大鼠初代星狀膠質細胞與人類星狀膠質細胞株之丙烯醯胺毒性分析

The Analysis of Acrylamide-induced Toxicity on Rat Primary Astrocyte and Human Glioblastoma Cells

王嬿琇(Yan-Shiu Wang)

指導教授 ： 周晉澄

國立臺灣大學/獸醫專業學院/獸醫學研究所/碩士(2012年)

https://doi.org/10.6342/NTU.2012.02026

摘要

丙烯醯胺 (Acrylamide；ACR) 為一具有神經毒性、基因毒性、繁殖毒性與致癌性的工業用化合物，廣泛運用於過濾飲水與蛋白質分析。由於胺基酸會與碳水化合物在高溫 (120－180oC) 下藉由梅納反應 (Maillard reaction) 生成副產物ACR，特別是富含天門冬胺酸與還原糖的日常食品中所產生的ACR更是一般飲水限制量的數百倍，此發現引發食品安全的疑慮。
在人類流行病學上以及實驗動物的證據皆能證實ACR能影響神經元並造成神經毒性，但對於膠細胞的研究卻十分局限。星狀膠質細胞成為近年來神經毒性研究的焦點，其在大腦的角色不再是被動、簡單的物理性支持，而是能影響神經生長、神經電位傳導以及控制整個大腦微環境 (micro-environment) 等等的重要細胞。Chen等 [2009, 2010]研究發現，ACR使人類膠胚母細胞株U-1240 MG的增生率受抑制、細胞存活率降低，造成基因毒性，進而影響DNA 傷害反應路徑相關蛋白如ATM/ATR、p53、p21、p27、Cdk2及cyclinD1，並使細胞週期停滯在G0/G1期，但ACR對不同細胞株 (U-1240 MG、U-87 MG與U-251 MG) 引發的效果不同，本研究因此探討上述細胞株與大鼠初代星狀膠質細胞經ACR暴露後之細胞毒理與凋亡反應之異同。
MTT assay結果顯示，四株細胞經1 mM ACR處理48及72小時與2 mM ACR處理 24至72小時，細胞存活率均顯著下降，但0.1 及0.5 mM低濃度僅對U-87 MG及初代星狀膠質細胞具有顯著影響。LDH leakage assay顯示，U-1240 MG與U-251 MG至72小時皆無顯著變化，但U-87 MG及初代星狀膠質細胞在2 mM ACR暴露48及72小時下顯著上升。流式細胞儀分析顯示，ACR引發所有細胞株與初代星狀膠質細胞之sub-G1期增加，且JC-1染色粒線體膜電位亦有明顯變化，意即具細胞凋亡反應。進一步利用西方墨點法偵測DNA傷害反應路徑 (如p53與pp53) 及細胞凋亡途徑相關蛋白分子 (如Bcl-2、Bax、pro-caspase 8、9與3)，U-1240 MG及U-251 MG細胞經2 mM ACR暴露48小時後，Bcl-2蛋白表現量顯著增加，而在U-87 MG及初代星狀膠質細胞中則顯著減少；初代星狀膠質細胞暴露不同ACR濃度，Bcl-2表現量也不同，其在低濃度組 (0.1及0.5 mM) 顯著增加，在高濃度組 (1及2 mM) 則顯著減少。Bax與pro-caspase 8、9及3蛋白表現量在不同細胞以及不同濃度皆無顯著差異，Bax蛋白為上升而pro-caspase 8、9及3蛋白皆為下降。而在DNA傷害反應路徑中，2 mM ACR 48小時的暴露也造成p53及pp53蛋白的增加，利用caffeine抑制劑共同處理抑制掉ATM/ATR後也可得到效果回復的結果。由於ACR引發U-87 MG之MTT、LDH、sub-G1 arrest、p53、pp53與Bcl-2蛋白表現等與初代星狀膠質細胞較相似，因此在考量動物犧牲與初代細胞的培養時間及相關技術要求前提下，建議以此細胞株取代星狀膠質細胞從事ACR之毒性機制研究。

關鍵字

丙烯醯胺 ； 神經毒性 ； 細胞凋亡 ； 初代星狀膠質細胞

並列摘要

Arylamide (ACR) is common used in industry for clarifying drinking water and protein analysis. ACR contains neurotoxicity, genotoxicity, reproductive toxicity and carcinogenicity. Under high-temperature (120－180oC) cooking, food rich with amino acid, particular asparagine, and reduced glucose can produce ACR through Maillard reaction more than 100 times the limits in drinking water which raises food safety concern.
There are several evdences showed ACR influenced neurons and induced neurotoxicity in human epidemiological studies and animal experiments. However, However, there are limited studies on ACR-induced toxicity of glia cell. Until recently, The role of astrocytes in the brain is no longer simple and passive but plays an active function including influencing neurite growth and transmission of nerve impulses, and controlling microenvironment. Chen et al. [2009, 2010]reported two studies about ACR-induced cytotoxicity on U-1240 MG cell. Further studies showed ACR caused genotoxicity, arrested cell cycle in G0/G1 phase and influenced protein associated with DNA-damage response pathway including ATM/ATR, p53, p21, p27, Cdk2 and cylinD1. Nevertheless, different cell lines (U-1240 MG, U-87 MG and U-251 MG) had different reactions after ACR exposure, for example, U-251 MG cells had no regulation of p53 and p21 protein under ACR treatment but U-1240 MG and U-87 MG cells did. The aim of this study determines which cell line has mostly similar toxic effect with rat primary astrocyte in apoptosis pathway in order to further study the ACR-induced astrocytoxic machnism.
The results in MTT assay showed decreasing cell viability was found after ACR 1 mM under 48 and 72 h and 2 mM under 24 to 72 h exposure among four cells in time-dependent and dose-dependent manners. Interestingly, there were changes in low concentration (0.1 and 0.5 mM) only on U-87 MG and primary astrocyte. Also, ACR didn’t induced LDH leakage on U-1240 MG and U-251 MG but on U-87 MG and primary astrocyte after 48 and 72 h 2 mM ACR exposure in LDH assay. ACR raised the sub-G1 phase analyzed by PI stain and change of mitochrondral potential analyzed by JC-1 stain among four cells. Moreover, the apoptotic mechanism was under explored by measuring the DNA damage response pathway proteins including p53 and pp53 and apoptosis pathway associated-molecular proteins including Bcl-2, Bax, pro-caspase 8, 9 and 3 through western blotting analysis. 2 mM ACR increased p53 and pp53 activity in DNA damage response pathway. The co-treatment of ACR with ATM/ATR inhibitor caffeine can attenuate the increase amount of p53 and pp53. Bcl-2 changed with different cells: up-regulation was found on U-1240 MG and U-251 MG and down-regulation was found on U-87 MG and primary astrocyte after 48 h 2 mM ACR exposure. Besides, different concentration ACR also caused different response: up-regulation of Bcl-2 was found in 0.1 and 0.5 mM concentration and down-regulation was found in 1 and 2 mM concentration on primary astrocyte. The up-regulation of Bax and down-regulation of pro-caspase 3, 8 and 9 were found at 0.1, 0.5, 1 and 2 mM ACR 48 h treatment. In conclusion, U-87 MG had the more similar response with primary astrocyte after ACR exposure. We advise the researchers subsequently studied the machanism of ACR-induced astrocytotocixity that U-87 MG cell line should be the best choice for saving the time and technique of the culture of primary astrocyte.

並列關鍵字

Acrylamide ； Neurotoxicity ； Apoptosis ； Primary astrocyte

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