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研究生:高偉智
研究生(外文):WEI-JHIH GAO
論文名稱:等鞭金藻(Isochrysis galbana tml)脂肪酸萃取物肥胖抑制機制之生化探討
論文名稱(外文):Studies on biochemical mechanism of anti-obesity ability of Isochrysis galbana tml fatty acid extracts
指導教授:吳希天
指導教授(外文):Hsi -Tien Wu
學位類別:碩士
校院名稱:國立嘉義大學
系所名稱:生物農業科技學系研究所
學門:農業科學學門
學類:農業技術學類
論文種類:學術論文
論文出版年:2019
畢業學年度:107
語文別:中文
論文頁數:159
中文關鍵詞:海洋微藻脂肪酸萃取物等鞭金藻肥胖
外文關鍵詞:MMFAIG-MMFAobesity
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非酒精性脂肪性肝病(non-alcoholic fatty liver disease, NAFLD)是一種常見的慢性疾病,其涵蓋範圍非常廣,從初期的脂肪變性到非酒精性脂肪性肝炎(non- alcoholic steatohepatitis, NASH)再進一步可導致肝纖維化及肝硬化,最終走向肝癌及死亡。超重、肥胖、糖尿病及老年人都屬於NAFLD高危險群,除了飲食控制與減重外,目前市面上有許多體重控制相關保健食品及藥物,如omega-3脂肪酸,為其中選項之一,且有研究證實可降低血脂及降低脂肪肝之進程。為分析海洋微藻脂肪酸萃取物(marine microalgae fatty acid, MMFA)作為抗肥胖之保健食品能力之前導實驗,利用HepG2細胞進行肝臟毒物硫代乙醯胺(thioacetamide, TAA)傷害,從而揀選出最適之MMFA進行動物實驗。結果顯示: 牟氏角毛藻(Chaetoceros muelleri, CM)、中肋骨條藻(Skeletonema costatum, SC)、等鞭金藻(Isochrysis galbana tml, IG)、魏氏海鏈(Thalassiosira weissflogii, TW)四種海洋微藻之MMFA具有清除自由基能力,且濃度與清除能力成正相關。MMFA可保護HepG2細胞免於TAA所造成的氧化壓力,與控制組相比,細胞提升了存活率、細胞內ROS濃度降低且CAT、SOD1、TNF及BCL2之mRNA表現恢復至與控制組相似且無顯著差異。綜合細胞之實驗數據,揀選IG藻進行後續實驗。動物實驗方面,利用C57BL/6J-Narl品系小鼠,飼餵含40%總脂肪之高脂飼料使其肥胖,並同時每日管餵IG-MMFA (125 mg/kg BW),為期四週,並檢測IG-MMFA對於肥胖進程的抑制效果。結果顯示,除血脂中的三酸甘油酯(triglycerides)些微下降,在生理學及病理分析中,體重增加幅度、腹股溝脂肪累積及BMI數值均減緩並與控制組無顯著差異,肝臟切片之脂肪累積及脂肪變性情形獲得改善,在基因層次分析中發現,由高脂飲食誘導肝臟之Cat、Il6、Tnf、Nfkb、Bax、Pparg及Acac表現量均回復至與控制組相同水平;心臟基因表現方面,由高脂飲食誘導之Cat、Tnf、Nfkb、Bax、Casp3及Ppara表現量均回復至與控制組相同水平;脂肪基因表現方面,由高脂飲食誘導之Cat、Il6、Tnf、Nfkb、Bax、Casp3、Ppara、Pparg、Cpt1a、Acox表現量均回復至與控制組相同水平。以上結果證實,IG-MMFA在肝臟、心臟及脂肪組織會抑制高脂飲食所誘導之氧化壓力、發炎反應及促凋亡反應,且脂肪酸代謝之相關基因獲得相對應改善。蛋白質表現部分在肝臟及心臟組織之CPT1及TNF蛋白質表現,得到與其mRNA表現相同之結果且達到顯著差異。值得注意的是,在單獨管餵IG-MMFA情況下,發現具有促進脂肪細胞發炎及提升肝臟脂肪酸代謝基因等情形。這些結果支持IG-MMFA作為抗肥胖保健食品之潛在能力,可提供後續食品開發、飼料添加劑等研究方向之基礎。
Non-alcoholic fatty liver disease (NAFLD) is a common chronic disease that covers a wide range of areas from early steatosis to nonalcoholic steatohepatitis (NASH), can lead to liver fibrosis and cirrhosis, eventually leading to liver cancer and death. Overweight, obesity, diabetes and the elderly are among the high-risk groups of NAFLD. In addition to diet control and weight loss, there are many healthy foods and drugs on the market, such as omega-3 fatty acids, and studies have confirmed that it can lower blood fat and reduce the progression of fatty liver. In order to analyze the marine microalgae fatty acid extract (MMFA) capable