探討大鼠發育腦幹與成年組織的脂肪酸成分與相關酵素基因表現量之相關性

Abstract

脂肪酸(fatty acids)是構成生物體磷脂膜（membrane phospholipids）的主要成分，會影響細胞膜的功能。長碳鏈脂肪酸可透過去飽和酶（FADSs）和延長碳鏈酵素（ELOVLs）經體內自行合成，在透過acyl-CoA synthetases（ACSLs）使長鏈脂肪酸活化接上CoA，並利用acyltransferases（AGPATs或LPCATs）將脂肪酸嵌入磷脂膜中。本論文欲透過相關性統計來探討60天大的大鼠組織和發育階段腦幹的參與合成相關的酵素和嵌入相關的蛋白質基因表現量，對於該組織的脂肪酸組成的影響。 本論文分析60天大的公鼠組織，以及0.5、7、14、21、28、60、210和360天大的公鼠大腦皮質和腦幹。利用氣相層析儀進行脂肪酸成分分析，與即時聚合酶鏈式反應分析參與合成相關之酵素和協助嵌入磷脂膜的蛋白質基因表現量，再利用Pearson correlation coefficient統計脂肪酸成份百分比與酵素基因表現量的相關性。以痛覺行為實驗分析21、28、60、210和360天大的公鼠其機械痛與熱痛敏感度的變化。 腦幹含有較高的非常長碳鏈飽和脂肪酸（20:0、22:0和24:0）與非常長碳鏈單元不飽和脂肪酸（20:1n-9、22:1n-9和24:1n-9）成分，18:2n-6主要富含在脂肪、血漿、肌肉和肝臟中，AA（20:4n-6）富含在腎上腺、腦垂體、肝臟和血漿中，DHA（22:5n-6）富含在視網膜、大腦皮質、海馬迴、下視丘、小腦和肌肉中。Fads2、Fads1、Elovl5 mRNA皆主要表現在肝臟和腎上腺，Elovl2 mRNA表現在視網膜和睪丸中，Elovl6 mRNA表現在腦部和腎上腺，Elovl1 mRNA表現在腦幹及肺臟中，Elovl7 mRNA表現在腦幹和腎上腺。參與活化以及嵌入磷脂膜相關的酵素mRNA表現量結果中，Acsl1 mRNA主要表現在肝臟和脂肪，Acsl3 mRNA和Acsl6 mRNA表現在腦部，Acsl4 mRNA表現在腎上腺，Acsl5 mRNA表現在肝臟和肺臟；Agpat4 mRNA、Agpat5 mRNA和Lpcat4 mRNA皆表現在腦部，Lpcat1 mRNA和Lpcat2 mRNA皆表現在視網膜，Lpcat3 mRNA主要表現在腎上腺和睪丸。組織中的DHA含量分別與Elovl2, Acsl3, Acsl6, Lpcat1, Lpcat2, Lpcat3, Agpat4和Agpat5 mRNA表現量皆有正相關性，而組織中的非常長鏈飽和與單元不飽和脂肪酸分別與Elovl1, Elovl7 與 Acsl6 mRNA表現量呈正相關性。 發育階段的腦幹中的非常長鏈飽和與單元不飽和脂肪酸，有隨年齡增加至21~28天時而含量逐漸增加，並在成年後維持plateau至360天大，而腦幹中參與其合成相關的酵素Elovl1和Elovl7基因表現量，亦隨出生至21天或28天大時表現量逐漸上升，並在成年後逐漸下降至210天大時持平。不同年齡腦幹的20:0、22:0、24:0、20:1n-9 和24:1n-9含量皆與Elovl1和Elovl7基因表現量呈正相關性。 在機械痛覺行為實驗以及熱痛檢測的溫水閃尾實驗皆顯示，210天至360天的大鼠其痛覺敏感度皆顯著低於21天至28天大的大鼠。 本論文推論，組織間有不同的參與脂肪酸合成相關酵素與嵌入磷脂膜相關的蛋白質表現量，而影響組織間的脂肪酸組成特性。隨著大鼠年齡增加，其機械痛與熱痛的敏感度皆逐漸下降。

