莊立民Chuang, Lee-Ming臺灣大學:分子醫學研究所陳冠勳Chen, Kuan-HsunKuan-HsunChen2010-05-042018-07-092010-05-042018-07-092009U0001-1201200915111400http://ntur.lib.ntu.edu.tw//handle/246246/178692第2型糖尿病現今已成為威脅全人類健康的一種疾病，目前第2型糖尿病已被廣泛的認為與肥胖所導致的輕微慢性發炎有關，此種發炎的特徵在於脂肪組織中出現大量巨噬細胞及其所分泌的炎症性細胞激素。介白素10是一種多功能性的細胞激素，可借由抑制炎症性的細胞激素來調控免疫及發炎反應，在脂肪組織中的巨噬細胞所釋放的介白素10可結抗發炎反應，並且保護脂肪細胞免於傷害。在本篇研究中，藉由PMA所誘導分化的人類巨噬細胞，吾人發現15-keto-PGE2 可誘使抗發炎的介白素10之生成，但不會誘使發炎反應的細胞激素生成。而15-keto-PGE2主要是藉由PI3K/AKT/GSK-3β的訊息傳遞途徑來誘使介白素10之生成。15-keto-PGE2 可藉由增強轉錄因子CREB及Sp1對於介白素10 promoter的結合能力來調控介白素10，除此之外，15-keto-PGE2 也經由過氧化氫受體γ之途徑來調控介白素10。綜合以上，這些結果開拓了15-keto-PGE2於調控抗發炎上之重要角色，以及增強了前列腺素代謝反應在免疫反應調控上之重要性。Type 2 diabetes (T2D) mellitus is currently one of the main health treats for humans. It is now broadly accepted that low-grade chronic inflammation associated with obesity leads to the onset of type 2 diabetes. Obesity-associated inflammation is characterized by an increased abundance of macrophages in adipose tissue along with the production of inflammatory cytokines. Interleuikin-10 (IL-10) is a pleiotropic cytokine that mediate immune and inflammatory responses by inhibiting the synthesis of pro-inflammatory cytokines. IL-10 secreted from macrophages in adipose tissue may counteract the inflammatory effects and protect adipose tissue from injuring. In this study, by using PMA-differentiated THP-1 macrophages, we demonstrated that 15-keto-PGE2 could induce the anti-inflammatory cytokine IL-10 production, but not the pro-inflammatory cytokines (TNF-α, IL-6 and IL-12). The capacity of 15-keto-PGE2 to elevate IL-10 expression level was mediated through PI3K/AKT/GSK-3β signaling pathway. 15-keto-PGE regulated IL-10 promoter activation by increasing the binding activities of transcription factors, CREB and Sp1. Furthermore, 15-keto-PGE2 also induced IL-10 production through partial PPARγ-dependent pathway. Taken together, these findings explore the crucial role of 15-keto-PGE2 in anti-inflammatory effect and enforce the importance of PGE2 catabolism in the immune response.謝誌 I文摘要 IIbstract IIIable of contents IVntroduction 1. Obesity, insulin resistance and Type 2 Diabetes 2I. Inflammation – linked obesity to Type 2 Diabetes 3he main origin of the systemic inflammatory response is located in adipose tissue 3he infiltration of macrophage causes local inflammation in adipose tissue 5II. Peroxisome proliferator-activated receptors (PPARs) 8acrophage PPARγ is a crucial regulator of inflammation and insulin resistance 9V. Anti-inflammatory cytokine - Interleukin-10 (IL-10) 11. Prostaglandins (PGs) 12rostaglandin E2 and its metabolites 13aterials and methods 15eagents 15ell culture 15repare whole cell extraction 15estern blot analysis 16NA isolation and Real-Time PCR 17nzyme-Linked Immunosorbent Assay (ELISA) 18hromatin Immunoprecipitation (ChIP) Assay 18tatistical Analysis 19esults 20MA induced differentiation of human THP-1 cells 205-keto-PGE2 induced the anti-inflammatory cytokine IL-10 mRNA and protein levels in human THP-1 derived macrophages 20ifferential effects of 15-keto-PGE2 and other PPARγ agonists on IL-10 production in human THP-1 cells 215-keto-PGE2 stimulated IL-10 expression through PPARγ-dependent pathway 22he effect of 15-keto-PGE2 on IL-10 production was mediated by PI3K/AKT pathway 22ole of MAPK family in the 15-keto-PGE2 activated IL-10 production 23ranscription factors binding to the IL-10 promoter in situ 24onclusion and Discussion 26PARγ ligands in immune regulation 26rostaglandin and its receptor on the cytokine regulation 28ranscriptional control of IL-10 production 28ur findings in drug discovery for the Type 2 diabetes mellitus therapy 30igures and Tables 31IG. 1 Characterization of human THP-1 derived macrophages. 31IG. 2 15-keto-PGE2 up-regulates the anti-inflammatory cytokine IL-10, but not pro-inflammatory cytokines in human THP-1 cells. 32IG. 3 15-keto-PGE2 induces IL-10 mRNA and protein expression in a dose- and time-dependent manner in human THP-1 derived macrophages. 33IG. 4 Effects of different PPARγ agonists on the IL-10 production by THP-1 cells. 34IG. 5 Effect of 15-keto-PGE2 on IL-10 production is mediated through PPARγ-dependent pathway. 35IG. 6 Effect of 15-keto-PGE2 on IL-10 production is mediated through PI3K/AKT but not PKA signaling pathway. 37IG. 7 Role of MAPK family in the 15-keto-PGE2 activated IL-10 production 39IG. 8 Activation of CREB and Sp1 binding at the IL-10 promoter by 15-keto-PGE2 in THP-1 derived macrophages. 41IG. 9 Proposed model for regulation of IL-10 production by 15-keto-PGE2 42upplemental Data 43igure S1. Effects of different PPARγ agonists in various dose on the IL-10 production by THP-1 derived macrophages. 43igure S2. The expression of PTGR-2 during THP-1 derived macrophage differentiation. 44able 1. Nucleotide sequence of the promoter region of hIL-10 gene 45eferences 46application/pdf1100319 bytesapplication/pdfen-US第2型糖尿病介白素1015-keto-PGE2巨噬細胞過氧化氫受體γInterleukin-10PPARγTHP-1 cellmacrophage[SDGs]SDG3http://ntur.lib.ntu.edu.tw/bitstream/246246/178692/1/ntu-98-R95448011-1.pdf