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研究生:潘俞文
研究生(外文):Yu-Wen Pan
論文名稱:甘藍及發酵甘藍萃取物對3T3-L1 前脂肪細胞生長和分化作用之影響
論文名稱(外文):The effect of cabbage and fermented cabbage extracts on growth and adipogenesis in 3T3-L1 preadipocytes
指導教授:徐源泰徐源泰引用關係
指導教授(外文):Yuan-Tay Shyu
口試委員:謝淑貞曾文聖吳思節
口試日期:2013-07-11
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:園藝暨景觀學系
學門:農業科學學門
學類:園藝學類
論文種類:學術論文
論文出版年:2013
畢業學年度:101
語文別:中文
論文頁數:92
中文關鍵詞:甘藍發酵甘藍肥胖3T3-L1 細胞分化作用
外文關鍵詞:CabbageFermented cabbageObesity3T3-L1 cellDifferentiation
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甘藍(Brassica oleracea L. var. capitata L.)富含機能性成分,是日常生活中不可或缺的蔬菜,傳統上常有產銷失衡情況。本研究以甘藍及發酵甘藍為材料進行水萃及甲醇萃取所得四種萃取物 (CW、FCW、CM、FCM),處理 3T3-L1前脂肪細胞,分析是否具有促進細胞凋亡以減少脂肪細胞數目,或可抑制前脂肪細胞分化成脂肪細胞以減少脂肪細胞體積。期望增加甘藍應用性。實驗分成三部分,第一部分測定萃取物處理 3T3-L1前脂肪細胞後其存活率,評估萃取物對細胞影響,處理後細胞存活率均大於 80%,選用 1、2、4 mg/mL 濃度處理之後實驗。第二部分測定萃取物是否可誘導 3T3-L1前脂肪細胞凋亡,四種萃取物中又以甘藍水萃物 (CW)增加細胞凋亡率較明顯,當濃度由 0 升至 4 mg/ml 細胞凋亡率從4.78±0.57% 提高至 8.46±1.56%;整體而言四種萃取物並沒有明顯促進 3T3-L1前脂肪細胞凋亡,顯示無法經由誘導 3T3-L1 前脂肪細胞凋亡而減少前脂肪細胞數目。第三部分測定萃取物是否可抑制前脂肪細胞分化成脂肪細胞,油紅染色結果顯示四種萃取物皆可抑制油滴累積,並可降低胞內三酸甘油脂含量,又以發酵甘藍水萃物 (FCW)效果最顯著,當濃度為 4 mg/ml 可降低 93.64% 三酸甘油脂含量;此部分顯示萃取物具有抑制前脂肪細胞分化作用。進一步探討萃取物是否經由調節分化轉錄因子 PPARγ 和 C/EBPα 的 mRNA 表現而抑制前脂肪細胞分化作用,結果顯示以發酵甘藍水萃取物濃度 4 mg/ml 處理分化三、六、九天細胞,分別會降低26.98% 、 47.87%、 50.33% C/EBP之 mRNA 表現量。實驗結果顯示發酵甘藍水萃物具有發展成為天然抗肥胖材料的潛力。

