花生四烯酸在大白鼠胚胎大腦皮質神經細胞與大白鼠腎臟髓質嗜鉻腫瘤細胞之神經毒性作用

The neurotoxicity of arachidonic acid on the primary neuronal cell culture and PC12 cells

林奎佑(Lin Kuei-Yu)

指導教授 ： 葉健全

臺北醫學大學/醫學院/醫學科學研究所/碩士(2001年)

摘要

中文摘要 花生四烯酸 (arachidonic acid)是許多重要不飽和脂肪酸之一，它是細胞膜之主要成分之一，也是重要的二級訊息傳遞物質，參與了許多生理及病理現象。過去的研究指出，在海馬迴神經細胞培養上,高劑量之AA其經由lipoxygenase pathway所產生的代謝會造成細胞死亡，由此可知AA在中樞神經細胞毒性上扮演了重要的角色。而本實驗室過去也在大腦皮質神經細胞培養上發現相似之情形，但是lipoxygenase抑制劑並無法完全阻斷AA的毒性。為了更加了解AA對神經細胞生存與發育的影響，與產生毒性作用的機轉和造成細胞死亡的方式，我們使用大白鼠胚胎大腦皮質細胞與PC12細胞來探討AA造成細胞毒性的機制，與是否走向apoptosis。結果發現，在大白鼠腦部皮質神經細胞體外培養3-5天AA並不會透過活化protein kinase C與cannabinoid receptor產生毒性作用，顯然AA是經由cycloxygenase，lipoxygenase，或epoxygenase其中一個pathway產生毒性，而在體外培養12-15天其部分毒性可能有透過活化protein kinase C的途徑。又AA會代謝成anandamide(ANA)，我們想知道ANA是否牽涉在AA的毒性機制中。而結果顯示ANA會造成神經毒性作用，在DIV3─5天細胞上顯然並不是經由活化protein kinase C或cannabinoid receptor之途徑，然而在DIV12─15天，抑制掉cycloxygenase，lipoxygenase，與epoxygenase pathway有增加毒性之現象。而抑制掉cannabinoid receptor或合併抑制cycloxygenase，lipoxygenase，epoxygenase pathway與PKC或cannabinoid receptor也是使毒性增加。 在AA對PC12細胞的影響方面，結果顯示在沒有神經生長因子 (NGF) 的狀態下，AA會造成細胞毒性作用(EC50=191.9±17.97)，劑量愈高毒性愈大且使細胞數減少(EC50=67.07±6.804)。而TUNEL assay也證實AA毒性作用會造成細胞走向apoptosis的趨勢。 　　[3H]thymidine　incorporation的數據顯示，AA 300mM顯著抑制細胞之[3H]thymidine　incorporation，而1-100mM之AA則增加[3Hthymidine incorporation。因此，1-100mM之AA可以促進細胞之DNA synthesis，也就是說1-100mM之AA可能可以促進細胞增生的作用。 在給予NGF之PC12細胞上，加入各濃度 AA處理後發現除了AA1mM之外，均隨劑量增加造成細胞死亡程度、走向apoptosis之比例增加(EC50=216.6±15.15)。 對於以NGF合併AA處理PC12細胞七天後，可以增加由NGF所誘發趨向分化細胞比例的這種現象，可能是由於細胞繼續複製增生的能力減少，而細胞趨向分化的能力增加，或是已分化完全的細胞進行apoptosis的速率降低所導致。而在給予NGF之PC12細胞上，AA並無法影響PC12細胞neurite outgrowth，原因可能是AA可以促進分化之initiation process，但卻比較無法影響分化之maturation of neurite framework。這與過去本實驗室發現naloxone對細胞之影響類似。

關鍵字

花生四烯酸

並列摘要

英文摘要 Polyunsaturated fatty acids are integral structural components of membrane phospholipids, where they play an important role in maintaining the structural and functional characteristics of bilayer cell membranes. Arachidonic acid (AA) is one of the most important fatty acids in biological systems including the brain, since it can serve as a precursor of a number of biologically active substances such as prostaglandins, leukotrines and thromboxanes. AA acts as an intracytoplasma second messenger in a variety of physiological or pathological condition, including the hypoxia-ischemia neuronal degeneration. Previous studies indicated that high concentration of AA produce overt neuronal cell death in culture and this toxic effect could be partly inhibited by lipoxygenase inhibitor. Our previous study also shows the similar data on primary neural cell culture, suggesting that the metabolites of AA in this pathway may be hazardous for neurons. Recently, anandamide has been identified as a brain AA derivative that actives cannabinoid receptors and protein kinase C. We further explore the mechanism underlying the AA-mediated toxicity using primary neuron cell culture and explore the effect of AA on cell survival, proliferation, and differentiation on pheochromocytoma PC12 cells. Mixed type cortical neuronal cultures were derived from E15-17 embryos of Spraque-Dawley rat embryos. On DIV3 and DIV12 cells were co-application of AA and various inhibitors for 2 hours. LDH in the culture medium were measured 24 hours after the treatment to serve as a quantification assay of cell death. Our results show that combine treatment with aspirin (cycloxygenase inhibitor), nordihydroguaiaretic acid (lipoxygenase inhibitor) and miconazole (epoxygenase inhibitor) could partly inhibit the AA induced toxicity in DIV3 cells but had no effect on DIV12 cells. Treatment with H7 (protein kinase C inhibitor) or AM281 (cannabinoid receptor antagonist) had no effect on AA or ANA induced toxicity on DIV3 cells. However,H7 partly decreased AA toxicity on DIV12 cells and no effect on DIV3 cells. We further determined the toxic effect of AA on the differentiated PC12 cell induced by nerve growth factor(NGF). AA concentration-dependent produced cell death. Tunnel assay studies showed that the AA-induced cell death including apoptosis. On the other hand, low concentration of AA (1mM) increased the induction of differentiation of PC12 cell by NGF stimulation but had no effect on the neurite growth of PC12 cells.

並列關鍵字

arachidonic acid

參考文獻

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