The effect of docosahexaenoic acid on the expression of hepatic genes in pigs

二十二碳六烯酸（Docosahexaenoic acid, DHA)為一種多不飽和脂肪酸，可降低轉錄因子固醇調節因子結合蛋白1 (sterol regulatory element-binding protein-1, SREBP-1) 的基因表現量。在哺乳動物，SREBP-1具有調控脂質生合成基因的功能。DHA可藉由降低脂質生合成基因的表現，減少動物體內脂質的蓄積。除了對於脂質生合成基因的調控，DHA對於動物體內的其他生理亦有所影響。 因此，本試驗目的首要探討DHA是否會影響豬隻肝臟中的肝臟型脂肪酸結合蛋白（liver fatty-acid binding protein , L-FABP）及白蛋白（albumin）的基因表現，進而影響脂肪酸的運輸。FABP是一種胞內蛋白，其分子量大約在12至15 kD，與脂肪酸具有高親和力的結合。Albumin在肝臟製造，其功能為運輸血液中的游離脂肪酸。在第一個試驗中，餵飼豬隻添加2％牛脂（飽和脂肪酸）或2％DHA油（n-3脂肪酸）之飼糧18天。在第二個試驗，餵飼豬隻添加10％DHA油、10％牛脂或10％大豆油（n-6脂肪酸）之飼糧2天。結果顯示不論是在餵飼2％不同脂肪酸的飼糧18天，或是餵飼不同脂肪酸10％的飼糧之2天，對於豬隻L-FABP及albumin的基因表現量都沒有顯著影響（P>0.05）。試驗結果顯示飼糧中不同脂肪酸的組成並不會影響豬隻肝臟中L-FABP及albumin的基因表現。 為了更進一步研究那些豬隻肝臟中的基因受到DHA飼糧的影響。從餵飼添加2％牛脂（飽和脂肪酸）或2％DHA油（n-3脂肪酸）之飼糧18天的豬隻肝臟中萃取其mRNA，利用抑制性雜合扣除法（Suppression subtractive hybridization, SSH）探究受DHA調控的特異性基因。進行完SSH後，得到288個來自於餵飼2％牛脂或2％DHA油所得的具特異性表現cDNA片段。所得基因序列與NCBI（http://www.ncbi.nlm.nih.gov/）中之基因資料進行比對，以確定其基因名稱。經菌株矩陣法的篩檢288個特異性表現cDNA片段後，僅有7個特異性表現基因需進一步使用北方吸漬法進行最後的篩檢。最後經北方吸漬法分析後發現飼糧中DHA油的添加可提升肝臟中血清澱粉狀蛋白A（serum amyloid A protein, SAA）mRNA表現量，與對照組相比，發現2％DHA油及10％DHA油可顯著的增加SAA基因表現量（P<0.05）。結果顯示DHA油可以顯著提高豬隻肝臟中SAA的表現，SAA表現的改變對生理的影響有待釐清。Docosahexaenoic acid (DHA) is one of the polyunsaturated fatty acids that can decrease gene expression of a transcription factor, sterol regulatory element-binding protein-1 (SREBP-1). In mammals, SREBP1 regulates the function of a group of lipogenic genes. Through the reduction of lipogenic genes, DHA may reduce lipid accumulation in animals. Besides the regulation on lipogenic genes, DHA may have other effects on animal physiology. Therefore, the first purpose of this study was to determine whether DHA influenced the expression of liver fatty-acid binding protein (L-FABP) and albumin to affect fatty acid transportation. FABP is a cytosolic protein which has low molecular weight (12 to 15 kD) and high affinity in binding fatty acids. Albumin is produced in the liver and functions as a carrier for free fatty acids in the blood stream. In the first experiment, pigs were fed with either 2% DHA oil (n-3 fatty acid) or 2% tallow (saturated fatty acid) containing diet for 18d. In the second experiment, pigs were fed with 10% DHA oil , 10% tallow or 10% soybean oil (n-6 fatty acid) containing diet for 2d.We found that neither 2% dietary DHA for 18d nor 10% dietary DHA for 2d had a significant effect on the expression of L-FABP and albumin genes. These data demonstrated that different dietary fatty acid composition did not differentially affect the expression of L-FABP and albumin in the livers of the pigs. In order to study which hepatic genes were affected by dietary DHA in pigs, we extracted mRNA from pig livers either fed with diets containing 2% DHA oil or 2% tallow for 18d. Suppression subtractive hybridization was used to explore genes that were specifically regulated by DHA. After subtraction, we got 288 cDNA fragments differentially expressed between livers from pigs either fed with 2% DHA oil or 2% tallow for 18d. The sequences of these specific cDNA fragments were compared with sequences from GenBank database of NCBI (http://www.ncbi.nlm.nih.gov/). After colony array screening, we found that 7 genes were differentially expressed. Northern analysis data showed that hepatic serum amyloid A protein (SAA) was upregulated by dietary DHA. We have confirmed that both treatments with 2% DHA oil for 18d and 10% DHA oil for 2d significantly increased the expression of SAA when compared with the control group （P<0.05）. The data showed that dietary DHA upregulated the expression of SAA in porcine liver. Whether elevated SAA has other physiological effect requires further demonstration.目錄----------------------------------------------------- i 表次---------------------------------------------------- ii 圖次----------------------------------------------------iii Ⅰ、摘要--------------------------------------------------1 Ⅱ、文獻檢討----------------------------------------------3 Ⅲ、試驗一-----------------------------------------------20 Ⅳ、試驗二-----------------------------------------------41 Ⅴ、結論------------------------------------------ ------51 Ⅵ、參考文獻---------------------------------------------52 Ⅶ、英文摘要---------------------------------------------65 Ⅷ、附錄-------------------------------------------------6