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研究生:黃嘉瑩
研究生(外文):Chia-Ying
論文名稱:麩胱甘肽轉移酶M2於肺癌細胞的表現量與減緩多環芳香烴DNA傷害之研究
論文名稱(外文):Study on expression of Glutathione S-transferase M2 gene in human lung cancer and alleviation of DNA damage exposed to benzo [a] pyrene
指導教授:柯俊良柯俊良引用關係
學位類別:碩士
校院名稱:中山醫學大學
系所名稱:醫學分子毒理學研究所
學門:醫藥衛生學門
學類:其他醫藥衛生學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:115
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麩胱甘肽轉移酶(Glutathione S-transferase, GST)是細胞毒物代謝機轉中的主要解毒酵素之ㄧ,它牽涉代謝細胞內的有毒物質,並減少毒性物質對細胞的傷害。過去本實驗室證實,穩定表現GST M2於H1355肺癌細胞株可有效的降低經由BPDE所造成的DNA adducts。本研究以RT-PCR與Real-time PCR分析正常肺細胞株(MRC-5、WI-38與Beas-2B)與肺癌細胞株(H1355、H1299、A549、Calu-1、CH27、H23、CL 1-0與CL 1-5)中GST M2 mRNA的表現。結果顯示,肺癌細胞株的GST M2基因表現量比正常肺細胞株低。但無論在正常肺細胞株或肺癌細胞株中,皆可發現另一GST isoform : GST Pi的表現,唯只有H1355肺癌細胞株偵測不到GST Pi的表現量。以Real-time PCR分析肺癌組織其GST M2基因表現量,結果發現比旁邊非腫瘤部分大約降低了7倍; GST Pi在肺癌組織的表現量比旁邊非腫瘤部分高大約2倍。當肺癌組織GST M2表現量喪失時,患者之預後有較差的趨勢。此外,GST M2基因並非因genomic DNA缺失而造成在肺癌細胞株中GST M2基因表現降低。將H1355細胞分別或共同處理(1)histone deacetylase(HDAC)inhibitor : TSA 及 demethylation agent : 5-aza-2’-deoxycytidine(5-aza-dC),皆無法回復GST M2基因的表現。因此,推測H1355細胞株中GST M2的不表現,並非因為啟動子的CpG發生甲基化或組織蛋白去乙醯化所造成。經Luciferase reporter 分析其promoter上多形性(C128T)位置,也與 GST M2 基因表現降低無關。因此H1355肺癌細胞株中GST M2表現量降低的機轉,仍待進一步釐清以彗星分析法的結果指出,GST M2可降低B[a]P對DNA的傷害。並且GST M2降低H1355細胞株經B[a]P處理後所造成細胞週期之S與G2/M phase堆積。綜言之,肺癌的成因與預後不佳可能與GST M2的喪失有關。



Glutathione S transferases(GSTs)are a family of phase II detoxification enzymes that catalyse the conjuation of glutatione(GSH)to a wide variety of endogenous and exogenous electrophilic compounds. In our previous studies, we found that the overexpression of GST M2 gene in H1355 cells prevented BPDE-mediated formation of DNA adducts. Furthermore, by using RT PCR and Real time-PCR, we found that the expression level of GST M2 was lower in lung cancer cells in comparison to normal lung cells. GST Pi expressed both in lung cancer cells and normal lung cells. However, in H1355 cells, GST Pi can not be detected by RT PCR. In comparison with adjacent normal tissue, expression level of GST M2, analyzed by Real Time-PCR, in lung tumor tissue was seven-times lower. However, expression of GST Pi in adjacent normal tissue was two-times higher than that in lung tumor tissue. Poor prognosis is associated with the low expression of GST M2 in lung tumor tissue. Undetectable expression of GST M2 in H1355 was not caused by the deletion of genomic DNA. The reduced expression GST M2 in H1355 cells can not be restored after treatments with histone deacetylase inhibitor: TSA or/and demethylation agent: 5-aza-dC. Neither the methylation of CpG in promoter nor the histone deacetylation contributed to the reduced expression of GST M2 in H1355 cells. The promoter activity of GST M2 was not associated with the polymorphism(C128T)in its promoter by using luciferase reporter assay. Further investigations will be needed to clarify the mechanisms of GST M2 down regulation in lung cancer cells. Comet assay demonstrated that overexpression of GST M2 in H1355 could alleviate B[a]P-mediated DNA damage. Additionally, overexpression of GST M2 in H1355 cells would alleviate B[a]P-mediated S and G2/M phase accumulation. Therefore, these results of this study suggest that lung carcinogenesis and poor prognosis of overall survival were associated with the reduced expression of GST M2 gene.




頁次
目錄 1
壹、 中文摘要 2
貳、 英文摘要 4
參、 縮寫表 6
肆、 緒論 8
伍、 研究動機 18
陸、 實驗設計 20
柒、 實驗材料 25
捌、 實驗方法 30
玖、 結果 57
拾、 討論 70
拾壹、 圖表說明 81
拾貳、 附圖與附表 106
拾參、 參考文獻 109



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