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  • 學位論文

Mitomycin C在培養細胞之細胞遺傳毒性與Luteolin在大白鼠缺血性傷害之心臟保護作用

Mitomycin C-induced Cytogenotoxicity in Cultured Cells and Cardioprotective Effect of Luteolin During Ischemia- Reperfusion Injury in Rats

指導教授 : 林瑞生

摘要


中國倉鼠卵巢細胞(CHO cell)、肺纖維母細胞(V79 cell)與人類T-淋巴細胞 (human T-lymphocytes) 常被用於探討化學物質在體外 (in vitro) 研究之細胞遺傳毒性,本研究所使用之細胞遺傳毒性測試法包括微核(micronuclei ; MN),姐妹染色分體交換(sister chromatid exchanges; SCEs)和染色體變異(chromosome aberrations; CAs)。絲裂霉素C (Mitomycin C;MMC)是一種使 DNA交互聯結的烷基化劑,臨床上用於抗癌,但過量或長期使用會致癌。本研究探討在MMC誘發此三種培養細胞細胞遺傳毒性與增加天冬氨酸特異性半胱胺酸蛋白酶 (cysteine aspartic acid-specific protease; caspase-3)活性之靈敏度差異性。本研究之三種培養細胞中,T-淋巴細胞對MMC所誘發的MN、CAs和SCEs細胞遺傳毒性皆展現出最高的靈敏度。CHO細胞和V79細胞則對此三種細胞遺傳毒性測試法的靈敏度呈現差異性;CHO細胞在MN測試法中的靈敏度高於V79細胞;在SCEs測試法之靈敏度則等於V79細胞;而在CAs測試法中則靈敏度高於V79細胞。進一步深入分析CAs顯示三種培養細胞之間亦有差異性:就染色分體斷裂 (chromatid breaks) 和雙中節 (dicentrics) 而言,T-淋巴細胞呈現最靈敏,而CHO細胞又比V79細胞靈敏;而就無中節 (acentrics) 和染色體間互換 (interchanges) 之變異,T-淋巴細胞的靈敏度大幅高於其他二種細胞。我們更進一步發現caspase-3活性增高對MMC誘發細胞遺傳毒性扮演著重要的角色, MMC誘發caspase-3活性增高之靈敏度T-淋巴細胞高於CHO細胞和V79細胞。 木犀草素 (Luteolin) 是一種存在於蔬果的類黃酮,為天然的多酚類化合物之一,如:豆科落花生(Arachis hypogaea)果實外殼,白毛夏枯草 (Ajuga decumbus) ,忍冬科忍冬 (Lonicera japonica Thunb),龍膽科植物濕生扁蕾(Gentianopsis paludosa),敗醬科黑水纈草(Valeriana amurensis Smir)均含有木犀草素。有學者研究顯示抗氧化物能有效減少缺血再灌流 (ischemia-reperfusion; IR) 的心肌傷害。木犀草素是具有抗氧化性質的類黃酮,本研究主要在探討木犀草素對於大白鼠心臟IR的損傷是否有保護作用?本研究使用大白鼠為標本,觀察短暫結紮冠狀動脈所造成心肌缺血和冠狀動脈血液再灌流之心肌傷害,以評估木犀草素之心臟保護作用。在冠狀動脈結紮前15分鐘,將木犀草素注入大白鼠靜脈,結果顯示在心肌缺血時,以木犀草素 (10μg/kg) 前處理可明顯降低心室心搏過速與心室顫動的發生率和持續時間以及大白鼠之死亡率。同樣地,木犀草素 (1μg/kg) 也降低冠狀動脈血液再灌流階段的死亡率及心室心律不整。利用木犀草素亦可減少血漿乳酸脫氫酶 (lactate dehydrogenase) 及一氧化氮 (NO) 含量。木犀草素 (10μg/kg) 具生物統計意義地降低心肌梗塞範圍大小,以及心肌IR傷害組織樣本的丙二醛 (malondialdehyde; MDA) 產量。我們更進一步證實木犀草素可減少誘導性NO合成酶 (iNOS) 之蛋白質和mRNA表現,但未明顯改變神經性NO合成酶 (nNOS) 或內皮細胞NO合成酶 (eNOS) 之表現。 結論:MMC在三種培養細胞至少部分可經由活化caspase-3增加MN、CAs和SCEs的頻率,由於T-淋巴細胞呈現最高之精確度、陽性預測值與靈敏度,使其成為最適合從事細胞遺傳毒性的標本。木犀草素能保護心肌對抗IR傷害。木犀草素保護心肌之作用機轉至少有一部分是藉由抑制iNOS降低NO形成和其本身所具有之抗氧化作用。

