臺灣博碩士論文加值系統

巨噬細胞的浸潤與活化在組織發炎的進程扮演一個相當重要的角色，特別是在肥胖相關的脂肪組織發炎，目前我們已經知道趨化素RANTES可以促進巨噬細胞的浸潤，而RANTES的接受器CCR5在巨噬細胞的活化也佔有重要的地位，但其調控的相關機轉還不是非常明瞭；在本實驗中，我們利用RAW cell小鼠巨噬細胞株及THP-1 cell人類巨噬細胞株，去探討RANTES是否會藉由影響葡萄糖攝取作用來調控巨噬細胞的浸潤，及影響巨噬細胞的活化。
實驗結果顯示，RANTES可以增加RAW cell的葡萄糖攝取作用，利用2-DG抑制巨噬細胞的葡萄糖攝取作用會降低RANTES促進的RAW cell巨噬細胞的移行作用；在細胞路徑的探討上，在觀察時間中，RANTES會增加p-AKT Thr308、p-AKT Ser473以及p-AMPK Thr172的磷酸化，以及增加GLUT1在細胞膜上的表現，p-p65-NF-κB以及p-ERK則是沒有顯著的變化，而抑制PI3K、AMPK以及PLC都會顯著地降低RANTES促進的巨噬細胞葡萄糖攝取上升以及巨噬細胞移行能力上升，在接受器的探討上，抑制CCR1、CCR3及CCR5都會抑制RANTES促進的巨噬細胞葡萄糖攝取上升，但只有CCR1及CCR5會抑制RANTES促進的巨噬細胞移行能力上升；更進一步的實驗結果顯示，抑制了PLC以及CCR5後，會降低RANTES促進的巨噬細胞葡萄糖攝取上升及巨噬細胞移行能力上升，也會降低p-AKT Thr308、p-AKT Ser473以及p-AMPK Thr172的磷酸化；在THP-1細胞上，RANTES可以增加M0、M1及M2的葡萄糖攝取作用，而抑制了PI3K、AMPK以及CCR5後，都會顯著地降低RANTES促進的葡萄糖攝取上升；在巨噬細胞的活化上，RANTES不會增加TNF-α、IL-6以及CD206的基因表現，而會增加IL-10的基因表現，另外在IL-6的分泌也沒有顯著的變化。
由目前結果顯示，在巨噬細胞，RANTES會藉由CCR5接受器，活化下游的PI3K/AKT、AMPK以及PLC路徑，使得細胞膜上的GLUT1表現上升，增加葡萄糖的攝取作用，最終影響了巨噬細胞的移行作用，而在巨噬細胞的活化上，RANTES沒有顯著的影響。

Macrophage infiltration and activation plays a critical role in the development of tissue inflammation, especially in obesity-associated adipose tissue inflammation. Chemokine RANTES (also known as CCL5) has been reported to promote macrophage infiltration and its receptor CCR5 is associated with macrophage activation, but the detailed mechanism is unclear. The aim of current study is to investigate the role of RANTES signaling in the regulation of macrophage migration and activation through RANTES-mediated glucose uptake.
The present result showed that RANTES increased macrophage migration and glucose uptake in a time and dose dependent manner in RAW cell model. Blockade of glucose uptake by 2-DG significantly inhibited RANTES -induced macrophage migration. RANTES could induce membrane GLUT1 expression as well as phosphorylation of AKT on Thr308、Ser473 and AMPK on Thr172 in RAW cells, but the phosphorylation of p65-NF-κB and ERK was unchanged during the observed period. In addition, inhibition of PI3K、 AMPK and PLC could significantly suppress RANTES-induced macrophage migration and glucose uptake. We also demonstrated that blockade of CCR1 and CCR5 but not CCR3 could inhibit RANTES -induced macrophage glucose uptake and also suppress RANTES -induced macrophage migration. Moreover, Inhibition of PLC and CCR5 could decrease the ability of macrophage migration and glucose uptake in a dose-dependent manner and decrease the phosphorylation of AKT on Thr308、Ser473 and AMPK on Thr172. In THP-1 macrophage cell line, RANTES could increase glucose uptake in M0、M1 and M2 type macrophage. Inhibition of PI3K、AMPK and CCR5 could suppress RANTES-induced macrophage glucose uptake. In respect of macrophage activation, there was not changed in the gene expression of TNF-α、IL-6 and CD206, but IL-10 gene expression was significantly increased after adding RANTES. Besides, the secretion of IL-6 which is M1 type marker was unchanged in treated RANTES group compared to untreated RANTES control.
Taken together, the present findings indicate that RANTES could be via CCR5 receptor to activate PLC signaling to increase phosphorylation of PI3K and AMPK thus increase GLUT1 membrane expression. As a result, macrophage glucose uptake was increased and then regulated macrophage migration. However, RANTES didn’t affect the process of macrophage activation.

