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研究生:吳雅玲
研究生(外文):Ya-Ling Wu
論文名稱:膳食油脂對大鼠脂肪組織中PPARγ與相關基因mRNA表現及化學組成影響之探討
論文名稱(外文):Effects of type and amount of dietary fat on the mRNA expression of PPARγand associated genes in relation to the chemical composition of epididymal adipose tissue in rats
指導教授:黃青真黃青真引用關係
指導教授(外文):Ching-Jang Huang
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:農業化學研究所
學門:農業科學學門
學類:農業化學類
論文種類:學術論文
論文出版年:1999
畢業學年度:87
語文別:中文
論文頁數:177
中文關鍵詞:過氧化體增殖劑活化受器體內模式膳食油脂北方墨漬法
外文關鍵詞:PPARin vivoDietary fatNorthern blot
相關次數:
  • 被引用被引用:10
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  • 收藏至我的研究室書目清單書目收藏:1
PPAR (Peroxisome Proliferator Activated Receptor) 是固醇類荷爾蒙核受器家族 (steroid hormone nuclear receptor superfamily) 的成員之一,具有α、β (δ) 及γ三種不同的isoform。分子生物學的研究結果顯示,PPARα主要表現於肝臟,調控了脂質代謝相關基因的轉錄作用。而PPARγ主要表現於脂肪組織,調控了與脂肪細胞分化有關的基因表現。因此,PPAR的傳訊途徑與人體膳食油脂的代謝及利用情形可能有著極大的關聯。目前已有文獻證實脂肪酸能夠活化PPAR,但對於膳食油脂是否影響PPAR的研究領域則著墨不多。另一方面,由本研究室先前的實驗中 (張氏,1998) 發現,餵予大鼠高脂飲食 (20%) 較正常量油脂飲食 (5%) 更能誘發肝臟中PPARα mRNA的表現,且高魚油組較其他高油飲食具有促進PPARα下游基因ACO mRNA (Acyl CoA oxidase) 表現的作用。因此我們便想知道在同樣的模式下,脂肪組織中PPARγ 及其下游基因的表現是否也會受到飲食油脂的影響。
為探討脂肪組織中PPARγ之mRNA是否受到飲食油脂影響,我們先建立偵測mRNA之工具及方法,由於PPARγ mRNA於脂肪組織中之表現很低,且γ2僅較γ1於N端多出了30個胺基酸,無法以常用之北方墨漬法(Northern blot)測出,故我們先建立RPA (RNase Protection Assay)法以同時偵測PPARγ1及γ2 mRNA。設計引子以RT-PCR法選殖涵蓋γ2 5''端轉譯起始點起273bp序列之cDNA為PPARγ2探針,此段亦涵蓋γ1轉譯起始點起183bp之序列。由於γ2比γ1多出了90 bps,故可以此區別二者。此外,為了解PPARγ下游基因表現受飲食油脂質與量之影響狀況,亦製備LPL、aP2 mRNA及18S rRNA探針,建立北方墨漬法偵測下游基因的表現。
本實驗採用Sprague-Dawley品系的離乳公鼠48隻,餵食NS、HS、SO、FO及CO等五組實驗飼料,飼養期為五週。五組飼料中NS組仿照AIN-76含5%紅花籽油為正常對照組,其餘四組均為含20%油脂之高油飼料。其中HS組含20%紅花籽油;SO組含20%高油酸紅花籽油;FO組含19.55%魚油;CO組含19.52%椰子油,FO及CO組分別添加0.45%及0.48%紅花籽油以防止必需脂肪酸缺乏。飼養五週後,在生長狀況方面,鼠體重至第五週時並無差異,飼料效率以HS組較高,每日熱量及飼料攝取則以攝食低油的NS組顯著較其他四組為高。
將五組大鼠之副睪脂肪塊總RNA抽出,以所建立之RPA法同時偵測PPARγ1與γ2 mRNA之表現量,結果顯示CO組無論在PPARγ1或γ2的mRNA均為五組中最高,而下游基因LPL與aP2之mRNA表現量,亦以CO組最高。此結果似乎顯示含高量中鏈飽和脂肪酸的高椰子油飼料 (CO組) 使大鼠副睪脂肪組織表現出較高之PPARγ mRNA,是否因而使PPARγ的表現及傳訊高於其他四組,而促進脂肪細胞分化﹖值得進一步探究。
五組大鼠之副睪脂肪組織也進行脂肪酸組成、每克脂肪組織中粗脂肪、蛋白質及DNA含量分析。結果顯示大鼠脂肪組織中各脂肪酸的分佈明顯反應了飲食油脂的組成特性。在每克脂肪組織DNA含量及蛋白質含量方面,攝食魚油的FO組顯著較其他四組為高 (p<0.05)。但是在粗脂肪含量方面,FO組則顯著低於其他四組 (p<0.05),顯示攝食魚油的鼠隻其脂肪細胞可能較其他四組為小,與文獻的結果一致。此外,我們也發現餵食魚油的大鼠其脂肪組織中β-actin mRNA顯著較高 (p<0.05),而LPL mRNA顯著較低。此現象是否與FO組鼠隻脂肪細胞的型態有關,值得進一步探究。
綜上所述,高椰油飼料似乎比其他高油飼料較能促進脂肪組織中PPARγ及其下游基因的表現。而高魚油飼料使每克脂肪組織的粗脂肪含量顯著降低,DNA及蛋白質含量顯著升高,改變了脂肪細胞的型態,顯示膳食油脂之種類與量確實會影響大鼠脂肪組織中PPARg及相關基因的表現。
