臺灣博碩士論文加值系統

辣椒素受器 (transient receptor potential vanilloid 1, TRPV1) 是一個鈣離子通道，其活性受到許多傷害性刺激所調節，例如辣椒素 (capsaicin)、溫度 (>43℃)，氫離子、花生四烯酸 (arachidonic acid) 之代謝產物以及多種炎症介質(inflammatory mediators)。辣椒素受器廣泛地分佈於全身，並且負責多種不同的細胞生理功能。近來有研究指出，辣椒素受器似乎會促進或減緩脂多醣 (lipopolysaccharide, LPS) 所以起的發炎現象，然而我們對於辣椒素受器在發炎反應中的明確角色仍然不甚清楚。由於巨噬細胞 (macrophage) 在先天免疫 (innate immunity) 中佔有重要的地位，而脂多醣主要透過第四型類鐸受體 (toll-like receptor 4, TLR4) 的訊息傳遞引發免疫反應，其下游的核因子 (nuclear factor-kappaB, NF-kappaB)負責許多發炎相關基因的表達。因此，本實驗目的即為研究巨噬細胞上的辣椒素受器是否能夠調節脂多醣引起之免疫反應，並且探討辣椒素受器否參與在第四型類鐸受體訊息傳遞路徑。我們採用從正常及辣椒素受器基因剔除小鼠 (gene knockout mice) 的骨髓取得並分化成的巨噬細胞，作為本研究的實驗模式。脂多醣會促使巨噬細胞增加腫瘤壞死因子-alpha (tumor necrosis factor-alpha, TNF-alpha), 第一介白質(interleukin-1 beta,IL-1 beta), 誘發性一氧化氮合成酶 (inducible nitric oxide synthase, iNOS) 的表現及其反應產物一氧化氮 (nitric oxide, NO) 的產量，然而這些反應在辣椒素受器基因剔除小鼠的巨噬細胞都下降。辣椒素受器基因剔除小鼠的巨噬細胞在受到脂多醣刺激造成的核因子�羠抑制蛋白 (inhibitor of NF-kappaB, IkappaB) 磷酸化、核因子位移 (translocation) 量以及絲裂原蛋白激酶(mitogen-activated protein kinase, MAPK) 的活化都較正常巨噬細胞減少。此外，從免疫沈澱法 (immunoprecipitation) 的結果發現，脂多醣刺激會使第四型類鐸受體訊息傳遞中的重要分子所形成的免疫複合物增加，其中包括第四型類鐸受體、髓樣分化因子88 (myeloid differentiation protein 88 , MyD88)、介白素1受體相關激酶1 (IL-1 receptor-associated kinase, IRAK) 及核因子kappa B抑制激酶（IkappaB kinase, IKK），然而此結合增加的程度在剔除辣椒素受器後會減少。我們更進一步發現在未受刺激時，辣椒素受器就與第四型類鐸受體及CD14形成複合物結合，且在受到脂多醣刺激後的5分鐘內增加。由本研究得知，辣椒素受器可能參與第四型類鐸受體及CD14所形成的複合物，並且經由第四型類鐸受體訊息傳遞路徑調節脂多醣所引起的發炎反應。

Abstract
Transient receptor potential vanilloid 1 (TRPV1) is a calcium channel activated by numerous noxious stimuli including capsaicin, heat (>43℃), protons, arachidonic acid metabolites and inflammatory mediators. TRPV1 is widely distributed over body and mediates various cell functions. Recent studies suggested that TRPV1 might positively or negatively regulate lipopolysaccharide (LPS)–induced inflammation; however, the exactly role of TRPV1 in inflammation is not fully understood. It is well established that macrophages play dominant roles in innate immune responses, and toll-like receptor (TLR) 4–mediated signaling transduction contributes largely to the inflammatory responses evoked by LPS. The mitogen-activated protein kinase (MAPK) and nuclear factor-kappaB (NF-kappaB) pathways are responsible for the expression of many inflammation-associated genes. Therefore, the aims of this study were to investigate the possibility that TRPV1 could regulate LPS-mediated inflammatory responses in macrophages, and the involvement of TRPV1 in TLR4 signaling pathway. To explore the hypothesis, bone marrow-derived macrophages harvested from wild type (WT) and TRPV1 knock out (TRPV1-/-) mice were used as cell models. The LPS-induced elevation of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), inducible nitric oxide synthase (iNOS) and subsequent product nitric oxide (NO) were attenuated in TRPV1-/- macrophages. Compared with WT macrophages, the phosphorylation of inhibitor of NF-kappaB (IkappaB), translocation of NF-kappaB and activation of MAP kinase were reduced in LPS-treated TRPV1-/- macrophages. Based on the results of immunoprecipitation, deficiency of TRPV1 in macrophages impaired the LPS-triggered immune-complex formation of myeloid differentiation protein 88 (MyD88), TLR4, IL-1 receptor-associated kinase (IRAK) and IkappaB kinases (IKK) that are important in TLR4 signaling pathway. Furthermore, the association between TRPV1, TLR4 and CD14 was observed in WT macrophages at basal condition and the interaction was increased within 5 minutes after LPS administration. In conclusion, TRPV1 might participate in the TLR4: CD14 complex formation and maintain the LPS-mediated inflammatory responses through TLR4 signaling pathway.

致謝....................................................IV
ABBREVIATIONS............................................V
ABSTRACT.................................................VI
摘要...................................................VIII
INTRODUCTION.............................................1
I. Lopolysaccharide (LPS)-induced inflammation ..........1
Sepsis...................................................1
Lipopolysaccharide.......................................2
Toll-like receptor.......................................2
Toll-like receptor 4 signaling pathway...................3
Nuclear factor-kappaB (NF-kappaB)........................4
Mitogen-activated protein kinase (MAPK)..................5
LPS-induced inflammatory cascade.........................6
II. Transient receptor potential vanilloid 1 (TRPV1).....7
The structure and ligand of TRP family...................7
Regulation of TRPV1 activity.............................9
Function of TRPV1........................................9
TRPV1 and inflammation..................................11
III. Objective..........................................12
MATERIALS AND METHODS...................................13
Drugs and antibodies....................................13
Mice....................................................14
Isolation of bone marrow-derived macrophages............14
Cell culture condition for J774 and 3T3L1...............15
Genome typing of WT and TRPV1-/- mice...................15
Isolation of RNA........................................16
Reverse transcriptase-PCR...............................17
Determination of cytokine concentrati...................18
Determination of nitrite production.....................19
Preparation of whole cell lysates.......................20
Preparation of nuclear extracts.........................20
Western blot............................................21
Immunoprecipitation.....................................21
Statistic...............................................22
RESULTS.................................................23
Genome typing and TRPV1 expression in macrophages.......23
LPS-induced TNF-alpha and IL-1beta production are attenuated in TRPV1-/- macrophages......................23
LPS-induced iNOS protein induction and NO production are decreased in TRPV1-/- macrophages.......................24
TRPV1 is involved in the regulation of LPS-induced IkappaBalpha phosphorylation and NF-kappaB activation in macrophages.............................................24
TRPV1 is involved in the regulation of LPS-induced MAPK activation in macrophages...............................25
TRPV1 is involved in the regulation of association between MyD88, TLR4, IRAK1 and IKKalpha/beta in macrophages ....26
Participation of TRPV1 in TLR4 complex was increased after LPS challenge...........................................27
DISCUSSION..............................................28
REFERENCES..............................................35
FIGURES.................................................60

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