臺灣博碩士論文加值系統

麩胱甘肽轉移酶 (Glutathione S-transferase, GST)在細胞的毒物代謝機制中扮演重要的角色，它可移除來自細胞外的毒性物質並減少毒性物質對細胞的傷害。 MDM2為一種致癌基因，腫瘤組織中除了表現完整的MDM2 mRNA之外也會有多種不同大小的MDM2 mRNA表現；而這類不同大小的MDM2 mRNA所轉譯的蛋白產物可能造成腫瘤發生的危險。李輝教授與柯俊良教授共同針對117位非小細胞肺癌病人及70位非肺癌病人組織中的MDM2 mRNA表現情形進行分析，證實肺癌病人組織中的MDM2 mRNA選擇性短小變異比例為26.4% (n=31)明顯高於非肺癌病人 (8.5%,n=6) (p＜0.001)；其中抽煙習慣及性別與MDM2 mRNA的選擇性短小變異發生有高度的正相關性，而MDM2 mRNA選擇性短小變異型態又以具有腫瘤惡質化特性的MDM2-657居多。有鑑於此，本研究欲進一步探討是否香菸中的毒性物質可造成MDM2 mRNA選擇性短小變異。本研究首先分析多株正常與肺癌細胞株中的MDM2 mRNA表現情形，發現大部分的正常肺細胞株只表現完整片段的MDM2 mRNA；而肺癌細胞株中除了H1355及CaLu-1細胞外，大多數細胞株均會有MDM2 mRNA選擇性短小變異的發生。進一步將H1355細胞處理於香菸中多環芳香烴化合物；Nested RT-PCR分析發現：B[a]P與BPDE於1μM及0.5、2μM可誘發三種選擇性短小變異的MDM2 mRNA表現。值得注意是，透過序列分析選擇性短小變異的MDM2 mRNA發現缺失部位存有某些特定序列，而體外轉錄轉譯蛋白實驗（in vitro transcription/translation）也證實，除了MDM2-966之外，其他選擇性短小變異的MDM2 mRNA均可轉譯出不同大小的蛋白產物。接著，RT-PCR分析發現，當MDM2 mRNA發生選擇性短小變異時，splicing factor-SR-A1的基因表現亦會增加。另外，Actinomycin D預處理可有效抑制BPDE誘發MDM2 mRNA發生選擇性短小變異。本研究結果證實香菸中所含的毒性物質-B[a]P及BPDE會造成MDM2 mRNA發生選擇性短小變異。為了要進一步探討由BPDE所造成的DNA adducts是否會增加MDM2 mRNA發生選擇性短小變異的機率；以及是否透過細胞中的解毒機制可有效避免DNA adducts形成，並進一步減緩MDM2 mRNA發生選擇性短小變異。因此，本研究分析多株正常肺細胞與肺癌細胞中毒物代謝酵素-麩胱甘肽轉移酶酵素活性，發現在正常肺細胞株中以MRC-5表現量最高，肺癌細胞株中以A549表現量最高而H1355表現量最低。RT-PCR分析上述細胞中麩胱甘肽轉移酶表現型，顯示肺癌細胞株內以GST-P1為主，但H1355細胞僅表現GST-M4；而正常肺細胞株除了GST-A1之外其它均有表現，但MRC-5不表現GST-M1。以MTS assay證實，BPDE-0.5μM劑量下對於H1355細胞有明顯的細胞毒性，但MRC-5細胞並不明顯；而BPDE-2μM劑量下的細胞毒性差異並不大。進一步比較H1355及MRC-5細胞在BPDE處理下，MDM2 mRNA發生選擇性短小變異的情形，結果發現僅H1355細胞發生MDM2 mRNA選擇性短小變異，而MRC-5細胞則無此現象。因此，以RT-PCR增幅MRC-5細胞中GST--M2及M4的基因表現，並將GST-M2或M4基因穩定表現於H1355肺癌細胞株。Nested-RT PCR及競爭性ELISA分析發現，穩定表現GST-M2之H1355細胞株可降低BPDE所造成的DNA-adducts，同時也可減緩經由處理BPDE後所誘發的MDM2 mRNA選擇性短小變異。此外，研究也發現GST-M2可減緩H1355細胞株在處理BPDE後所造成細胞週期之S phase堆積。綜合以上結果，本研究證實穩定表現GST-M2於H1355肺癌細胞株可有效的降低經由BPDE所造成的DNA adducts，並減緩MDM2 mRNA發生選擇性短小變異；同時也說明了GST-M2酵素活性對於降低BPDE造成之DNA 傷害可能扮演重要的角色。

