臺灣博碩士論文加值系統

化學治療是肺癌常見的治療方式，抗藥性的產生是化學治療失敗的主要原因。當產生抗藥性時，常會發生對於其他種化療藥物產生交叉抗性的情形，在先前的實驗中，我們利用A549細胞篩選出對歐洲紫杉醇(Doxetaxel；剋癌易)產生抗藥性的細胞株A549/D16及A549/D32進行研究。初步的結果發現對歐洲紫杉醇產生抗藥性的細胞株比起親代細胞A549，對於葉酸類化療藥愛寧達(Alimta)，不但沒有交叉抗性的情形產生，反而較為敏感。因此想要探討A549/D16及A549/D32對於愛寧達主要的標的基因及與藥物代謝相關的基因的蛋白表現以及愛寧達的敏感性產生的機制。我們在實驗中使用MTT、Clonogenic assay、Western blot等分析方式深入探討。在抗藥株細胞中愛寧達主要的標的基因Thymidylate synthase (TS)、dihydrofolate reductase (DHFR)及與藥物代謝相關的基因gamma-glutamyl hydrolase (GGH)與親代細胞株A549相比較表現均較低。在此我們證明對歐洲紫杉醇產生抗藥性的細胞株對愛寧達產生敏感是因為TS表現較低的關係；經過轉染表達TS後會減少對愛寧達敏感性。我們也發現A549細胞處理歐洲紫杉醇後，會導致p53上升，同時TS也下降，所以p53似乎會使得TS表現降低。希望我們的結果對於歐洲紫杉醇產生抗藥性的病人接受下一步的化學治療有更客製化的選擇。

Chemotherapy is the common treatment for lung cancer patients. The development of drug resistance is the most important cause of treatment failure. When cancer resistant to chemotheraputic drugs, it is commonly developed cross-resistance to other anti-cancer drugs. In previous experiments, we have used docetaxel to select A549 cells and two sublines of A549/D16 and A549/D32 have been established. We have found these two sublines were more sensitive to Alimta than the parental A549 cells. Therefore, we plan to further characterize the mechanism of Alimta sensitivity in these two A549/D16 and A549/D32 cells. We used MTT assay, Clonogenic assay, Western blot to determine the Alimta sensitivity and protein expression in these docetaxel-resistant cells. The data showed that thymidylate synthase (TS), dihydrofolate reductase (DHFR) and gamma-glutamyl hydrolase (GGH) protein expression were downregulated. We further showed that the docetaxel resistant lung cancer line with low TS determined higher Alimta sensitivity. When TS was overexpressed by transfection resulted in lower Alimta sensitivity. In addition, we also found that when A549 cells treated with docetaxel, the upregulated p53 was associated with a decline in the TS. The data indicated that p53 may downregulate TS expression. The genes of p53 and TS that mediate Alimta sensitivity in docetaxel-resistant A549 cells could be used as a biomarker for the second line chemotherapy. Our study may benefit those docetaxel-resistant lung cancer patients to have a tailed-made anti-cancer therapy.

壹、 中文摘要..........................1
貳、 英文摘要..........................2
參、 縮寫表............................3
肆、 緒論
一、 肺癌
1. 肺癌流行病學.......................5
2. 肺癌分類..........................6
3. 非小細胞癌分期.....................7
二、 抗藥性............................8
三、 化療藥物 Tubulin Binding Agents...8
四、 歐洲紫杉醇 Docetaxel................................9
五、 抗代謝藥物 Antimetablite...........9
六、 愛寧達 Alimta, Pemetrexed.........11
七、 其他相關基因.......................12
伍、 研究動機..........................14
陸、 實驗材料..........................15
柒、 實驗方法..........................19
捌、 實驗結果..........................33
一、 A549/D16及A549/D32細胞比親代細胞株對Pemetrexed較敏感.......................................33
二、 與親代細胞株相比，Pemetrexed對A549/D16及A549/D32細胞有較好的抑制群落生長的能力..................34
三、 與親代細胞株相比，Pemetrexed的標的蛋白在A549/D16及A549/D32表現都較低........................34
四、 利用anti-flag抗體確定在A549/D16中，短暫轉染TS會大量表現.......................................35
五、 Docetaxel對A549、H460、CL1-0、H1299、H1355細胞的p53及TS的影響................................36
六、 Wild type p53在TBAs上，似乎可以抑制TS的表現....37
玖、 討論..............................38
壹拾、 圖表及圖表說明
圖一、利用Clonogenic assay分析Docetaxel對A549、A549/D16及A549/D32細胞之群落生長的能力................44
圖二、使用MTT方法分析A549、A549/D16及A549/D32細胞株對Pemetrexed的敏感性.........................46
圖三、利用Clonogenic assay分析Pemetrexed對A549、A549/D16及A549/D32細胞之群落生長的能力................47
圖四、利用Western blot偵測A549、A549/D16及A549/D32的相關蛋白變化......................................49
圖五、利用Western blot偵測短暫轉染Flag、TS的表現…....50
圖六、利用MTT assay分析A549/D16及轉染vector及TS後的對於Pemetrexed的細胞存活率……………………………...51
圖七、利用Western blot偵測A549、H460、H1299、CL1.0及H1355經由Docetaxel刺激後的相關蛋白變化..........................52
圖八、利用Western blot偵測A549及H460細胞經由Docetaxel及Vincristine刺激後的相關蛋白變化.......................53
圖九、比較A549細胞經由Docetaxel及Vincristine刺激後的相關蛋白變化…………………………………………………...54
表一、A549、A549/D16及A549/D32對Docetaxel、Vincristine、Doxorubicine的IC50.....................55
表二、A549、A549/D16及A549/D32對Pemetrexed的IC50..56
表三、A549/D16、A549/D16/V/C及A549/D16/TS對Pemetrexed
的IC50.............................56
壹拾壹、 附表及附圖.......................57
附圖一、2012年全球癌症發生率……………………………...57
附圖二、民國102年台灣男女性十大癌症死因比較…………57
附圖三、Docetaxel結構式……………………………………...58
附圖四、Pemetrexed及Folic acid結構式……………....……..58
附圖五、Pemetrexed作用機制………………………………....59
附圖六、TS open reading frame質體之建築…………………..60
附表一、分析A549/D16及A549/D32細胞與親代細胞株中Pemetrexed藥物作用之相關基因的表現………………………61
壹拾貳、 參考文獻…………………………………………………..62

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