並列摘要


CHO cell, V79 cell and human T-lymphocytes are often used for investigating the cytogenotoxicity of cells in chemical substance in vitro studies. The cytogenotoxicity test methods applied in this study include micronuclei (MN), sister chromatid exchanges (SCEs) and chromosome aberrations (CAs). Mitomycin C (MMC) is an alkylating agent that causes DNA to cross-link and it is used for fighting cancer clinically but may lead to cancer in the long term or in excessive amounts. This study discusses the sensitivity difference of the three cultured cells with MMC-induced cytogenotoxicity and the addition of cysteine aspartic acid-specific protease (caspase-3). Among the three cultured cells, T-lymphocytes presented the highest sensitivity to the MMC-induced MN, CAs and SCEs cytogenotoxicity. CHO cell and V79 cell were different toward the sensitivity of the three test methods: CHO cell was more sensitive than V79 cell in MN and CAs tests, and had sensitivity equal to V79 cell in SCEs test. The three cultured cells also showed differences after CAs was further analyzed: in chromatid breaks and dicentrics, T-lymphocytes were the most sensitive and CHO cell was more sensitive than V79 cell; in acentrics and chromosome interchanges, the sensitivity of T-lymphocytes were much higher than the other two cells. We further discovered that the increment of caspase-3 activity played an important role in MMC-induced cytogenotoxicity. The MMC-induced increment of caspase-3 activity led to higher sensitivity in T-lymphocytes than CHO cell and V79 cell. Luteolin is a flavonoid in vegetables and is one of the natural polyphenol compounds. For example, Luteolin exists in fruit shell of Arachis hypogaea, Ajuga decumbus, Lonicera japonica Thunb, Gentianopsis paludosa, and Valeriana amurensis Smir. Previous studies indicated that antioxidants can reduce effectively myocardial ischemia-reperfusion (IR) injury. Luteolin is an antioxidative flavonoid, thus this study mainly investigates whether luteolin has protective effects on IR injuries of rats’ hearts. Using rats as samples, this study observed the myocardial injuries of ischemia and coronary artery reperfusion caused by ligation of coronary artery to evaluate the heart protecting effect of Luteolin. Luteolin was injected into a rat's veins 15min before the coronary artery ligation, and the results showed that, when ischemia occurred, the pretreatment by luteolin (10μg/kg) can significantly decrease the incidents and duration of ventricular tachycardia (VT) and ventricular fibrillation (VF) and the death rate of rats. The luteolin (1μg/kg) also lowered the death rate and ventricular arrhythmia in the coronary artery reperfusion phase. Using luteolin can also reduce the contents of lactate dehydrogenase and NO. In biological statistics, the Luteolin (10μg/kg) narrowed the range of myocardial infarction and reduced the malondialdehyde (MDA) yield of myocardial IR injury tissue samples. We further confirmed that luteolin can decease inducible NO synthase (iNOS) proteins and mRNA (imRNA) expression, but cannot change significantly the expression of neuronal NO synthase (nNOS) or endothelial NO synthase (eNOS). Conclusion:The MMC in the three cultured cells can at least partially increase frequencies of MN, CAs and SCEs by activating caspase-3. As T-lymphocytes presented the highest precision, positive prediction and sensitivity, they are the most suitable samples for studying genotixicity. Luteolin can protect cardiac muscles against IR injuries. At least part of the myocardial protecting effect of Luteolin is through restraining iNOS to reduce formation of NO and by its own antioxidative effect.

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