Cellular detoxification is important for the routine removal of environmental and dietary carcinogens. Glutathione S-transferases (GST) is major cellular phase II detoxification enzymes. In addition to full length MDM2 transcript, multiple spliced forms of MDM2 transcripts have been observed in human tumors and may contribute toward tumorigenesis. The collective results of Huei Lee, Ph.D and Jiunn-Liang Ko, Ph. D. have shown that the frequency of MDM2 splice variants in lung cancer patients (31 of 117; 26%) was significantly higher than that of nontumor patients (6 of 70; 8.57%). Interestingly, there was a higher frequency of MDM2 splice variants among smoker and sex. The major splice variant was MDM2-657, a splice variant that was significant associations with tumor malignant. First of all, the study investigates whether the toxinogens of cigarette smoking would induce alternative splicing of MDM2 mRNA. Furthermore, most normal lung cell lines examined possessed only full-length MDM2 mRNA, while among several lung cancer cell lines; only H1355 and CaLu-1 cells lacked alternatively spliced MDM2 transcripts. Among H1355 cells treated in vitro with PAHs, B[a]P or the B[a]P metabolite benzo[a]pyrene diolepoxide (BPDE)-mediated MDM2 splicing were detected by nested RT-PCR. To deserve to be mentioned that splicing occurred at cryptic splice donor and acceptor sites in regions with high sequence homology. All of the MDM2 splicing variants were translated into protein in vitro, except MDM2-995. Furthermore, RT-PCR have manifested that alternative splicing of MDM2 was mediated by splicing factor, such as SR-A1. In addition, BPDE-induced MDM2 splicing was prevented with the actinomycin D treatment prior to BPDE exposure. Finally, to investigat whether the formation of DNA adducts increase frequency of MDM2 splicing and what’s the factor in cells would prevent from DNA adduct formation and further alleviate MDM2 splicing. Therefore, the enzyme activities of total GST were analyzed among normal and tumor lung cell lines. The GST activities of MRC-5 was highest than other normal lung cells, then A549 was expressed highest GST activities than other lung cancer cells. Interesting, the catalytic activity of H1355 was few to undetectable. Furthermore, RT-PCR shown that the phenotypes of GST in MRC-5 were involved GST-P1, GST-M2 and GST-M4, but in H1355 cells only expressed GST-M4. MTS assay found that the cell toxicities of BPDE-0.5μM was significant difference between MRC-5 and H1355 cells, but not different from BPDE-2μM treatment. Therefore, on the same dosage of BPDE-2μM treated, the alternative splicing of MDM2 was only detected by nested RT-PCR in H1355 cells but not in MRC-5 cells. Further, GST-M2 or GST-M4 was overexpressed in H1355 cells. The nested RT-PCR and competitive ELISA were demonstrated that overexpression of GST-M2 would prevent from DNA adduct formation and further alleviate MDM2 splicing. The additional finding, overexpression of GST-M2 in H1355 cells would alleviate BPDE-mediated S phase accumulation. Experiments were performed to define the overexpression of GST-M2 in H1355 cells would prevent from BPDE-mediated DNA adduct formation and further alleviate MDM2 splicing. The catalytic activity of GST-M2 might play an important future role in lowering the incidence of BPDE-induced DNA damage.

目錄
謝誌…………………………………………………………………….. 2
目錄…………………………………………………………………….. 4
縮寫表………………………………………………………………….. 7
論文題目……………………………………………………………….. 8
中文摘要……………………………………………………………….. 9
英文摘要……………………………………………………………….. 11
一、前言與MDM2致癌基因的背景…………………………………. 13
二、 MDM2致癌基因與腫瘤形成的關係…………………………… 14
三、選擇性短小變異的MDM2 mRNA與腫瘤的關係……………… 15
四、香菸曝露與肺癌之間的關係……………………………………… 17
五、選擇性短小變異的MDM2 mRNA表現與肺癌發生之間的關係 18
六、暴露BPDE與MDM2 mRNA選擇性短小變異的關係………… 19
七、外來毒性物質於細胞內的移除機制……………………………… 19
八、麩胱甘肽轉移酶的功能與重要性………………………………… 21

研究動機……………………………………………………………….. 24

研究材料與方法……………………………………………………… 25
組織收集…………………………………………………………… 25
試劑及細胞培養材料……………………………………………… 25
Nested RT-PCR分析腫瘤組織中選擇性短小變異MDM2 mRNA表現…………………………………………………………….. 26
分析選擇性短小變異的MDM2序列……………………………... 27
處理不同種類之香菸中多環芳香烴……………………………… 28
分析H1355細胞內之MDM2 mRNA表現………………………. 28
以定量PCR分析H1355細胞內之MDM2基因表現量………… 28
細胞培養及致突變藥劑與抑制劑處理、RNA萃取與RT-PCR分析……………………………………………………………… 30
以體外蛋白轉錄/轉譯系統進行MDM2蛋白轉譯……………….. 31
統計分析…………………………………………………………… 31
以MTS Assay分析BPDE對MRC-5及H1355細胞造成之細胞毒性…………………………………………………………… 31
以RT-PCR分析正常及肺癌細胞中的麩胱甘肽轉移酶表現情形. 32
構築麩胱甘肽轉移酶-M2與M4 表現載體並穩定表現於H1355細胞中……………………………… 32
麩胱甘肽轉移酶 (GST)之活性測定……………………………... 33
處理non-substrate ligand之麩胱甘肽轉移酶 (GST)抑制劑…….. 34
以西方點墨法分析GST -M2及M4之蛋白表現………………… 35
萃取genomic DNA………………………………………………… 35
BPDE-DNA 製備………………………………………………….. 36
競爭性ELISA……………………………………………………… 36
分析細胞週期變化………………………………………………… 38
西方點墨法分析細胞週期之相關蛋白變化……………………… 38

