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研究生:李怡儒
研究生(外文):Yi-Ru Li
論文名稱:Aciculatin對3T3-L1脂肪細胞的脂質生成及脂解作用之研究
論文名稱(外文):Effect of aciculatin on the adipogenesis and lipolysis in 3T3-L1 cells
指導教授:陳建志陳建志引用關係邱文慧邱文慧引用關係
指導教授(外文):Chien-Chih ChenWen-Fei Chiou
學位類別:碩士
校院名稱:弘光科技大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
畢業學年度:101
語文別:中文
論文頁數:78
中文關鍵詞:Aciculatin脂質生成脂解作用PPARγC/EBPαHSL
外文關鍵詞:AciculatinadipogenesislipolysisPPARγC/EBPαHSL
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Aciculatin是臺灣民間草藥竹節草 (Chrysopogon aciculatis) 的主要化學組成之一,本論文研究目的是為了探討aciculatin對於脂肪細胞的脂質生成以及脂解作用的影響,並探討其可能的分子作用機制。利用含有3-isobutyl-1-methylxanthine、dexamethasone和insulin的 (MDI) 分化劑刺激3T3-L1細胞分化為成熟脂肪細胞,可以偵測到明顯增加細胞內三酸甘油酯的堆積、分化相關轉錄因子的蛋白(C/EBPβ、C/EBPα和PPARγ) 表現以及mRNA (C/EBPα和PPARγ)的表現量。而這些脂質生成的活化指標皆可以被濃度相關性地aciculatin 所抑制,顯示aciculatin可有效抑制脂肪細胞的分化。
另外,在培養成熟脂肪細胞的期間,將β-腎上腺性致效劑(β-adrenergic agonist) isoproterenol或aciculatin與成熟脂肪細胞共同培養,均可以明顯增加脂解產物的釋放,甘油及游離脂肪酸釋出到培養基中,顯示aciculatin可以刺激脂肪細胞進行基礎的脂肪分解。西方點墨法分析結果發現在3T3-L1細胞幾乎偵測不到 (hormone sensitive lipase;HSL) 蛋白質,然而在成熟脂肪細胞中卻大量的表現HSL蛋白,顯示飽含油滴 (三酸甘油酯) 的脂肪細胞才會需要HSL來進行脂解作用;若將成熟脂肪細胞與isoproterenol或aciculatin共同培養,發現可輕微增加原態HSL蛋白的表現。進一步分析p-HSL表現,相較於未分化細胞,分化九天的成熟脂肪細胞可以表現出少量p-HSL,然而在isoproterenol的刺激下,顯著提高p-HSL的表現量,顯示此p-HSL蛋白質與脂解作用關係密切。有趣的是aciculatin同樣可以顯著提高p-HSL,且呈現濃度相關性。
綜合上述結果顯示,aciculatin一方面可藉由抑制相關轉錄因子(C/EBPβ、C/EBPα和PPARγ) 的表現而抑制脂肪細胞分化,另一方面還能透過活化p-HSL蛋白來刺激成熟的脂肪細胞進行脂肪分解,更詳細的作用機制有待進一步深入研究。

關鍵字:Aciculatin、脂質生成、脂解作用、PPARγ、C/EBPα、HSL


Aciculatin is the main constitute existed in Chrysopogon aciculatus. This study is aimed to investigate the effects of aciculatin on the adipogenesis and lipolysis and further elucidate the possible mechanisms of action. Experiment is conducted in 3T3-L1 cells cultured with medium containing 3-isobutyl-1-methylxanthine, dexamethasone and insulin (MDI) to induce adipocyte differentiation. Significant accumulation of triglyceride and increased expression of transcription factors both at protein (C/EBPβ, C/EBPα and PPARγ) and mRNA (C/EBPα and PPARγ) levels were observed in mature adipocyte and such responsiveness were clearly repressed in the presence of aciculatin concentration- dependently. Basal release of glycerol and free fatty acid to culture medium was detected in mature adipocyte. Both the -adrenergic agonist isoproterenol and aciculatin significantly accelerated the release of above lipolytic metabolites.Results showed that none detectable level of HSL was observed in 3T3-L1 cells. Nevertheless, a dramatically increased in HSL protein expression was observed in mature adipocyte after differentiated for 9 day. Western blot analysis also demonstrated that mature adipocyte had higher level of phosphorylated HSL when compared to 3T3-L1 cells. In the presence of isoprorerenol, protein level of phosphorylated HSL was significantly increased suggesting activated HSL may participate in regulation of lipolysis. Interesting, same phenomenon was observed when cells treated with aciculatin. In conclusion, the results showed that aciculatin was able to inhibit preadipocyte differentiation via inhibiting the expressions of transcription factors characteristic to adipocyte and also stimulate lipolysis probably mediated by activation of HSL phosphorylation and will further understanding of its mechanism.

