穀胱甘肽轉移酶（GST）在生物體內能對許多內源性及外源性的有毒物質進行解毒作用，是真核細胞中不可缺乏的重要解毒酵素。而在過去本實驗室的研究中發現，日本血吸蟲（Schistosoma japonicum）穀胱甘肽轉移酶會影響人類乳癌細胞MDA-MB 435s的生長，為了進一步去分析SjGST對MDA-MB 435s的生長及遷移的影響，我們首先將日本血吸蟲（Schistosoma japonicum）穀胱甘肽轉移酶的基因轉入人類乳癌細胞MDA-MB 435s中，進而分析SjGST-轉化細胞株在細胞生長及遷移的影響，結果顯示轉化細胞與未轉化細胞在生長上並沒有差異，基因分析顯示外源具有推架漂移突變基因。因而基因不能成功的表達。因此，我們另外利用重組SjGST蛋白進行體外細胞增生實驗，結果顯示SjGST顯著的促進乳癌細胞株MDA-MB 435s的生長。而傷痕癒合實驗結果證明，在SjGST存在的形況下細胞的遷移結果明顯比沒有外加SjGST的對照增高。此外，脫毒實驗證明SjGST能消除紫草素對細胞的毒殺作用，阻斷毒性物質對癌細胞的毒殺，在乳癌細胞外形成有效的保護作用避免細胞的死亡。本研究的結果指出，SjGST對人類乳癌細胞的增生與遷移具有顯著性的促進效果，同時也能有效阻斷毒性物質對癌細胞的毒殺作用，然而詳細的機制還需要進一步的研究。

Glutathione S-transferase (GST) is an essential detoxification enzyme in eukaryotic cells, by catalysing the conjugation of electrophilic substrates to glutathione (GSH) and detoxification both in external and internal cellular enviornments. Previous studies of our team suggested that SjGST had profound on the growth of human breast cancer cells in vitro. In order to analyse the effect of SjGST on the proliferation and migration of human breast cancer cell line, MDA-MB 435s , we initially constructed plasmid containing gst fragment in pAcGFP-N1 and transfected into MDA-MB 435s cells. Assays for proliferation and migration of the transfected cells showed that no difference between transfected and non-transfected cells was found. Analysis of the transfected gene found a frameshift mutation occurred in the plasmid, and thus no expression was detedted. Thus, we carried out assays for proliferation and migration of the cancer cells using recombiation SjGST instead. Results from assays for proliferation and migration showed that SjGST enhance both proliferation and migration of MDA-MB 435s. Furthermore, the recombinant protein was a strong inhibitor to cell death, probably by detoxification pathway. But the mechanism of action of detoxification of shikonin by the recombinant protein, however remains unknow.

圖錄 І
附圖表錄 Ⅲ
縮寫表 Ⅳ
中文摘要 Ⅵ
英文摘要 Ⅶ
第一章　緒 論 1
1.1　穀胱甘肽轉移酶GST介紹 1
1.2　穀胱甘肽轉移酶的生理功能 2
1.2.1 內外毒物的解毒代謝 2
1.2.2　高氧壓造成的有毒物質代謝 4
1.3　SjGST的結構 5
1.4　研究動機 7
第二章　材料與方法 8
實驗材料 8
實驗方法 12
2.1　SjGST基因工程改造 12
2.1.1　SjGST重組基因的製備 引子的設計 12
2.1.2　聚合酶鏈反應 12
2.1.3　瓊脂糖凝膠電泳分析 13
2.1.4　TA cloning 14
2.1.5　勝任細胞的製備 14
2.1.6　轉化作用 14
2.1.7　SjGST質體的純化 15
2.1.8　限制酶切割及質體片段從凝膠中的回收 16
2.1.9　連接反應 17
2.1.10　細胞培養(Cell culture) 18
2.1.11　乳癌細胞MDA-MB-435s的轉染 19
2.1.12　DNA定序分析 20
2.2　SjGST蛋白的表現與萃取 21
2.2.1　SjGST蛋白的純化 21
2.2.2　SDS- PAGE 22
2.2.3　SjGST蛋白質液含量的確定 23
2.3　細胞株的細胞學測試 23
2.3.1　細胞增生分析 (cell proliferation assay) 23
2.3.2　細胞遷移分析 (cell migration assay) 24
2.3.3　SjGST對紫草素脫毒實驗 25
2.4　統計分析 26
第三章　結果 28
3.1　SjGST基因工程改造 28
3.2　SjGST蛋白對癌細胞生長及遷移的影響 30
3.2.1　SjGST蛋白的純化 30
3.2.2　細胞增生結果 30
3.2.3　細胞遷移結果 34
3.3　SjGST對紫草素脫毒實驗 35
第四章　討論 37
4.1　SjGST基因工程改造 37
4.2　SjGST對癌細胞生長及遷移的影響 37
4.3　SjGST紫草素脫毒實驗 39
參考文獻 41
圖 47
附圖表 81

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