臺灣博碩士論文加值系統

近年來，在中國大陸曾記載，攝取到發霉的甘蔗，而導致了類亨汀氏舞蹈症（Huntington’s disease；HD）的症狀。學者進行分析研究後發現，發霉甘蔗內的Arthrinum黴菌，所產生毒性物質三硝基丙酸（3-nitroproponic acid；3-NP），會造成紋狀體神經細胞的退化，導致類似亨汀氏舞蹈症的病理學特徵。動物的研究顯示出，雌性素（estradiol，E2）能夠減弱三硝基丙酸所誘發的類舞蹈症的運動症狀。然而，目前並無直接的證據指出雌性素能夠抑制三硝基丙酸對紋狀體細胞所造成之毒性，本論文的目的為探討在初級紋狀體細胞中雌性素對於三硝基丙酸所引發之毒性是否具有保護或治療的作用。實驗材料為取自懷孕14~17天的大白鼠胚胎紋狀體窿突，經初級培養後的紋狀體細胞。利用WST-1檢測來分析三硝基丙酸及雌性素對於細胞存活率的影響情形，並結合DAPI（4’，6-diamidino-2-phenylindole） 及麩胺酸脫酵素（GAD）免疫細胞化學染色來觀察三硝基丙酸對紋狀體伽瑪胺基丁酸（γ-aminobutyric，GABA）毒性之反應狀況。實驗結果顯示，單獨給予10-5 M雌性素會明顯增加紋狀體細胞的存活率（148±3.0%，P<0.05），而藉由DAPI染色結果也發現，經雌性素處理的細胞會減少伽瑪胺基丁酸細胞凋亡的現象。另外，單獨加入3 mM 三硝基丙酸處理後經由WST-1分析及DAPI免疫化學染色的結果顯示，3mM三硝基丙酸作用6小時即有毒性反應，至加入後48小時其毒性可達最大作用。另一方面，預先加入10-5M 雌性素 48小時候再加入3mM 三硝基丙酸藉由WST-1分析、DAPI及GAD免疫細胞化學染色發現細胞存活率（103.16±4.6%）幾乎到達沒有加藥處理的控制組（100±4.5%）水準，並且細胞死亡的程度也明顯（P<0.05）減少。然而本實驗中亦發現到當施以3mM 三硝基丙酸前處理6小時造成細胞損傷後再加入雌性素處理，其WST-1結果發現仍具保護作用，但是經觀察GABA神經元的DAPI及免疫細胞化學染色後仍發現其具有顯著（P<0.05）之毒性反應，顯示當三硝基丙酸造成神經損傷後，雌性素對GABA神經細胞並不具有保護效果。另外，將雌性素及三硝基丙酸同時處理6小時後的分析結果也發現，雌性素可抑制三硝基丙酸之毒性作用而具有神經保護作用。根據上述結果顯示，雌性素處理能夠抑制初級紋狀體培養細胞的凋亡，並能保護三硝基丙酸對於紋狀體細胞的毒性作用。

In recent years, ingestion of mildewed sugarcane caused Huntington's disease (HD) like syndrome in Southern China. It prompted further investigation and the corresponding results showed that was due to the toxicity of 3-nitroprpionic acid (3-NP) produced by Arthrinum in mildewed sugarcane. Metabolic compromise with systemic 3-NP resulted in the degeneration of striatal cell, mimicking the pathology of HD. In vivo study had shown that estradiol （E2） was able to attenuate HD like syndrome, which induced by 3-NP. However, there was no direct evidence to indicate that E2 could prevent 3-NP-induced toxicity on the striatal cells. In this study aimed to test the protecting and/or therapeutic effect of E2 to 3-NP-induced toxicity on primary striatal cell culture. The primary striatal cell cultures were prepared from E14-17 embryos. The WST-1 test was examined for the viability of normal, E2 and 3-NP treated on the striatal cell culture. DAPI and GAD immunocyto-chemistry double stains were applied to reveal 3-NP toxicity. The data showed that cell viability of striatal cells were obviously increased when singly treated with 10-5M E2. It could be also discovered that according to the corresponding result of DAPI stain, apoptosis rate were depressed for cells treated with E2. Besides, the results of WST-1 assays and DAPI stain showed that if 3-NP was solely treated with dose of 3mM, toxicity response was detected after 6 hours and extended to the max degree after 48 hours. Furthermore, when pretreated with 10-5M E2 and then treated with 3mM 3-NP after 48 hours, cell viability was increased almost to the degree of control level and apoptosis rate was also apparently decreased. However, it could also be found in our experiment that if E2 was added after pretreated with 3mM 3-NP for 6 hours, the corresponding data of WST-1 assays would showed that the protection from E2 was still valid, while observable toxicity response was also detected form the DAPI stain of GABA neurons. These phenomena described above could be concluded that E2 will not protect neurons when 3-NP induced injury was activated on neurons. On the other hand, after concurrently treated with E2 and 3-NP for 6 hours, we could observe that 3-NP induced toxicity would be inhibited due to the protection of E2. Hence, E2 could protect striatal cells against 3-NP toxicity via various modulating mechanism.

目 錄 I
表 目 錄 Ⅳ
圖 目 錄 Ⅴ
中 文 摘 要 Ⅶ
英 文 摘 要 Ⅸ
緒 論 1
壹、亨汀氏舞蹈症 1
貳、三硝基丙酸 3
一、三硝基丙酸的化學成分 3
二、三硝基丙酸的動物模式 4
三、三硝基丙酸的神經毒性 5
參、雌性素的簡介 8
肆、雌性素對於中樞神經系統的保護作用 9
伍、雌性素與三硝基丙酸 12
陸、細胞凋亡 13
柒、研究目的 16
實驗設計 17
實驗材料 18
壹、細胞來源 18
貳、細胞培養液的製備 18
參、免疫細胞化學染色抗體 18
肆、其他化學試劑 19
伍、儀器設備 21
實驗方法 22
壹、神經組織的製備 22
貳、胚胎細胞的初級培養 22
參、實驗藥物之給予 24
肆、免疫細胞化學染色 25
伍、WST-1分析法 26
陸、統計分析 26
結果 27
壹、初級培養神經細胞之型態與純度鑑定 27
貳、雌性素對初級培養紋狀體細胞的作用 28
參、三硝基丙酸對初級紋狀體細胞的作用 28
肆、雌性素增加三硝基丙酸處理之細胞存活率 30
伍、雌性素於三硝基丙酸造成細胞毒殺作用後仍可減輕細胞死亡 31
陸、雌性素可抑制三硝基丙酸之毒性作用 32
討論 34
結論 40
參考文獻 66

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