Cabbage (Brassica oleracea L. var. capitata L.) is rich in functional compounds, and it is one of the most important vegetables in our daily life. In recent years, market imbalance on supply and demand of cabbage has led to unstable price of cabbage. In this study, cabbage and fermented cabbage were used as a materials for water and methanol extraction to obtain four different extracts (CW-Cabbage Water extracts、FCW-Fermented Cabbage Water extracts、CM-Cabbage Methanol extracts、FCM-Fermented Cabbage Methanol extracts). 3T3-L1 preadipocyte treated with four cabbage extracts to determine their functionality on fat cells or inhibition, i.e. the blockage on the differentiation of preadipocytes into adipocytes. Hope this finding would be useful for increasing the application of cabbage. The experiment was divided into three parts. At the first part, we evaluated the impact of extracts on 3T3-L1 preadipocyte viability, it was observed cell viability greater than 80% and further optimized three concentration 1, 2, 4 mg/mL for further experiments. At the second part, to find out extracts effectiveness on 3T3-L1 preadipocyte apoptosis, we applied extract concentrations from 0 to 4 mg/mL, the apoptosis rate increased from 4.78±0.57% to 8.46±1.56%. Overall, treatment with four extracts did not increase 3T3-L1 preadipocyte apoptosis rate significantly, which imply that extracts was not effective to reduce preadipocyte number by promoting cell apoptosis. At the third part, we have tested the effects of extracts on differentiation of preadipocyte into adipocyte. Oil red o staining indicated that the cell treated with four extracts displayed reduced formation of fat droplets and accumulation of intracellular triglyceride. Among the extracts, FCW gave a great reduction up to 93.46% of intracellular triglyceride content when cells treated with 4 mg/mL. Subsequently, determined whether the extracts inhibit the differentiation of preadipocyte by regulating the mRNA expression of transcription factors(PPARγ、C/EBPα). The results showed that treatment with 4 mg/mL of water extract from fermented cabbage down-regulated the mRNA expression of C/EBPα by 26.98%, 47.87% and 50.33%, respectively. These results are found positive for potential application as a natural anti-obesity food.

中文摘要……………………………………………………………………………….I
英文摘要……………………………………………………………….........................II
目錄……………………………………………………………………………………IV
圖目錄…………………………………………………………………………………VII
表目錄………………………………………………………………............................X
壹、 前言…………………………………………………………………………….1
貳、 文獻回顧
一、 甘藍
(一) 甘藍簡介…………………………………………………………2
(二) 甘藍中機能性成份………………………………………………3
(三) 甘藍功效…………………………………………………………7
二、 肥胖
(二) 肥胖的定義………………………………………………………8
(三) 治療肥胖的方法…………………………………………………9
三、 脂肪細胞
(一) 脂肪細胞的特性與種類………………………………………...21
(二) 前脂肪細胞……………………………………………………...21
(三) 3T3-L1脂肪細胞………………………………………………..22
(四) 脂肪細胞生命週期及分化過程………………………………...23
(五) 調節脂肪細胞轉錄因子介紹…………………………………...26
参、材料與方法
一、 實驗目的……………………………………………………………….29
二、 實驗架構……………………………………………………………….29
三、 細胞培養
(一) 細胞繼代………………………………………………………….30
(二) 細胞低溫保存(凍管)……………………………………………. 32
(三) 細胞活化………………………………………………………….33
(四) 細胞分化………………………………………………………….34
四、 樣品製備
(一) 材料萃取前製備…………………………………………………35
(二) 材料萃取……………………………………................................36
五、 樣品分析
(一) 3T3-L1前脂肪細胞存活率之測定……………………………..37
(二) 3T3-L1前脂肪細胞凋亡率之測定……………………………..38
(三) 3T3-L1前脂肪細胞分化率之測定-Oil red O 染色…………...39
(四) 3T3-L1前脂肪細胞分化率之測定-Triglyceride 含量測定…...40
(五) 3T3-L1前脂肪細胞分化率之測定-抽取RNA………………...42
(六) 3T3-L1前脂肪細胞分化率之測定-反轉錄聚合酶鏈反應……43
(七) 3T3-L1前脂肪細胞分化率之測定-即時定量聚合酶鏈反應…44
六、 統計分析……………………………………………………………….45
肆、結果與討論
一、萃取物之萃取率…………………………………………….....................46
二、3T3-L1前脂肪細胞存活率之測定(MTS 試驗)………………………...48
三、3T3-L1前脂肪細胞凋亡率之測定………………………………………54
四、3T3-L1前脂肪細胞分化率之測定………………………………………65
(ㄧ) 前脂肪細胞分化成脂肪細胞過程……………………………….65
(二) Oil red O染色…………………………………………………..65
(三) 三酸甘油脂含量………………………………………................66
(四) 調節分化之轉錄因子mRNA表現量…………………………..68
伍、總結………………………………………………………………………………80
陸、參考文獻…………………………………………………………………………81


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