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研究生:莊淳涵
研究生(外文):JHUANG, CHUN-HAN
論文名稱:探討棕色脂肪細胞中第三型血清澱粉樣蛋白在小鼠攝食誘導的產熱機轉中所扮演的角色
論文名稱(外文):The Role of Brown Adipocyte Derived Serum Amyloid A3 Protein in Regulation of Diet-induced Thermogenesis in Mice
指導教授:謝博軒
指導教授(外文):HSIEH PO-SHIUNA
口試委員:郁兆蘭王志豪阮琪昌
口試委員(外文):YU CHAO-LANWANG CHIN-HAOJUAN,CHI-CHANG
口試日期:2022-05-11
學位類別:碩士
校院名稱:國防醫學院
系所名稱:生理學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2022
畢業學年度:110
語文別:中文
論文頁數:92
中文關鍵詞:適應性產熱第三型血清澱粉樣蛋白棕色脂肪細胞攝食誘導的產熱作用
外文關鍵詞:Serum amyloid A3Diet-induced thermogenesisBrown adipose tissue
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近年研究發現,棕色脂肪組織除冷刺激外也參與攝食誘導的適應性產熱作用。本實驗利用專一性棕色脂肪細胞剔除SAA3小鼠(UCP1-SAA3基因剔除鼠)來探討SAA3是否參與小鼠的攝食誘導產熱作用及影響總能量消耗,最後利用棕色脂肪前驅細胞株 (WT-1)及全身性剔除SAA3小鼠的初級棕色脂肪 細胞 探討其機轉。動物實驗結果 顯示UCP1-SAA3基因剔除鼠與對照組小鼠相比, 在空 腹時期能量消耗無顯著差異,但在隨意餵食和空腹後再餵食中,總能量消耗及攝食誘導的產熱作用皆顯著降低。此外,我們發現再餵食後 UCP1-SAA3基因剔除鼠的棕色脂肪組織的 UCP1產熱蛋白表現降低,吸收組織外游離脂肪酸和葡萄糖功能增加且脂肪酸生合成途徑亦增加。然而 三酸甘油脂含量顯著 降低 且脂解作用沒有顯著差異 。 高脂飼料餵食十四天後的 UCP1-SAA3基因 剔除 鼠 不僅體重顯著高於對照組小鼠,其總能量消耗 顯著低於對照組與其正常飼料組別 。 WT-1細胞在無血清或不同
葡萄糖和胰島素之培養基中放置 24小時後結果顯示胰島素濃度可顯著誘發 SAA3和UCP1蛋白表現量皆增加,但利用全身性剔除SAA3小鼠的初代細胞中,再給予胰島素後,UCP1表現量並無增加 。
根據目前實驗結果顯示, 飯後血中胰島素濃度升高可能經由刺激 棕色脂肪細胞SAA3活性參與小鼠 飯後攝食誘導的產熱作用,其機轉可能與調控棕色脂肪組織中的脂解作用 及 UCP1依賴 適應性產熱作用有關進而影響總能量消耗。
Recent studies have reported that brown adipose tissue (BAT) is not only participates cold-induced thermogenesis but also involved in diet-induced thermogenesis (DIT). In this experiment, the specific brown adipocyte knockout SAA3 mice (UCP1-SAA3 knockout mice) were used to explore whether SAA3 participates DIT in mice and total energy expenditure (TEE). Finally, we use brown adipocyte precursor cell line (WT-1) and primary brown adipocytes from SAA3 global knockout mice to elucidate the possible underlying mechanism.
The results of animal experiments showed that UCP1-SAA3 knockout mice had no significant difference compared with control mice in energy consumption during the fasting period, but in ad libitum feeding and fasting then refeeding, TEE and DIT were significantly reduced. Moreover, we found that the BAT of UCP1-SAA3 knockout mice showed the thermogenic protein UCP1 expression decreased, and BAT uptake free fatty acids and glucose significantly increase and significantly increased fatty acid synthesis pathway after refeeding. In vitro experiments,WT-1 cells were placed in serum-free、 high or low glucose medium and insulin for 24 hours, the datas showed that insulin could increase the expression of SAA3 and UCP1 proteins. However, after insulin administration, the expression of UCP1 did not increase in SAA3 global knockout mice primary brown adipocyte.
Collectively, it is suggested that postprandial hyperinsulinemia could promote brown adipocyte SAA3-mediated adaptive thermogenesis through regulating lipolysis and UCP1-dependent adaptive thermogenesis in BAT and eventually contributes to the regulation of energy balance.
