2026-11-092026-11-09https://scholars.csmu.edu.tw/handle/123456789/37909Dietary lipid plays important roles in physiology, including energy supply, component of membrane phospholipids, and precursors of prostaglandins and leukotrienes. Differences in quantity and quality of dietary lipid modulate gene expression directly or indirectly, and affect cell metabolism, growth and differentiation. Dietary lipid plays an important role in diabetes, hypertension, cardiovascular disease, and cancer, and those diseases are closely related to obesity. Excessive dietary lipid intake is one of the causes of obesity. Recently, by molecular biotechnology, the effect of dietary lipid on adipocytes, hepatocytes, immune cells, tumor cells, and/or hormone, growth factor, and nerve transducer secretion has been investigated. The role of peroxisome proliferator-activated receptors in fatty acid synthesis and oxidation gene expression is one example. We have studied the effect of dietary lipid on hepatocarcinogenesis and hepatic cytochrome P-450 gene expression, and results showed that n-3 lipid inhibited hepatic foci development induced by diethylnitrosamine initiation/phenobarbital promotion compared to n-6 lipid and n-3 lipid increased cytochrome P-450 1A, 2B, and 3A activity and expression compared to n-6 lipid. In liver, cytochrome P-450 superfamily are involved in xenobiotic detoxification, and endogenous fat-soluble material and procarcinogen biotransformation. So cytochrome P-450s are considered to closely relate to cancer development. There are many studies about dietary lipid and detoxification enzymes, but most studies focus on cytochrome P-450 4A. However, few studies about dietary lipid and cytochrome P-450 1-3 families, especially cytochrome 2B. Cytochrome P-450 2B is involved in oxidation of testosterone, arachidonic acid, lauric acid, drugs, and environmental chemicals, and bioactivation of alfatoxin B1 and benzo[a]pyrene. It has been shown that alpha-tocopherol increases cytochrome P-450 2B1 expression via modulation of PGE2 synthesis. The mechanism for dietary lipid regulation of gene expression may not be single. Based on the relationship among dietary lipid, tumor development, and cytochrome P-450, clarification of the role of dietary lipid in cytochrome P-450 expression may help understand the relationship between nutrients and cancers. The objective of this study was to study the effects of linoleicacid, arachidonic acid, EPA, and DHA on cytochrome P-450 2B1 expression, and the possible role of PGE2 involved. We plan to use primary hepatocyte culture model to study the effects of linoleicacid, arachidonic acid, EPA, and DHA on cytochrome P-450 2B1 expression, and the possible mechanisms involved in one year. The items we are going to finish includes: 1. primary hepatocyte culture: establish cytochrome P-450 2B1 expression in hepatocytes. 