探討二十二碳六烯酸在人類神經細胞株與發育大鼠對視網酸誘導之神經元軸突生長和相關蛋白質的影響

Abstract

二十二碳六烯酸 ( Docosahexaenoic acid, DHA ) 和視網酸 ( Retinoic acid, RA ) 在大腦神經發育過程皆扮演重要角色，具有促進神經細胞生成 ( neurogenesis )、分化 ( differentiation )、神經元軸突生長 ( neurite outgrowth ) 等功能。RA 可透過 RAR-RXR受體影響基因轉錄活性，DHA 也是 RXR的配體之一，故本論文以 RA 的面向切入，使用人類神經母細胞瘤 SH-SY5Y 細胞株，探討神經細胞發育過程 DHA 對由 RA 所誘導的細胞分化之形態和神經相關蛋白質表現的影響；結果顯示，隨著 RA 處理天數增加 ( 3 - 12 天 ) 及濃度 ( 0 - 10 μM ) 增加，最大神經元軸突長度 ( maximum neurite length ) 及神經元軸突長度總和 ( total neurite length ) 有顯著增加情形，在 RA 誘導分化的情況，添加 DHA 可使最大神經元軸突長度及神經元軸突長度總和顯著增加；蛋白質表現方面，RA 誘導分化下添加 DHA 可使 GAP-43、BDNF、TrkB 及 CaMKIIα 等蛋白質表現量顯著增加。 利用雌鼠在懷孕泌乳期給予缺乏 n-3 PUFA 飲食 ( 占總攝取熱量的 0.13 % ) 或足夠 n-3 PUFA 飲食 ( 占總攝取熱量的 0.6% )，探討其子代在大腦發育時期 DHA 缺乏情況對腦部皮質與海馬迴內神經相關蛋白質表現的影響；結果顯示，皮質與海馬迴內神經相關蛋白質表現會隨著出生天數而增加；缺乏 n-3 PUFA 飲食組別的腦部 DHA 含量比起足夠 n-3 PUFA 飲食組別顯著較低，且海馬迴 GAP-43、TrkB、PSD-95 與皮質 Synapsin I 的蛋白質表現量也顯著較低。 本論文發現，DHA 可促進由 RA 所誘導的神經元軸突生長，並增加神經分化指標、生長相關及突觸功能性蛋白質的表現；大鼠大腦發育時期若缺乏 DHA會造成腦部神經分化指標、生長相關及突觸功能性蛋白質的表現降低。

Retinoic acid ( RA ) is essential for neuronal development. Docosahexaenoic acid ( DHA, 22:6n-3 ) accumulates rapidly during brain development and is one of ligand of retinoic receptors. The aim of this study was to examine the effect of DHA on RA-induced neurite outgrowth and neuronal protein expression in human neuroblastoma SH-SY5Y cell line and the developing rats. SH-SY5Y was cultured in 0 - 10 μM RA or / and 10 μM DHA in 1% FBS with DMEM / F12 medium for 3 - 12 days. The RA-induced total neurite length and maximum neurite length were significantly increased with increasing incubation time and RA concentration. The GAP43, BDNF, TrkB, synapsin I and CaMKIIα protein expression were significantly increased with increasing RA concentration. DHA significantly enhanced RA-induced total neurite length and maximum neurite length as well as GAP43, BDNF, TrkB, PSD-95 and CaMKIIα protein expression in SH-SY5Y. In studying the developing brain, rats exposed to a corn oil-based n-3 fatty acid-deficient diet or the same diet supplemented with n-3 fatty acid-enriched fish oil as an n-3 fatty acid-adequate diet from in utero via maternal intake. The pups were sacrificed at age of 0, 7, 14 and 21 days old. The expression of GAP-43, TrkB, NR2A, GluR1, PSD-95, synapsin I, CaMkIIα were significantly increased gradually from age of 0 to 21 days old in cortex and hippocampus. The rats exposed to n-3 fatty acid-adequate diet showed significantly higher GAP43, TrkB and PSD-95 in hippocampus compared to the rats exposed to n-3 fatty acid-deficient diet. This study suggests that RA and DHA are important for, especially, DHA enhanced RA-induced neurite outgrowth and neuronal protein expression during neuronal developing.