了探究其機制，我們從正常人及第一、第二型糖尿病患(T1DM, T2DM)抽出全血，再以五種TLR agonists與Mycobacterium bovis bacillus Calmette-Gu�臆in (BCG)刺激周邊白血球誘發免疫細胞激素反應。藉由偵測不同細胞激素(IL-12,IL-1β, IL-6, TNF-α, IFN-γ and IL-10)濃度，評估正常人與糖尿病患的免疫反應。我們發現T1DM病患，其白血球產生的較少，而T2DM病患的白血球受到TLR4 agonist刺激所誘發的IL-1β, IL-6, TNF-α, IFN-γ and IL-10也都比正常人少。DM病患的白血球受BCG刺激誘發的細胞激素也較正常人少。糖尿病患免疫力低落顯然是由於體內白血球分泌細胞激素功能受損，導致免疫系統異常。另外，胰島素可同樣降低T1DM與T2DM體內IL-1β的生成。顯示IL-1β的改變並未受到的T2DM的胰島素拮抗影響。DM病患的細胞激素分泌降低可能是受到病患的血糖過高症所影響。我們進一步藉由觀察THP-1細胞的GSK3在高糖濃度的環境下，受lipopolysaccharide (LPS)引發的磷酸化情形，研究高血糖症是否影響TLR的訊息傳遞。我們發現在高糖環境下，有無LPS刺激都可誘發GSK3的磷酸化。而在高糖環境下，胰島素則可以促進LPS誘發的GSK3的磷酸化。我們同樣發現在高糖環境下reactive oxygen species (ROS)的生成也會增加。這些結果顯示，糖尿病患的高血糖症可能導致TLR誘發細胞激素的反應異常，進而導致先天免疫系統的缺失。

Diabetes mellitus (DM) is one of the most common metabolism diseases in the world. Previously, we found that DM patients were susceptible to Mycobacterium tuberculosis infection and innate immune responses may be defective in DM patients. Toll-like receptors (TLRs) can recognize pathogens and can induce cytokine production to initiate innate immune responses. Resent studies indicated that TLR-induced cytokine production is negatively regulated by glycogen synthase kinase 3 (GSK3), which is a critical component in glucose metabolism. However, the innate immune defects and the influence of TLR-signaling in DM patients are not fully understood. In this study, we used five different TLR agonists and Mycobacterium bovis bacillus Calmette-Gu�臆in (BCG) to induce cytokine production in peripheral blood isolated from normal volunteers and patients with type I or type II DM to investigate the influence of DM in TLR-induced cytokine production. IL-12,IL-1β, IL-6, TNF-α, IFN-γ and IL-10 were measured. Leukocytes from Type I DM patients showed decreased IL-12 production after TLR agonist stimulation. TLR4 induced cytokine productions were defective in leukocytes isolated from type II DM patients, except for IL-12 production. Moreover, innate immune cytokine productions induced by BCG were decreased and insulin can down-regulate IL-1β production in leukocytes from both type 1 DM and type 2 DM patients. These findings suggested that hyperglycemia, the common feature of type I and type II DM, may be the cause of inhibited innate immune cytokine production in patients. We further investigated GSK3β phosphorylation after lipopolysaccharide (LPS) in THP-1cells cultured in high glucose condition to mimic the hyperglycemia. We found that the phosphorylation of GSK3β was increased in high glucose condition with or without LPS stimulation. Moreover, LPS induced GSK3β phosphorylation was promoted by insulin in high glucose condition. We also found that LPS-induced reactive oxygen species (ROS) production was increased in high glucose condition with or without insulin stimulation. These findings suggest that defective TLR-induced cytokine production may be due to hyperglycemia in diabetes patients.

ABSTRACT I
中文摘要 III
誌謝 IV
TABLE OF CONTENTS V
LIST OF ABBREVIATIONS VIII
1. INTRODUCTION 1
1.1 Diabetes mellitus 1
Type 1 diabetes mellitus 1
Type 2 diabetes mellitus 1
1.2 Innate immunity 2
1.3 Cytokines in innate immunity 3
1.4 The role of toll-like receptor in innate immunity 4
TLRs and their specific ligands 5
MyD88-dependent signaling pathway 6
MyD88-independent signaling pathway 6
1.5 Diabetes, toll-like receptor and glycogen synthase kinase 3 (GSK3) 7
1.6 The aim of this study 8
2. MATERIALS AND METHODS 10
2.1 Patients and healthy donors 10
2.2 Peripheral blood mononuclear cells (PBMC) isolation 10
2.3 BCG culture 10
2.4 Cell culture 11
2.5 Toll-like receptor agonists and BCG stimulation of PBMCs 11
2.6 Human IL-12 enzyme-linked immunoassay (ELISA) 12
2.7 Multiple cytokine assay by Luminex beadlyte suspension array system 12
2.8 High glucose stimulated THP-1 cells and human PBMCs 13
2.9 LPS stimulation of THP-1cells for GSK3 phosphorylation 14
2.10 Western blot 14
2.11 ROS production assay 15
3. RESULT 17
3.1 The cytokine responses of leukocytes induced by TLR agonists were decreased in DM patients 17
3.2 The cytokine production of leukocytes induced by BCG were decreased in DM patients. 18
3.3 The GSK3 phosphporylation induced by LPS in THP-1 cells was enhanced by high glucose concentration. 19
3.4 High glucose concentration enhanced ROS production of human monocytes and THP-1 cells 21
DISCUSSION 23
4.1 The TLRs-induced cytokine responses were defective in DM patients. 24
4.2 The cytokine responses of leukocytes induced by BCG were decreased in DM patients. 25
4.3 GSK3β phosphorylation was regulated by insulin signaling and high glucose 27
4.4 High glucose enhanced the ROS production of monocytes may result in chronic inflammation 28
CONCLUSION 30
REFERENCE LIST 31
RESULTS 41
Figure 1. 41
Figure 2. 45
Figure 3. 49
Figure 4. 52
Figure 5. 53
Figure 6. 54
Figure 7. 55

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