Peroxisome proliferator-activated receptors (PPAR) are members of steroid nuclear receptor superfamily of ligand-activated transcription factors. To date, three isoforms have been identified, α, β (δ) and γ, encoded by three seperate genes. PPARa is expressed predominantly in lipid metabolizing tissue, such as liver where it plays a role in lipid catabolism. PPARγ, on the other hand, is mainly expressed in adipose tissue and has a critical role in adipocyte differentiation and lipid storage. Thus, the PPAR family is implicated in mediating the expression of fat-specific genes and lipid metabolism in the body.
Although a variety of fatty acids have been reported to activate PPAR in vitro, little is known about whether dietary fat might regulate PPAR gene expression in vivo. Since a previous study in our lab demonstrated that hepatic PPARα mRNA was increased by high fat feeding and ACO (acyl CoA oxidase) mRNA was induced to a greater extent in rats receiving high fish oil diet, the aim of this study was to investigate the effect of dietary fat on the expression of PPARγ in adipose tissue.
For simultaneous detection of mRNAs of both PPARg1 and g2 isoforms (only 90 bps discrepancy in 5'' translated region) encoded by PPARγ gene, we first cloned a partial rat PPARγ cDNA by RT-PCR and developed an RNase protection assay. For the preparation of probes for the detection of aP2, LPL mRNA and 18S rRNA by Northern blot, cDNA fragments of these genes were also cloned.
To explore the effects of type and amount of dietary fat on PPARγ expression in adipose tissue, 5-week-old male Sprague-Dawley rats were assigned to one of the following groups: NS (containing 5% safflower oil and served as the normal diet control), HS (containing 20% safflower oil), SO (containing 20% high oleic safflower oil), FO (containing 19.55% refined edible fish oil plus 0.45% safflower oil) and CO (containing 19.52% coconut oil plus 0.48% safflower oil). After feeding for five weeks, the mean body weight was similar among the dietary groups. Rats of the HS group had the highest feed efficiency among five groups, while rats in the NS group showed the highest daily food and energy intake (p<0.05). Results of the RNase protection analysis on total RNA of epididymal fat revealed that rats fed the CO diet showed a modest but significant increase in both PPARγ1 and PPARγ2 mRNA in epididymal fat pad, and a corresponding increase in LPL and aP2 mRNA as shown by Northern blot analysis. These observations suggest that the MCFA-rich coconut oil might enhance the expression of PPARγ gene. Further studies will be required to address this issue.