Fatty acids are the most important and common component of lipids and are presented in phospholipids which are the major component of cell membranes. Long-chain fatty acids are biosynthesized via fatty acid desaturases (FADSs) and very long chain elongases (ELOVLs), following by converted to acyl-Co-A by long-chain acyl-CoA synthetases (ACSLs) and then incorporated into lyso-phosphatidic acid or lyso-phospholipids by 1-acyl-sn-glycerol-3-phosphate acyltransferases (AGPATs) in de novo synthesis or by lyso-phospholipid acyl transferases (LPCATs) in the deacylation-reacylation process, respectively, to produce the diversity of membrane phospholipids. The aim of this thesis was to examine fatty acid metabolism in tissues by conducting the correlation of fatty acid levels and its enzyme expression involved in fatty acid biosynthesis and incorporation into phospholipids in a chow diet fed 8 weeks old male rats and in developing rat brain stem. The fatty acid composition by GC and mRNA expression by RT-qPCR were analyzed. Correlation analysis was performed by Pearson correlation coefficient. The mechanical and thermal pain were also measured in rats at age of 21, 28, 60, 210 and 360 days old. Very long chain saturated (20:0, 22:0 and 24:0) and unsaturated (20:1n-9, 24:1n-9) fatty acids were mainly rich in brain stem. Linoleic acid (18:2n-6) were enriched in adipose and plasma, muscle and liver; arachidonic acid (20:4n-6) were in adrenal gland, pituitary gland, liver and plasma; docosapentaenic acid (22:5n-6) were in testis; docosahexaenoic acid (22:6n-3) were in retina, cortex, hippocampus, cerebellum and muscle. The enzymes involved in fatty acid biosynthesis, Fads2 mRNA and Fads1 mRNA were mainly expressed in liver and adrenal gland; Elovl2 mRNA were in retina and testis; Elovl1 mRNA was in brain stem and lung; Elovl7 mRNA were in brain stem and adrenal gland. The enzymes involved in fatty acid incorporation into phospholipids, Acsl1 mRNA were mainly expressed in liver and adipose; Acsl3 mRNA and Acsl 6 mRNA were in brain; Acsl4 mRNA were in adrenal gland; Acsl5 mRNA were in liver and lung, Agpat4 mRNA, Agpat5 mRNA and LPACAT4 mRNA were in brain, Lpcat1 mRNA and Lpcat2 mRNA were in retina; Lpcat3 were in adrenal gland and testis. The 22:6n-3 levels were significantly positively correlated with the mRNA expression of Elovl2, Acsl3, Acsl6, Lpcat1, Lpcat2, Lpcat3, Agpat4 and Agpat5 in tissues. The levels of very long chain saturated and unsaturated fatty acids were significantly positively associated with Elovl1, Elovl7 and Acsl6 in tissues. The brain stem 20:0, 22:0, 24:0, 20:1n-9 and 24:1n-9 levels were increased during brain development up to age of 21-28 days old and then plateau up to the examined age at age of 360 days old. The enzymes involved its biosynthesis Elovl1 mRNA and Elovl7 mRNA expression were increased in the developing brain up to age of 21-28 days old, then dropped down to 210 days old. The levels 20:0, 22:0, 24:0, 20:1n-9 and 24:1n-9 were significantly positively associated with the mRNA expression of Elovl1 and Elovl7 in developing brain stem. The mechanical pain by Von Frey filament as well as the thermal pain by tail flick via warm water and plantar test were significantly higher at age of 21-28days old compared to the age of 210-360 days old. It was concluded that fatty acid distribution was different among the tissues that may resulted from the enzymes involved its fatty acid biosynthesis and incorporation into phospholipids. The pain sensitivity is decreased in aging.