實驗結果……………………………………………………………….. 41
一、Nested RT-PCR分析選擇性短小變異MDM2 mRNA於肺細胞 中的表現情形…………………..…………………………….. 41
二、H1355細胞處理不同種類的香煙中多環芳香烴………………… 41
三、H1355細胞處理B[a]P及BPDE誘發選擇性短小變異的MDM2 mRNA表現……………………………………………………. 42
四、RT-PCR分析經由BPDE所誘發之MDM2 mRNA表現……….. 42
五、MDM2 序列分析與比較………………………………………… 43
六、利用in vitro translation分析選擇性短小變異的MDM2蛋白表現情形……………………………………………………..… 44
七、BPDE透過PI3K 或 MAPK路徑誘發MDM2 發生選擇性短小變異………………………………………………………..… 45
八、Splicing factor SR-A1調控BPDE或B[a]P所誘發之MDM2 mRNA選擇性短小變異……………………………………… 46
九、MDM2 splicing的現象是否為de novo RNA synthesis？……… 46
十、RT-PCR分析正常及肺癌細胞株之GST基因表現…………….. 47
十一、分析正常及肺癌細胞株之麩胱甘肽轉移酶(GST)酵素活性表現…………………………………………………………… 47
十二、MRC-5與H1355對於BPDE暴露後之毒性分析
十三、MRC-5細胞可抵抗BPDE所誘發的MDM2 mRNA選擇性短小變異………………………………………………………. 48
十四、利用西方點墨法分析GST-M2及M4穩定表現於H1355細胞株的情形………………………………………………….. 49
十五、分析穩定表現GST-M2及M4的H1355細胞株之麩胱甘肽轉移酶(GST)酵素活性……………………………………... 49
十六、穩定表現GST-M2於H1355細胞株可有效減緩經由處理BPDE所誘發的MDM2 mRNA選擇性短小變異……….... 50
十七、體外實驗證實GST-M2可降低經由BPDE所造成之DNA傷害…………………………………………………………….. 50
十八、處理non-substrate ligand inhibitor可有效抑制GST-M2的酵素活性……………………………………………………….. 51
十九、穩定表現GST-M2可減緩BPDE所誘發之S phase accumulation………………………………………………… 52
二十、穩定表現GST-M2可影響與細胞週期進行之相關蛋白表現.. 52

討論……………………………………………………………………. 54
未來展望……………………………………………………………….. 67
圖表與附圖表…………………………………………………………. 69
表一與表二………………………………………………………… 70
圖一……………………………………………………………….. 71
圖二A………………………………………………………….…. 72
圖二B……………………………………………………………… 73
圖二C……………………………………………………………… 74
圖三A……………………………………………………………… 75
圖三B…………………………………………………………..…. 76
圖四A………………………………………………………….…... 77
圖四B………………………………………………………….….. 78
圖四C………………………………………………………….…. 79
圖五A………………………………………………………….….. 80
圖五B……………………………………………………………… 81
圖五C……………………………………………………………… 82
圖六.……………………………………………………………..…. 83
圖七A………………………………………………………….….. 84
圖七B……………………………………………………………… 85
圖八………………………………………………………………… 86
圖九……………………………………………………………….... 87
圖十A………………………………………………………….… 88
圖十B……………………………………………………………… 89
圖十一……………………………………………………………… 90
圖十二……………………………………………………………… 91
圖十三A…………………………………………………………… 92
圖十三B…………………………………………………………… 93
圖十三C…………………………………………………………… 94
圖十四A…………………………………………………………. 95
圖十四B…………………………………………………………… 96
附表一……………………………………………………………… 97
附表二……………………………………………………………… 98
附表三……………………………………………………………... 99
附圖一……………………………………………………………… 101
附圖二……………………………………………………………… 102
附圖三……………………………………………………………… 103
附圖四……………………………………………………………… 106
附圖五……………………………………………………………… 107
附圖六……………………………………………………………… 108
參考文獻……………………………………………………………….. 109
個人資料表…………………………………………………………….. 118
已發表之論文抽印本………………………………………………….. 120

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