探討DHA抵抗類澱粉蛋白誘發之神經毒性以預防阿茲海默氏病之機轉

Abstract

本論文在探討二十二碳六烯酸(Docosahexaenoic acid,DHA,22:6ω3)對阿茲海默氏病(Alzheimer’s disease, AD)可能的預防機轉。AD是一種神經退化性疾病，好發於老年人，在患者腦部有老年斑塊(senile plaque)與神經纖維纏結(neurofibrillary tangle)等特徵。老年斑塊是由類澱粉蛋白(β-amyloid peptide, Aβ)在神經細胞外堆積而成，而神經纖維纏結是由過度磷酸化的Tau蛋白在神經細胞內堆積而成，並造成細胞內微小管(microtubule)斷裂、動態(dynamics)降低的現象。DHA是神經細胞膜上最重要的多不飽和脂肪酸，對大腦的發育及正常功能的維持極為重要。 利用懷孕期第十八天胎鼠的海馬回做初級細胞培養，在培養液中添加0~50uM DHA，發現：（1）以細胞型態完整性分析，當添加10μM DHA時細胞有最高存活率。當添加25μM DHA時最能抵抗Aβ42的毒性並有較高細胞存活率。（2）以氣相層析儀(Gas chromatograph,GC)分析，發現DHA添加量越多，細胞內DHA含量也越多，呈現濃度依賴效應(dose-dependent effect)，當添加至25μM時，神經細胞內DHA含量達到高原期(plateau)。（3）以西方點墨法(Western blot)分析，發現添加DHA的海馬回細胞其代表微小管動態上升的tyrosinated tubulin蛋白質表現在添加DHA 5μM時上升61 %，代表動態下降的acetylated tubulin蛋白質表現在DHA 25μM上升9 %，表示DHA可調控微小管動態，其變化正與添加DHA存活率趨勢相符。此外，actin蛋白質表現在添加25μM DHA時下降37 %。 綜合以上結果，我們發現DHA可以經由培養液併入海馬回神經細胞，並增加微小管動態以抵抗類澱粉蛋白引起的的神經毒性，提高神經細胞的存活率。證明DHA對預防阿茲海默氏病的確有相當的效果。

Alzheimer’s disease (AD) is a progressing neurodegenerative disease characterized by dementia to a degree that it infers with person’s ability and function. The aim of the study is to understand the mechanism of docosahexaenoic acid (DHA, 22:6ω3) on the progression of AD by conducting studies in primary hippocampal cells supplemented with 0~50 uM DHA. We hypothesized that increased DHA level in membrane structure of hippocampal neuron would build up a barrier to against neurotoxicity induced by fibrillar amyloid b peptide (Ab42), and increased microtubules dynamics. It was found that: （1）The optimal concentration of DHA for the survival of cultiured primary hippocampal cells was 10μM. （2）The DHA supplemented to the medium did increase the DHA level in hippocampal cells as a dose-dependent level. （3）The increased DHA level in hippocampal cells did against neurotoxicity induced by fibrillar Ab42 to increase its cell viability. （4）The 5uM but not 25uM DHA supplemented to medium did increase 61% tyrosinated tubulin expression, a maker of dynamic microtubules, compare with controls. （5）The 25uM but not 5uM DHA supplemented to medium did upregulate 9% acetylated tubulin expression, a maker of stabilized microtubules, compared with controls. （6）The 25uM but not 5uM DHA supplemented to medium did downregulate 37%β-actin compared with controls. We concluded that the supplemented DHA can be incorporated into the hippocampal cells to increase its survival, did against Aβ42 induced neurotoxicity and regulate microtubule dynamics, suggesting DHA is a potential supplement for AD prevention.