塑化劑鄰苯二甲酸二（2-乙基己基）酯對人類神經幹細胞之多巴胺性神經元分化的影響

Effects of Di- (2-ethylhexyl) phthalate on dopaminergic neurons differentiation of human neural stem cells

高紀雅(Ji-Ya Kao)

指導教授 ： 林壯澔

高雄醫學大學/醫學院/醫學系生理學科碩士班/碩士(2015年)

摘要

塑膠是我們生活中重要組成成分，其在2006年的生產量就高達2.45億噸，且數量每年持續的增加，而塑膠成分中產量最為豐富的便是鄰苯二甲酸二 （2-乙基己基）酯（Di（2-ethylhexyl）phthalate，DEHP），每年至少有200萬噸的產量，其主要做為聚氯乙烯 （polyvinyl chloride，PVC）產品的塑化劑。以PVC為原料的產品，在日常生活中隨處可見，如食品包裝、化妝品、衣服、兒童玩具及醫療設備等。DEHP添加至塑膠中並未形成鍵結，因此非常容易釋出到環境中，而目前DEHP已被證實具有生殖毒性及致畸胎作用，甚至引發癌症，另外，目前也有研究發現 DEHP 暴露後造成大鼠腦部許多區域出現神經退化，胚胎或哺乳時期受到DEHP暴露後，幼鼠大腦中多巴胺神經元的發育和酪胺酸羥化酶（Tyrosine hydroxylase，TH）的表達明顯受到抑制，顯示DEHP對神經細胞或多巴胺性神經元可能具有毒性。因此我們想探討DEHP對分化中多巴胺性神經元相關蛋白包括神經絲中鏈蛋白（Neurofilament-Medium，NF-M）、TH、Bcl-xL和Hypoxia-inducible factor 1 alpha （HIF-1α）表達的影響。在實驗設計中，我們建立以人類神經幹細胞（Human neural stem cells，hNSC）分化成多巴胺性神經元的模式，利用細胞影像分析和西方墨點法，去確立我們的實驗模式建立和觀察DEHP對神經元分化聚集的影響以及其分化成多巴胺性神經元蛋白質NF-M、TH、Bcl-xL和HIF-1α的影響。本實驗以人類神經幹細胞為材料，建立多巴胺性神經元分化模式，並於第9天、第15天和第18天觀察細胞聚集。其結果顯示隨著天數增加，細胞聚集的面積越大。而NF-M的表達，亦隨著分化時間的增加而增加。而實驗結果亦顯示分化第18天的TH、Bcl-xL和HIF-1α表達量較第15天增加，顯示人類神經幹細胞分化成多巴胺性神經元與分化時間有關。以上述細胞培養模式觀察DEHP對多巴胺性神經元分化的影響，發現隨著DEHP劑量增加，分化第15天和第18天的細胞聚集面積越大，而NF-M、TH、Bcl-xL和HIF-1α表達在分化15天時，隨著DEHP的劑量增加而增加。但NF-M和TH的表達在分化18天時，則隨DEHP的劑量增加而降低，而Bcl-xL和HIF-1α的表達在分化18天時，隨著DEHP的劑量增加而增加，這些顯示出DEHP可能會透過HIF-1α和Bcl-xL影響多巴胺性神經元發育的成熟度，且隨著分化時間的不同而有所差異。

關鍵字

塑化劑鄰苯二甲酸二（2-乙基己基）酯 ； 人類神經幹細胞 ； 多巴胺性神經元 ； 分化

並列摘要

Plastics is commonly used in the world. The plastic production reached 245 million tons in 2006 and increased each year. Di- (2-ethylhexyl) phthalate (DEHP), manufactured at an annual rate of 2 million tons, is mainly plasticizer in polyvinyl chloride (PVC) plastic products. PVC-based products can be seen everywhere in our daily life, such as food packaging, cosmetics, clothes, children products, and medical devices. DEHP doesn’t bind covalently to the plastic molecules, therefore, it is easy migrating into the environment. It is well known the reproductive toxicity and teratogenesis of DEHP, and its potential on carcinogenesis. Previous studies revealed that developing rat brain occurred neurodegeneration after DEHP administration. Other evidences also showed that the number of tyrosine hydroxylase (TH) – positive expression neuron in rat brain was obviously decreased after prenatal or fetal DEHP exposure. Therefore, we investigated the effects of DEHP on dopaminergic neurons differentiation. The human neural stem cells (hNSCs) were used to study the effect of DEHP on dopaminergic neurons differentiation. We observed the effect of DEHP on cell aggregation during neurons differentiation. The western blot was used to observe the change of dopaminergic neurons marker protein including, NF-M, TH, Bcl-xL, and HIF-1α. The results showed that the size of cell aggregation was increased during differentiation at 9th day, 15th day, and 18th day. The level of NF-M protein also was increased after 10th day, 15th day, and 18th day differentiation. Compared with 15th day and 18th day, the level of TH, Bcl-xL, and HIF-1α protein also were increased. After various concentration of DEHP (10-5μM, 10-2μM) treatment for 15 and 18 days, the cell aggregation was increased does-dependently and time- dependently. The NF-M, TH, Bcl-xL, and HIF-1α also were elevated after various concentration of DEHP treatment for 15 days. But the expression of NF-M and TH protein were decreased after various concentration of DEHP treatment for 18 days. The expression of HIF-1α and Bcl-xL protein still was increased after various concentration of DEHP treatment for 18 days. The effect of DEHP on NF-M, TH, Bcl-xL, and HIF-1α was does-dependent. These results showed that DEHP could affect the dopaminergic neurons differentiation via HIF-1α and Bcl-xL.

並列關鍵字

Di- (2-ethylhexyl) phthalate ； human neural stem cells ； dopaminergic neurons ； differentiation

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