as an anti-obesity health food, HepG2 cells were used to detect liver toxic damage by thioacetamide (TAA), then selected the most suitable MMFA for animal experiments. The results showed that Chaetoceros muelleri (CM), Skeletoneema costatum (SC), Isochrysis galbana tml (IG), Thalassiosira weissflogii (TW) four MMFAs had the ability to scavenge free radicals, and the concentration is positively correlated with scavenging ability. MMFA can protect HepG2 cells from the oxidative stress caused by TAA. Compared with the control group, the cells improved the survival rate, decreased the intracellular ROS concentration and the mRNA expressions of CAT, SOD1, TNF and BCL2 were not significantly different from the control group. For animal experiments, the inhibitory effect of IG-MMFA on the obesity process was examined. C57BL/6JNarl mice were fed a high-fat diet containing 40% total fat to induce obesity, and IG-MMFA (125 mg/kg BW) was administered daily simultaneously for a period of 4 weeks. The results showed that the triglyceride in the blood lipid decreased slightly. In the physiological and pathological analysis, cumulative and steatosis were improved. The weight gain, the inguinal fat accumulation, and the BMI were not significantly different from the control group. In the gene level analysis, the mRNA genes expression of Cat, Il6, Tnf, Nfκb, Bax, Ppara, Pparg and Acac in the liver induced by high-fat diet returned to the same level as the control group. The expression of Cat, Tnf, Nfκb, Bax, Casp3, and Ppara in the heart induced by high-fat diet returned to the same level as the control group. The expression of Cat, Il6, Tnf, Nfκb, Bax, Casp3, Ppara, Pparg, Cpt1a and Acox in the fat tissue induced by high-fat diet returned to the same level as the control group. The results confirmed that IG-MMFA inhibits oxidative stress, inflammatory response and pro-apoptotic response which induced by high-fat diet in liver, heart and fat tissue, and genes related to fatty acid metabolism are improvement relatively. CPT1 and TNF protein expression in liver and heart tissue showed the same results as their mRNA expression and with significant differences compared to the control group. It is worth noting that in the case of feeding IG-MMFA alone, it was found to promote adipose cells inflammation and increase liver fatty acid metabolize. These results support the potential of IG-MMFA as an anti-obesity health food, providing the basis for research on subsequent food development, feed additives and more.
