探討大鼠發育過程及人類細胞株中參與二十二碳六烯酸合成及嵌入細胞膜磷脂質酵素基因表現

Abstract

二十二碳六烯酸（Docosahexaenoic acid, DHA, 22:6n-3）富含在大腦細胞膜磷脂質之中，可維持神經細胞之正常結構及功能。DHA主要在大腦發育時期快速堆積在腦部，成年後則維持穩定的含量。DHA透過前驅物α-次亞麻油酸（α-linolenic acid, ALA, 18:3n-3）在肝臟中透過去飽和（Desaturation）及加長碳鏈（Elongation）合成，經由Fatty acids-transport proteins（FATPs）及Fatty acid-binding proteins（FABPs）協助體循環中DHA進入大腦，透過Long-chain fatty acid acyl-CoA synthetase （ACSLs）活化形成DHA-CoA，最後經由Acyltransferase嵌入細胞膜磷脂質之中。本論文欲探討為何DHA主要富含在大腦，且為何DHA主要在大腦發育時期堆積在大腦中。 本論文第一部分利用母鼠懷孕第一天給予corn oil-based 或sunflower oil-based n-3脂肪酸缺乏飲食，或n-3脂肪酸缺乏飲食給予每日管餵魚油做為n-3脂肪酸充足飲食，子代離乳後持續給予該飲食至犧牲。取出生子代出生0.5、7、14、21、28、90、210及360天之大腦皮質及肝臟。探討在大腦發育期至成年過程與攝取n-3脂肪酸缺乏飲食，是否會影響大腦皮質及肝臟之中DHA含量、參與DHA合成及嵌入細胞磷脂膜之相關蛋白及酵素之基因表現。以氣相層析儀分析脂肪酸組成，即時聚合酶連鎖反應分析mRNA表現量。 本論文第一部分發現大腦皮質之DHA含量在大腦發育時期逐漸增加，成年之後DHA含量維持穩定，肝臟DHA含量在發育至成年過程無變化，且肝臟DHA含量約為大腦皮質的50 %。大腦皮質中參與DHA合成酵素FADS2、Elovl5、FADS1及Elovl2 mRNA表現量在大腦發育初期表現量較高，且此時大腦皮質與肝臟的表現量相當；成年後大腦皮質FADS2、Elovl5、FADS1及Elovl2 mRNA表現量下降，而肝臟表現量則隨年齡增加而上升，並顯著高於大腦皮質。大腦皮質中，協助DHA運輸至大腦的蛋白質FABP7及FABP5 mRNA表現量在大腦發育初期表現量較高，出生14天至成年後逐漸下降，成年後表現量皆很低。而大腦皮質中參與DHA嵌入相關酵素ACSL3、ACSL6、LPEAT2及AGPAT4 mRNA表現量在大腦發育時期逐漸增至出生21天，並且顯著高於肝臟。在大腦發育及成年過程，n-3脂肪酸缺乏飲食不影響這些酵素及蛋白質的表現。本論文第二部分利用shRNA分別抑制人類細胞株SH-SY5Y及HEK293T細胞中LPEAT2、ACSL3、ACSL6或FABP5表現量，探討抑制參與DHA嵌入細胞膜磷脂質相關酵素或蛋白質之表現，是否會影響細胞對DHA嵌入能力。本論文第二部分發現無論是在SH-SY5Y或HEK293T細胞中，抑制LPEAT2、ACSL3、ACSL6或FABP5表現，皆不影響細胞對DHA嵌入能力。 本論文推論，大腦在大腦發育期有較高FADS2、Elovl5、FADS1及Elovl2 mRNA表現量，協助大腦自行合成部分DHA，而大腦在大腦發育期亦有較高的FABP7及FABP5 mRNA表現量，協助體循環中的DHA進入大腦，故DHA在大腦發育期快速堆積在大腦中。而大腦皮質中ACSL3、ACSL6、LPEAT2及AGPAT4 mRNA表現量顯著高於肝臟，協助大腦中DHA嵌入細胞膜磷脂質，故DHA主要富含在大腦而非肝臟中。且大腦皮質ACSL3、ACSL6、LPEAT2及AGPAT4 mRNA表現量隨大腦發育逐漸增加，成年後維持高量表現，協助成年後大腦DHA含量維持plateau。

Docosahexaenoic acid (DHA, 22:6n-3) is mainly enriched in the brain and is primary anchored into neuronal membrane phospholipids for normal neurological function. Most DHA accumulation in brain takes place during brain development and DHA is biosynthesized from its precursor, -linolenic acid (18:3n-3) via desaturases and elongases following by converted to DHA-Co-A by long-chain acyl-CoA synthetase (ACSLs) and then incorporated into phospholipids by Lyso-phosphatidate acyltransferase (LPAATs). The aim of this thesis was to examine the effect of age and n-3 fatty acid-deficient diet on the enzyme involved in DHA biosynthesis and incorporation into membrane phospholipids during brain development, in adult rats and human cell lines. Rats exposed to a sunflower oil-based n-3 fatty acid-deficient diet or sunflower oil mixed with fish oil as an n-3 fatty acid-adequate diet from in utero via maternal intake. After weaning, pups were maintained on the same diet as their dams till sacrificed at age of 0.5, 7, 14, 21, 28, 90, 210 and 360 days old. The fatty acid composition and mRNA expression in cortex and liver were analyzed by GC and RT-qPCR, respectively. The enzymes involved in DHA biosynthesis, FADS2, FADS1, ELOVL5 and ELOVL2 mRNA expression were no difference between cortex and liver during brain development up to age of 7-14 days old, then they were increased in liver but not in cortex up to 28 days old and then plateau. The DHA levels and mRNA expression of ACSL3, ACSL6, LPEAT2, and AGPAT4 in brain were increased but remained constant in liver during brain development. The mRAN expression of FADS2, ELOVL5, FADS1, DBP, SCPx, FATP1, FATP5, FABP1, FABP3, ACSL1, ACSL4, and ACSL5 in liver were increased during brain development up to age of 28 days old and then plateau up to examined age of 360 days old. The mRNA expression of FABP5 were high at newborn and FABP7 in liver were increased during brain development up to age of 14 days old following decreased and remain constant low at adult 90-360 days old. No main effect of n-3 fatty acid deficient diet on above enzymes mRNA expression during developing and in adult liver. The knockdown expression of LPEAT2, ACSL3, ACSL6 and FABP5 in SH-SY5Y or HEK293T cells had no effect on DHA incorporation into cells. It was concluded that the brain DHA levels were accumulated rapidly during brain development. The expressing of the enzyme involved in DHA biosynthesis in brain and the fatty acid binding protein 5 and 7 was high at new born, suggesting they may plan important roles for DHA accumulation during brain development.