臺灣博碩士論文加值系統

肥胖乃因身體的能量平衡失常造成。當能量吸收大過於能量消耗，過剩的能量以三酸甘油脂的形式堆積在脂肪細胞內，導致脂肪細胞體積過大，同時也影響了脂肪細胞正常的分泌功能，使得脂肪細胞無法正常的分泌調控身體內能量平衡的激素，以及發炎因子的大量分泌造成慢性發炎的情況產生，也因此促成了惡性的循環。之前研究指出細胞膜上具有一種富含膽固醇的結構，稱為lipid raft，對於細胞的胞吞作用、分泌和訊息傳遞上扮演重要的角色，但是其相關機制並不清楚；而在肥胖造成體積過大的脂肪細胞則有細胞膜上的膽固醇含量減少的情況產生。結合以上的結果，我們推論脂肪細胞的細胞膜上膽固醇的減少，可能造成肥胖時的脂肪細胞分泌功能不正常的原因之一。
我們利用一種對膽固醇具有高度親和力的藥物methyl--cyclodextrin (MCD)處理脂肪細胞，模擬脂肪細胞肥大時細胞膜膽固醇含量下降的情況，觀察其對於脂肪細胞瘦素和趨化素 monocyte chemoattractant protein 1 (MCP-1)分泌的影響。實驗結果發現，處理MCD會減少脂肪細胞瘦素的分泌，也會降低胰島素訊息傳遞當中Akt的磷酸化；相反地，減少細胞膜上的膽固醇，會增加MCP-1的分泌，並且增加ERK/MAPK磷酸化，暗示發炎反應的上升。
由於之前文獻指出lipid raft結構之一的caveola在脂肪細胞的訊息傳遞上扮演著一定的角色存在，於是我們利用RNA interference 減少內生性caveola的結構蛋白caveolin-1來觀察其對脂肪細胞分泌的影響。由實驗結果發現，減少caveolin-1也會減少脂肪細胞瘦素分泌，但是並不影響MCP-1的分泌。因此，減少細胞膜上的膽固醇可能會影響著脂肪細胞的分泌功能。希望未來實驗進一步了解其作用機制，以利將來可能成為一個治療相關疾病的新方向。

Obesity is caused by the disorders of body energy balance. Excess energy accumulates in the form of triglycerides in adipocytes when energy absorption is more than energy depletion, not only resulting in increased adipocyte volume, but also leading to the aberrant secretion of adipokines, the cytokines secreted by adipocytes. Lipid raft is a membrane structure enriched in cholesterol. It plays an important role in cellular endocytosis, secretion and signal pathway. However, its role in adipokine secretion remains unknown. Moreover, hypertrophied adipocytes were characterized by decreased membrane cholesterol in obesity. Therefore, we hypothesize that reduction of cholesterol in the plasma membranes of adipocytes, which leads to aberrant function of lipid raft, may cause abnormal function of adipokine secretion in obesity.
We applied methyl--cyclodextrin (MCD), a drug with high affinity for cholesterol, on differentiated 3T3-L1 adipocytes to reduce the cholesterol content of plasma membrane and examined the secretion of adipokines. We found that MCD treatments reduced basal and insulin-stimulated leptin secretion. MCD treatments also reduced insulin-stimulated Akt phosphorylation. In contrast, MCD treatment increased the secretion of a chemokine monocyte chemoattractant protein 1 (MCP-1), and also increased phosphorylation of extracellular signal-regulated kinases.
Caveola, a kind of lipid raft, plays an role in signal pathway in adipocytes in the previous studies. We depleted endogenous caveolin-1, a structure protein of caveola, by RNA interference and investigated adipocyte secretion. We found that decreased caveolin-1 levels reduced leptin secretion but had no effect on MCP-1 secretion.
Taking together, reducing membrane cholesterol by MCD treatment may influence secretory function in adipocytes. Further experiments will be required to elucidate the mechanism, so as to provide new directions for treating metabolic diseases in the future.