目錄
圖目錄 IV
縮寫表 VI
摘要 VIII
ABSTRACT IX
第一章 、前言 1
第一節 、 體重控制與能量平衡 1
第二節 、 總能量消耗的組成和調控機轉 2
壹、 總能量消耗的組成 2
貳、 總能量消耗的調控機轉 3
第三節 、攝食誘導的產熱作用與肥胖的發展 4
第四節 、棕色脂肪組織在攝食誘導產熱作用中所扮演的角色 5
第五節 、棕色脂肪組織參與食物誘導產熱作用和可能的調控機制 6
第六節 、SAA3與棕色脂肪細胞的產熱作用 7
壹、 血清澱粉樣蛋白3 7
貳、 第三型血清澱粉樣(SAA3)蛋白在棕色脂肪組織中的角色 8
第七節 、假說 8
第二章 、實驗目的 9
第三章 、材料與方法 10
第一節 實驗材料 10
壹、 實驗動物與細胞系 10
貳、 儀器設備 11
參、 化學製劑 12
肆、 實驗試劑與配方 15
第二節 、實驗設計 16
壹、 動物實驗 16
貳、 細胞實驗 20
第三節 、 實驗方法 21
壹、 全身能量代謝測量 21
貳、 動物採血與犧牲流程 21
參、 小鼠棕色脂肪前驅細胞株 WT-1 培養 22
肆、 小鼠初代棕色脂肪前驅細胞培養 23
伍、 mRNA 基因萃取與測定 23
陸、 動物棕色脂肪組織和棕色脂肪細胞的蛋白質萃取及分析 25
柒、 組織三酸甘油脂含量測定 28
捌、 胰島素測定 29
玖、 數據分析 29
第四章 、研究結果 30
第一節 、禁食與再餵食對棕色脂肪組織中 SAA3蛋白和產熱蛋白UCP1表現量的影響 30
第二節 、UCP1-SAA3 KO組織特異性剃除基因小鼠,其脂肪組織中的SAA3蛋白表現及其他SAA亞型基因表現 31
第三節 、UCP1-SAA3 KO小鼠與對照組小鼠之生長曲線與進食熱量和食物利用率 31
第四節 、UCP1-SAA3 剔除在隨意餵食與禁食下之能量代謝的影響 32
第五節 、UCP1-SAA3 KO小鼠與對照組小鼠在攝食誘導產熱作用中能量代謝的差異 33
第六節 、UCP1-SAA3 KO小鼠與對照組小鼠之攝食誘導產熱作用占總能量消耗及能量攝入的比例 34
第七節 、棕色脂肪組織SAA3再餵食2小時後,小鼠組織重量及棕色脂肪組織三酸甘油脂含量 35
第八節 、棕色脂肪組織SAA3空腹後再餵食2小時後,棕色脂肪組之產熱蛋白、游離脂肪酸轉運蛋白和脂解作用蛋白表現 36
第九節 、棕色脂肪細胞SAA3影響小鼠再餵食兩小時後棕色脂肪組織的產熱作用與脂質代謝 36
第十節 、UCP1-SAA3 KO小鼠與對照組小鼠在不同飲食型態中體重增加量、熱量攝取量和食物利用率 37
第十一節 、UCP1-SAA3 KO小鼠與對照組小鼠在不同飲食型態中的代謝差異 38
第十二節 、在離體模式中胰島素對棕色脂肪細胞SAA3誘導的產熱作用的影響 39
第十三節 、UCP1KO小鼠與對照組小鼠在給予葡萄糖後血中血糖和胰島素表現 40
第十四節 、UCP1KO小鼠與對照組小鼠在給予葡萄糖後棕色脂肪組織SAA3與產熱相關蛋白表現 41
第五章 、討論 42
第一節 、攝食誘導的產熱作用中,產熱蛋白UCP1和SAA3之關係 42
第二節 、對照組小鼠與UCP1-SAA3KO小鼠在空腹和自由進食狀態下期間總能量消耗差異所代表的意義 43
第三節 、棕色脂肪細胞中SAA3影響小鼠攝食誘導產熱作用的可能因素 44
第四節 、剔除SAA3基因可能誘發UCP1不依賴產熱途徑 45
第五節 、營養成分對攝食誘導的產熱作用影響 46
第六節 、SAA3促進的攝食誘導的產熱作用會受到飯後血中胰島素濃度升高影響 47
第七節 、脂質與葡萄糖攝食對棕色脂肪細胞SAA3表現之影響 48
第八節 、探討SAA3在體重控制和能量代謝上可能的角色和臨床應用價值 48
第九節 、實驗限制 49
第六章 、結論 50
參考文獻 51
實驗結果附圖 58


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