目錄
圖目錄 IV
中文摘要 VII
Abstract IX
第一章、前言 1
第一節 肥胖的定義 1
第二節 巨噬細胞浸潤與肥胖 2
第三節 巨噬細胞活化與肥胖 3
第四節 趨化素(chemokine)與巨噬細胞浸潤 3
第五節 趨化素(chemokine)與巨噬細胞活化 5
第六節 RANTES (CCL5)與其接受器 5
第七節 RANTES (CCL5)路徑與巨噬細胞浸潤和活化 6
第八節 葡萄糖攝取作用與巨噬細胞浸潤和活化 7
第九節 假說 9
第二章、 實驗目的 10
第三章、材料與方法 11
第一節、實驗材料 11
第二節、儀器設備 11
第三節、化學製劑 12
第四節、實驗設計 15
第五節、實驗方法 17
RAW cell、 THP-1 cell培養及繼代 17
活化THP-1 human monocyte 17
葡萄糖攝取實驗 17
細胞移行實驗 18
mRNA基因表現測定 18
膜蛋白萃取 20
西方墨點法 21
細胞激素測定(ELISA) 24
第六節、統計方法 25
第四章、實驗結果 26
一、RANTES對於小鼠巨噬細胞株RAW 264.7葡萄糖攝取作用的影響 26
二、葡萄糖攝取作用對於小鼠巨噬細胞株RAW 264.7移行作用的影響 26
三、RANTES對於RAW 264.7 之AKT磷酸化之影響 27
四、RANTES對於RAW 264.7 之AMPK磷酸化之影響 27
五、RANTES對於RAW 264.7 之GLUT1表現之影響 28
六、PI3K/AKT及AMPK在RANTES訊息傳遞中可能的交互影響 29
七、RANTES對於RAW 264.7 之NF-κB及ERK表現之影響 29
八、PI3K/AKT及AMPK路徑在RANTES促進之巨噬細胞葡萄糖攝取上升及移行能力上升的機轉中所扮演的角色 30
九、CCR1、CCR3及CCR5在RANTES促進之巨噬細胞葡萄糖攝取上升及移行能力上升的機轉中所扮演的角色 30
十、PLC路徑在RANTES促進之巨噬細胞葡萄糖攝取作用上升及移行能力上升的機轉中所扮演的角色 31
十一、RANTES/CCR5路徑在RANTES促進之巨噬細胞葡萄糖攝取上升及移行能力上升的機轉中所扮演的角色 32
十二、RANTES/CCR5路徑對RANTES促進之AKT、AMPK磷酸化上升及膜上GLUT1表現上升的機轉中所扮演的角色 32
十三、PLC路徑對RANTES促進之AKT、AMPK磷酸化上升的機轉中所扮演的角色 33
十四、RANTES對於人類單核球細胞株THP-1分化之M0、M1、M2巨噬細胞葡萄糖攝取作用的影響 33
十五、PI3K/AKT及AMPK路徑在RANTES促進之THP-1分化之M0、M1、M2巨噬細胞葡萄糖攝取作用所扮演的角色 34
十六、RANTES/CCR5路徑在RANTES促進之THP-1分化之M0、M1、M2巨噬細胞葡萄糖攝取作用所扮演的角色 35
十七、RANTES對巨噬細胞活化的影響 35
第五章、討論 37
第六章、結論 48
參考文獻 49

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