Chemical components, including DNA, protein, fat and fatty acid composition of the epididymal fat were also determined. Total fatty acid composition from adipose tissue in five groups of rats all reflected their dietary fat sources precisely. Total DNA and protein content per gram of adipose tissue were significantly higher in the FO diet fed rats than in the remaining four groups, whereas FO group also showed the lowest fat content among five groups (p<0.05). These data suggested that adipocytes of the FO diet fed rats were smaller in size. Furthermore, rats fed the FO diet showed significantly higher b-actin mRNA (p<0.05) and a lower LPL mRNA in their epididymal fat pad. Further studies are needed to clarify the relationship between the adipocyte development and gene expression in the fish oil fed rats.
In conclusion, feeding a high coconut oil diet resulted in a higher expression of PPARγ and its downstream genes in the epididymal fat of rats. Rats receiving high fish oil diet seems to have different size distribution of adipocyte. These results suggest that the expression of PPARγ and associated genes may be altered by the type and amount of dietary fat.
縮寫對照表
中文摘要 1
英文摘要 3
緒言 5
第一章 文獻回顧 7
第一節 PPAR的功能與介紹 7
一、總論 7
二、PPRE與標的基因 8
三、活化劑及ligands 9
第二節 PPAR傳訊途徑的生理效應 11
一、PPARα 於脂質代謝之角色 11
二、PPARγ 於脂肪細胞分化之角色 12
第三節 PPAR基因表現之調控 18
一、PPARα 18
二、PPARγ 19
第四節 偵測mRNA的工具及其應用 21
一、北方墨漬法 21
二、反轉錄-聚合連鎖反應 22
三、RPA (Ribonuclease Protection Assay, RNase Protection Assay) 24
第二章 實驗中所需cDNA質體之構築 30
第一節 前言 30
第二節 材料與方法 30
一、副睪脂肪塊總RNA抽取 31
二、RNA電泳 32
三、反轉錄--聚合連鎖反應(Reverse Transcriptase Polymerase Chain Reaction, RT-PCR) 34
四、DNA電泳 41
五﹑重組DNA之製備 41
六﹑宿主轉型與菌落篩選 41
七﹑質體之純化製備 42
八﹑限制圖譜鑑定 46
九﹑DNA序列分析 46
第三節 結果與討論 53
一、 RT-PCR產物之確認 53
二、 限制圖譜之鑑定 53
三、 DNA序列之判讀 54
第三章 以RNase Protection Assay (RPA) 法偵測PPARγ1及γ2 mRNA之方法建立 68
第一節 前言 68
第二節 材料與方法 69
一、實驗流程 69
二、探針之製備 70
三、帶測樣品之製備 73
四、雜交反應 73
五、RNase分解作用 74
六、電泳分析 75
七、影像量化處理 76
第三節 結果與討論 77
第四章 膳食油脂對大鼠脂肪組織中PPARγ mRNA表現及相關現象之探討 92
第一節 前言 92
第二節 材料與方法 93
一、實驗設計 93
二、動物飼養、飲食處理及組織取樣 94
三、PPARγ及下游基因表現量之測定 95
四、脂肪組織之化學分析 101
五、統計分析 106
第三節 實驗結果 109
一、 動物攝食及生長情形 109
二、 副睪脂肪塊中mRNA之表現 109
三、 脂肪組織之化學分析結果 110
四、 各基因表現及各組成之相關性分析 113
第四節 討論 140
一、 動物攝食及生長情形 140
二、 膳食油脂對副睪脂肪塊中mRNA之表現 140
三、 脂肪組織之化學分析結果 144
四、 魚油於體內的生理效應 146
五、 綜合討論 148
總結 151
參考文獻 153
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