博碩士論文 105821018 詳細資訊




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姓名 董孟融(Meng-Rong Dong)  查詢紙本館藏   畢業系所 生命科學系
論文名稱 環狀核苷酸磷酸二脂酶4B對內毒素刺激小鼠樹突細胞表現NOD1與CXCR4的影響
(Effects of phosphodiesterase 4B in endotoxin-induced NOD1 and CXCR4 expression in mouse bone marrow-derived dendritic cells)
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摘要(中) Pattern recognition receptors (PRRs)在先天免疫中扮演重要的角色。已知在免疫細胞中,活化PRR訊息傳導可誘導許多不同的免疫發炎反應。環狀核苷酸磷酸二脂酶4 (phosphodiesterase 4;PDE4)為免疫細胞內主要分解cAMP的酵素,抑制PDE4活性以提升細胞內cAMP濃度,可減緩多種免疫發炎反應,然而PDE4對於樹突細胞的影響目前仍知之甚少。為此,本研究使用小鼠骨髓細胞分化的樹突細胞(bone marrow-derived dendritic cell;BMDC)進行實驗,並以細菌的成分lipopolysaccharide (LPS)誘導細胞成熟,且在成熟過程中檢測PDE4對NOD1、NOD2與TLR4三種PRRs及趨化激素受器CXCR4 mRNA表現的影響。由定量PCR結果顯示,細胞在LPS刺激4小時後,NOD1與NOD2 mRNA的表現會顯著增加,而TLR4與CXCR4 mRNA則會下降。當以PDE4抑制劑rolipram共同處理細胞時, LPS所誘導的NOD1 mRNA表現會明顯被抑制,然而NOD2、TLR4及CXCR4 mRNA表現量則會上升。在LPS處理BMDC 24-36小時後,四個蛋白質受體的表現皆會降回或低於基礎值。進一步以PDE4基因剔除的BMDC進行實驗,我們發現rolipram對於NOD1 mRNA表現的抑制作用主要是由於抑制了PDE4B及小部分抑制PDE4A所致,而對於CXCR4 mRNA表現的提升則是經由抑制PDE4B所致。至於在LPS對NOD2與TLR4 mRNA的調控中,PDE4的參與相當有限。綜上所述,在LPS誘導BMDC成熟的過程中,PDE4B為主要參與調控NOD1與CXCR4 mRNA表現的PDE4亞型。因此我們認為,PDE4B選擇性抑制劑應可有效降低LPS在樹突細胞內所誘導的NOD1訊息傳導及其發炎反應,同時可增強由CXCR4所引導的移走能力,以提升樹突細胞吞噬病原菌的效率。
摘要(英) Pattern recognition receptors (PRR) are important in innate immunity. Activation of PRRs initiates inflammatory responses in various immune cells, including dendritic cells (DCs). Elevation of cAMP by inhibition of phosphodiesterase 4 (PDE4), enzyme that specifically hydrolyze cAMP, has been shown to suppress a variety of inflammatory response in most inflammatory cells, such as macrophages and T cells, but such information in DCs is less documented. In this study, we investigated the effect of PDE4 in the mRNA expression of the PRRs NOD1, NOD2 and TLR4 and chemokine receptor CXCR4 in bone marrow-derived DCs (BMDCs) during lipopolysaccharide (LPS)-stimulated maturation. Quantitative PCR analysis revealed that the mRNA expression of NOD1 and NOD2 was markedly upregulated, while TLR4 and CXCR4 expression was downregulated at 4h of LPS stimulation. Inhibition of PDE4 with rolipram resulted in a significant reduction of NOD1 but increase in NOD2, TLR4 and CXCR4 mRNA expression. At 24-36h of LPS exposure, the mRNA levels of the four receptors were return to or below the basal levels. Further analysis, using PDE4-/- BMDCs indicated that the effect of rolipram on NOD1 gene expression was mediated by inhibition of PDE4B and, to a lesser extent, PDE4A, while rolipram upregulation of CXCR4 was mediated by inhibition of only PDE4B. The impact of PDE4 on NOD2 gene expression was limited, when LPS is present. Taken together, these finding demonstrate that PDE4, mainly the PDE4B isoform, is involved in the expression of NOD1 and CXCR4 in BMDCs during LPS-induced maturation. This suggests that PDE4B-selected inhibitors may inhibit LPS-elicited NOD1 signaling and its inflammatory responses as well as promote CXCR4-directed DC migration for pathogen capturing.