Keywords: Aciculatin; adipogenesis; lipolysis; PPARγ; C/EBPα; HSL

中文摘要...................................................................................................1
英文摘要...................................................................................................2
縮寫對照表...............................................................................................3
第一章 緒論.............................................................................................4
第二章 文獻回顧.....................................................................................6
第一節、脂質生成................................................................................6
第二節、脂肪細胞的分化時期.............................................................8
一、細胞增殖期 (cell proliferation) ................................................8
二、細胞接觸性生長期抑制/生長停滯期 (cell contact inhibition/ growth arrest) .................................................................................8
三、細胞擴張期 (clonal expansion)...............................................9
四、永久性生長停滯期 (permanent growth arrest)........................9
五、脂質堆積期 (lipid accumulation) …....…………...................9
第三節、調控脂肪細胞生長分化之轉錄因子…..............................10
一、ADD1/SREBP1………………………………...……………10
二、C/EBP family…………………………………...……………11
三、PPAR family ……………………………….......……………12
第四節、脂質分解…………..………………………………………13
第五節、竹節草Chrysopogon aciculatis介紹……….……………16
第三章 研究目的……………....……………………………………17
第四章 材料與方法………….……………………………………18
第一節、實驗材料…………....……………………………………18
一、實驗樣品……..………………………………………………18
二、實驗藥品與試劑..……………………………………………19
三、實驗儀器………..……………………………………………22
四、實驗耗材………..……………………………………………22
第二節、實驗方法..…………………………………………………23
一、3T3-L1前驅脂肪細胞解凍與保存………….………………23
二、3T3-L1前驅脂肪細胞繼代培養……….……………………23
三、脂肪細胞脂質生成之實驗方法……………..………………24
1. 3T3-L1前驅脂肪細胞分化培養…………......……………24
2. Aciculatin對3T3-L1細胞之毒性分析 (MTT assay)..…..24
3. 油紅染色測定 (Oil Red O stain) ………..………………25
4. 細胞內三酸甘油脂含量分析 (Triglyceride assay) ………26
5. 蛋白質的萃取與定量……………...………………………27
6. 西方墨點法 (Western Blot)………………………………27
7. 萃取Total RNA與即時定量PCR (Quantitative Real
Time PCR) ……………………........…………………………28
四、脂肪細胞脂質分解之實驗方法…………..…………………30
1. 成熟脂肪細胞之獲得………...............…………………30
2. 細胞存活率分析 (MTT assay)....…….…………………30
3. 油紅染色測定 (Oil Red O stain)................….…………31
4. 細胞外甘油釋出含量分析 (Glycerol release assay) ........31
5. 游離脂肪酸含量分析 (Free fatty acid assay)..................32
五、統計分析……………………....….………………….............33
第五章 結果…………………………..……………………………34
第一節、Aciculatin對於脂肪細胞脂質生成之影響…..……...........34
一、Aciculatin對於3T3-L1前驅脂肪細胞分化之影響以及細胞毒性分析…………….....…………………….......………………34
二、Aciculatin對3T3- L1前驅脂肪細胞分化後細胞內三酸甘油 酯堆積之影響………............……………………………..34
三、Aciculatin對脂質生成相關轉錄因子蛋白質表現量之影響..…………………………........………………………………..35


四、Aciculatin對脂質生成相關轉錄因子mRNA表現量之影
響..…………………………........………………………………..36
第二節、Aciculatin對於脂肪細胞脂解作用之影響......................37
一、Aciculatin對於成熟脂肪細胞內三酸甘油酯含量之影響以及
細胞毒性分析…………….……………………………………37
二、Aciculatin對於脂肪分解產物之影…………………………37
三、Aciculatin對於脂解酶HSL蛋白質表現之影響……………38
第六章 討論…………………………………………………………40
第一節、Aciculatin對於脂肪細胞脂質生成之作用機制…….........40
第二節、Aciculatin對於脂肪細胞脂解作用之機制.........................41
第七章 結論.......................................................................................44
第八章 參考文獻.................................................................................45
第九章 表與圖.........................................................................51



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