指導教授推薦書
口試委員會審定書
授權書 iii
誌謝 iv
中文摘要 v
Abstract vii
目錄 ix
圖表目錄 xi

緒論 - 1 -
肥胖與代謝性症候群 (Metabolic Syndrome and Obesity) - 1 -
脂肪細胞 (Adipocytes) - 2 -
肥胖、脂肪細胞與能量代謝 (Obesity, Adipocytes and Energy Balance) - 4 -
脂肪細胞與脂肪激素 (Adipocytes and Adipokines) - 5 -
脂肪激素與能量代謝 (Adipokine and Energy Metabolism) - 6 -
肥胖、瘦素與能量代謝 (Obesity, Leptin and Energy Metabolism) - 7 -
發炎反應與肥胖 (Inflammation and Obesity) - 8 -
單核球趨化蛋白-1與發炎反應 (MCP-1 and Inflammation) - 9 -
細胞膜之膽固醇與脂筏 (Membrane Cholesterol and Lipid Raft) - 10 -
動機 (Rationale) - 12 -
研究方向 (Specific Aims) - 13 -
材料與方法 - 14 -
細胞培養及分化 (cell culture and differentiation) - 14 -
Protein Assay - 14 -
Leptin ELISA (Leptin Enzyme-linked immunosorbent assay) - 15 -
MCP-1 ELISA (MCP-1 Enzyme-linked immunosorbent assay) - 16 -
西方點墨法（Western blotting） - 17 -
電穿孔（Electroporation） - 19 -
RNA萃取（RNA extraction） - 19 -
反轉錄（Reverse transcription） - 20 -
即時定量PCR（Real time PCR） - 21 -
MTT assay - 21 -
實驗結果 - 22 -
處理MCD使3T3-L1脂肪細胞leptin分泌的降低 - 22 -
處理MCD使胰島素的訊息傳遞受到影響 - 23 -
處理MCD降低3T3-L1脂肪細胞的leptin mRNA - 23 -
處理MCD使3T3-L1脂肪細胞基本的MCP-1分泌上升 - 24 -
處理MCD增加3T3-L1脂肪細胞的MCP-1 mRNA - 25 -
處理MCD使3T3-L1脂肪細胞內的MCP-1蛋白表現量上升 - 26 -
處理MCD會增加ERK的磷酸化 - 27 -
Knockdown of caveolin-1 - 29 -
減少caveolin-1對脂肪激素分泌的影響 - 29 -
討論 - 31 -
細胞膜上膽固醇對3T3-L1脂肪細胞分泌leptin之影響 - 31 -
細胞膜上膽固醇對3T3-L1脂肪細胞分泌MCP-1之影響 - 33 -
Lipid raft當中的caveolae是否參與脂肪細胞的分泌 - 37 -
參考資料 - 39 -

圖表目錄
Table.1 處理MCD對leptin的影響 - 42 -
Table.2 處理MCD對MCP-1的影響 - 43 -
Table.3 knockdown caveolin-1對adipokines的影響 - 44 -
Fig.1 處理24小時MCD 2.5 mM降低3T3-L1脂肪細胞leptin的分泌 - 45 -
Fig.2 處理MCD 2.5 mM影響胰島素的訊息傳遞 - 46 -
Fig.3 處理MCD 2.5 mM降低3T3-L1脂肪細胞leptin mRNA - 47 -
Fig.4 處理MCD 2.5 mM增加3T3-L1脂肪細胞MCP-1的基本分泌 - 48 -
Fig.5 處理MCD 2.5 mM 增加3T3-L1脂肪細胞MCP-1 mRNA - 49 -
Fig.6 處理MCD 2.5 mM四小時會增加3T3-L1脂肪細胞MCP-1的蛋白表現量 - 50 -
Fig.7 處理MCD 2.5 mM二十四小時會增加3T3-L1脂肪細胞MCP-1的蛋白表現量 - 51 -
Fig.8 處理MCD 2.5 mM促進3T3-L1脂肪細胞ERK的磷酸化 - 52 -
Fig.9 處理MCD 2.5 mM透過ERK-MAPK增加MCP-1的分泌 - 53 -
Fig.10 利用RNA interference 減少內生性caveolin-1 的表現 - 54 -
Fig.11 減少caveolin-1的表現量會降低leptin的分泌 - 55 -
Fig.12 降低caveolin-1的表現量不影響MCP-1的分泌 - 56 -

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