關鍵字(中) ★ 環狀核苷酸磷酸二脂酶
★ 樹突細胞
★ 環腺苷酸
★ 脂多醣
關鍵字(英) ★ phosphodiesterase,PDE
★ PDE4
★ cAMP
★ dendritic cell
★ LPS
★ NOD1
★ NOD2
★ TLR4
★ CXCR4
論文目次 中文摘要 ……………………………………………………………… i
英文摘要 ……………………………………………………………… ii
誌謝 ……………………………………………………………… iv
目錄 ……………………………………………………………… v
圖目錄 ……………………………………………………………… viii
縮寫檢索表 ……………………………………………………………… ix
一、 緒論………………………………………………………… 1
1-1 樹突細胞(Dendritic cell)…………………………………... 1
1-2 樹突細胞的成熟作用……………………………………… 1
1-3 樹突細胞的種類與特性…………………………………… 2
1-4 樹突細胞與自體免疫疾病………………………………… 3
1-5 Pattern recognition receptor (PRR)………………………… 3
1-5-1 Toll-like receptor (TLR)…………………………………… 4
1-5-2 Toll-like receptor 4…………………………………………. 4
1-5-3 Nucleotide-binding oligomerization domain-like receptor (NOD-like receptor;NLR)……………………………......
5
1-5-4 NOD1與NOD2…………………………………………… 6
1-6-1 腺嘌呤環狀核苷酸( cyclic adenosine monophosphate;cAMP)訊息傳導……………………………………………
7
1-6-2 cAMP與免疫發炎反應………………………………….... 8
1-7 環狀核苷酸磷酸二脂酶(Cyclic nucleotide Phosphodiesterase;PDE) …………………………………
8
1-7-1 PDE4……………………………………………………….. 10
1-7-2 PDE4抑制劑與免疫功能調節……………………………. 11
1-8 C-X-C chemokine receptor type 4 (CXCR-4)……………... 12
二、 研究動機與目的…………………………………………… 14
三、 實驗材料與方法…………………………………………… 15
3-1 實驗材料…………………………………………………… 15
3-1-1 實驗小鼠…………………………………………………… 15
3-1-2 實驗用藥品………………………………………………… 15
3-1-2-1 細胞培養相關溶液………………………………………… 15
3-1-2-2 處理細胞之小分子藥劑…………………………………… 16
3-1-2-3 萃取RNA與qPCR相關試劑……………………………. 16
3-2 實驗方法…………………………………………………… 16
3-2-1 配製C10培養液…………………………………………... 16
3-2-2 分離小鼠骨髓細胞………………………………………… 16
3-2-3 小鼠骨髓細胞分化為未成熟樹突細胞…………………… 17
3-2-4 未成熟樹突細胞分化為成熟樹突細胞…………………… 17
3-2-5 萃取樹突細胞內RNA…………………………………….. 18
3-2-6 反轉錄作用………………………………………………… 18
3-2-7 cDNA cleaning…………………………………………….. 18
3-2-8 定量聚合酵素連鎖反應(Quantitative PCR)……….... 19
四、 實驗結果…………………………………………………… 20
4-1 NOD1、NOD2、TLR4及CXCR4在未成熟樹突細胞中的表現………………………………………………………
20
4-2 PDE4抑制劑可降低樹突細胞內LPS誘導NOD1 mRNA的表現………………………………………………………
20
4-3 剔除PDE4A與PDE4B可抑制樹突細胞內LPS誘導NOD1 mRNA的表現………………………………….......
21
4-4 PDE4抑制劑對樹突細胞內LPS誘導NOD2 mRNA表現的影響……………………………………………………
22
4-5 在LPS刺激樹突細胞成熟過程中抑制PDE4B可誘導NOD2基因的表現………………………………………....
23
4-6 LPS與PDE4對樹突細胞內TLR4基因表現的調控…… 23
4-7 在樹突細胞中剔除PDE4B可減緩LPS對TLR4基因表現的抑制作用………………………………………………
24
4-8 在LPS刺激樹突細胞成熟過程中PDE4對CXCR4 mRNA表現的影響………………………………………...
25
4-9 在LPS活化的樹突細胞中剔除PDE4B可增加CXCR4 mRNA的表現……………………………………………...
25
五、 討論………………………………………………………… 27
六、 圖與圖解…………………………………………………… 32
參考文獻 ……………………………………………………………… 43
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指導教授 金秀蓮(Shiow-Lian Catherine Jin) 審核日期 2019-1-23
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