目錄

摘要 1
Abstract 4
誌謝 6
目錄 7
圖次 13
表次 15
壹、文獻回顧 16
一、海洋微藻 16
(一) Chaetoceros muelleri 17
(二) Skeletonema costatum 18
(三) Isochrysis galbana tml 18
(四) Thalassiosira weissflogii 18
二、內生性抗氧化酵素 19
(一) Catalase(CAT) 19
(二) Superoxide dismutase(SOD) 20
(三) Glutathione peroxidase(GPX) 21
三、脂肪酸 21
(一) 飽和脂肪酸 21
(二) 不飽和脂肪酸 22
(三) 二十碳五烯酸(EPA) 24
(四) 二十二碳六烯酸(DHA) 24
四、肝臟功能及脂肪酸代謝 25
(一) 脂肪酸合成 26
(二) 脂肪酸代謝與β氧化 26
五、非酒精性脂肪性肝病 28
六、AMPK路徑與脂肪酸代謝關係 30
貳、研究動機與目的 32
參、材料與方法 34
一、試驗流程 34
二、海洋微藻之培養 34
三、MMFA組成份分析及清除自由基能力試驗 35
(一) 脂肪酸萃取 35
(二) 脂肪酸前處理 35
(三) 氣相層析法 35
(四) DPPH 自由基清除能力試驗 36
四、HepG2細胞培養條件及氧化壓力模式建立 36
(一) HepG2培養與繼代 36
(二) 氧化壓力模式建立及MMFA處理 37
五、細活性試驗 37
六、細胞活性氧化物分析 38
七、細胞RNA基因表現量分析 38
(一) Total RNA抽取 38
(二) cDNA製備 39
(三) 及時定量聚合酶連鎖反應 39
八、肥胖小鼠模式建立及萃取物處理 40
(一) 小鼠品系及飼育條件 40
(二) 肥胖小鼠模式建立及萃取物處理 40
九、血液生化分析 40
(一) 肝功能之AST、ALT 41
(二) 血脂之TC、TG、HDL、LDL 41
(三) 動脈硬化指數(AI) 41
十、生理學分析 41
(一) 體重變化 41
(二) 相對肝重 42
(三) 腹股溝脂肪相對重量 42
(四) BMI 42
十一、肝臟組織病理學分析 42
(一) 石蠟切片 42
(二) 冷凍切片 43
十二、肝臟、心臟、脂肪組織RNA基因表現量分析 43
(一) Total RNA抽取 43
(二) cDNA製備 43
(三) 及時定量聚合酶連鎖反應 43
十三、西方墨點法 44
(一) 臟器總蛋白萃取 44
(二) 西方墨點法 44
十四、資料分析 45
十五、藥品配製 45
(一) Oil Red染劑配製 45
(二) Paraformaldhyde配製 45
(三) Permeabilization solution 配製 45
(四) 20μM DCFDA配製 46
(五) Phosphate-Buffered Saline配製 46
(六) SDS gel配製 46
(七) PVDF transfer buffer配製 46
(八) 5%blocking buffer配製 46
(九) Washing buffer配製 46
肆、結果 47
一、海洋微藻脂肪酸萃取物(MMFA)組成分分析 47
二、DPPH 自由基清除能力試驗 47
三、TAA及MMFA處理下HepG2細胞存活率 47
四、細胞氧化應激試驗 48
五、TAA及MMFA處理下HepG2細胞基因表現 48
(一) 細胞內CAT、SOD1、GPX1抗氧化基因表現 48
(二) 細胞內TNF發炎基因表現 48
(三) 細胞內BCL2抗凋亡基因表現 49
六、血液生化分析 49
(一) 肝功能之 AST、ALT 49
(二) 血脂之TC、TG、HDL、LDL 49
(三) 動脈硬化指數 (AI) 50
七、小鼠肥胖誘導及萃取物處理之生理學變化 50
(一) 小鼠及肝臟外觀 50
(二) 體重變化 50
(三) 相對肝重變化 51
(四) 相對脂肪重變化 51
(五) 身體質量指數 (BMI) 51
八、肝臟組織病理學分析 52
(一) 肝臟石蠟包埋切片 52
(二) 肝臟冷凍切片 52
九、臟器組織RNA層次基因表現量之變化 52
(一) 肝臟組織RNA層次基因表現量之變化 52
(二) 心臟組織RNA層次基因表現量之變化 54
(三) 脂肪組織RNA層次基因表現量之變化 55
十、肝臟組織蛋白質表現量分析 57
(一) TNF發炎蛋白質表現量變化 57
(二) CPT1A脂肪代謝蛋白質表現量變化 57
十一、心臟組織蛋白質表現量分析 57
(一) TNF發炎蛋白質表現量變化 57
伍、討論 121
一、海洋微藻脂肪酸(MMFA)組成分之探討 121
二、MMFA抗氧化能力之探討 121
三、MMFA保護肝臟細胞能力之探討 122
四、MMFA減少細胞氧化應激與抗氧化、發炎基因能力之探討 122
五、IG-MMFA影響高脂飼料誘導B6小鼠血液生化分析變化探討 124
六、IG-MMFA影響高脂飼料誘導B6小鼠生理學變化之探討 125
七、IG-MMFA對高脂飼料誘導B6小鼠之組織病理學影響探討 126
八、IG-MMFA影響高脂飼料誘導B6小鼠基因表現之探討 127
九、IG-MMFA影響高脂飼料誘導B6小鼠蛋白質表現量之探討 131
陸、結論 134
柒、參考文獻 137
捌、附錄 156
柒、參考文獻
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