2. Cox mRNA expression: use RT-PCR and Northern blot to analyze Cox-1 and/or Cox-2 mRNA expression in hepatocytes. 3. PGE2 receptors identification. 4. Effect of different fatty acids on cytochrome P-450 2B1 protein and mRNA expression. 5. Effect of different fatty acids on PGE2 secretion. 6. Investigate PGE2-mediated signal pathway.關鍵詞： 飲食脂肪酸、前列腺素、解毒酵素、肝細胞 飲食油脂的生理角色，除了可是能量來源、細胞膜磷脂質原料、前列腺素與白三烯素先質外，也已知油脂可透過其中脂肪酸質與量的差異，直接或間接調控基因的表現，影響細胞的代謝、生長以及分化。所以，可以想像飲食油脂的生理意義及其扮演角色，要比傳統認知複雜得多。因此，適量或適當種類的飲食油脂絕對與每一個人的身體健康狀況有密切關係。現代人聞之色變的多種慢性病，比如，糖尿病、高血壓、心血管疾病、腫瘤等，多少都與肥胖有關，而肥胖即是油脂過度攝食的結果之一。除了間接由流行病學探討脂質與健康相關性外，直接藉由分子生物學方法研究油脂或脂肪酸對脂肪細胞（adipocytes）、肝細胞、免疫細胞、腫瘤細胞或其它細胞中荷爾蒙/生長因子/神經傳導物質的活性或分泌之影響，或對細胞發育、分化、能量代謝、癌化相關基因的調控等，已成為近年來脂質的主要研究方向，轉錄因子—peroxisome proliferator-activated receptors (PPARs)在脂肪酸合成與氧化相關基因調控上即是一個例子。本實驗室過去數年針對飲食油脂種類對肝癌生成與肝臟解毒酵素表現之效應進行過系列研究，結果顯示：相較玉米油，攝食富含n-3不飽和脂肪酸之魚油組大鼠，其Diethylnitrosamine (DEN)/Phenobarbital (PB)誘發肝前癌細胞（g-glutamyl-cysteine-positive preneoplastic foci）發生明顯較少，且魚油組大鼠肝臟中細胞色素P450（cytochrome P450, CYP）1A、2B、3A活性與表現也較玉米油組高，另外，高玉米油組CYP表現也較低玉米油組高（23.5% vs. 5%）。肝臟中，CYP此一超級家族蛋白，不但是生物體中負責外來毒物的解毒作用與內生性脂溶性物質的生物轉化作用（biotransformation）的重要酵素，也是體內活化致癌物的催化酵素，因此一般認為CYP活性與腫瘤發生間也有密切相關性。有關飲食油脂與解毒酵素活性之報告並不少見，但是，就作用機轉而言，除了CYP 4A家族外，油脂或脂肪酸如何影響CYP 第1至第3家族，相關研究就少得多，尤其針對CYP 2B家族更是少見，值得研究。CYP 2B家族在生物體中，除參與睪脂酮、花生四烯酸、月桂酸、藥物和環境化學物質的氧化作用外，也是黃麴毒素B1和benzo[a]pyrene等致癌物的活化酵素。我們曾在初代肝細胞實驗中指出a-tocopherol可以增加CYP 2B1表現，原因可能與其抑制PGE2合成有關；雖然後續實驗已有初步資料支持PGE2的確極可能參與其中，不過仍須進一步證據。當然，飲食油脂或脂肪酸調控基因表現不會是單一途徑，比如，透過a-Tocopherol的抗氧化作用改變胞內氧化還原狀態（redox status），也是可能機轉之一。有鑑於油脂、腫瘤發生與CYP酵素三者相關性，飲食油脂調控CYP表現機制之釐清或許有助於瞭解營養因子與癌症間關係。本計畫主要目的將是比較亞麻油酸（linoleic acid）、花生四烯酸（arachidonic acid）、eicosapentaenoic acid（EPA）、docosahexaenoic acid（DHA）等四種不飽和脂肪酸對CYP 2B1表現之差異，並探討PGE2-mediated pathway在其中扮演之角色。 參、計畫內容 頁數限制：5頁 一、研究主旨：請分點具體列述本計畫所要達成之目標以及所要完成之工作項目，應避免空泛性之敘述。屬中長期計畫者，應列述全程計畫之總目標及分年計畫之目的。 本計畫預定以一年時間利用大鼠初代肝細胞作為實驗模式，研究亞麻油酸（linoleic acid）、花生四烯酸（arachidonic acid）、eicosapentaenoic acid（EPA）、docosahexaenoic acid（DHA）等四種不飽和脂肪酸對解毒酵素—CYP 2B1表現之影響及可能機制探討，工作內容及預期成果分述如下； 工作項目: (1) 初代肝細胞培養：確立肝細胞正常表現CYP 2B1。 (2) COX mRNA之表現：利用RT-PCR and Northern blot分析確定COX-1 and/or COX-2 mRNA於肝細胞之表現。 (3) PGE2接受器種類之鑑定 (4) 不同脂肪酸對肝細胞CYP 2B1蛋白質與mRNA表現之影響 (5) 不同脂肪酸對肝細胞分泌PGE2之影響 (6) PGE2–mediated signal pathway之探討：此步驟將分別使用PGE2、COX抑制劑-indomethacin、EP2 agoinst-butaprost、adenylate cyclase inhibitor-SQ22536、protein kinase A inhibitor-H89驗證n-3與n-6脂肪酸對肝細胞CYP 2B1基因表現之差異性與PGE2之生合成有關，然後PGE2再透過EP2接受器傳遞此一訊號。飲食油脂前列腺素解毒酵素肝細胞dietary lipidprostaglandindetoxification enzymehepatocyteProject