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研究生:丘翎燕
研究生(外文):Ling-Yen
論文名稱:維管束結合藥物對肺癌細胞的抗藥性基因之研究
論文名稱(外文):Genomic study of the tubulin-binding agent(TBA)-resistant A549 cells
指導教授:許國堂
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:2008
畢業學年度:96
語文別:中文
論文頁數:48
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肺癌是致死率最高的癌症,主要分成兩大類,一為非小細胞肺癌,其發生率為85﹪;另一類為小細胞肺癌,佔15﹪。非小細胞肺癌治療以手術切除、放射線治療和化學治療為主,臨床上可經開刀的早期病例僅約四分之一,可切除的病例不過15~18%,而不能開刀的病例使用化療藥物又常常會產生抗藥性,因此本篇研究,利用Tubulin-Binding Agents (TBAs)篩選出抗藥株,來研究抗藥性的機制。本實驗利用MTT毒性測試法來分析抗藥株的抗藥程度,發現不論是A549 Taxotere resistant cell line或A549 Vincristine resistant cell line對Taxotere和Vincristine均有抵抗性,且24小時的IC50比親代A549最少上升兩百倍,在48小時的IC50也比親代A549最少上升六十倍。
因為已知對TBAs產生抗藥性的機制主要有二種,第一種機制是腫瘤細胞出現multi-drug resistant (MDR;ABCB1) phenotype,此磷糖蛋白位於細胞膜上,是輸出藥物的幫浦,而Taxanes和Vinca alkaloids皆為此幫浦很好的受質,因此很容易經由此幫浦被送出細胞外而降低細胞中藥物的含量,造成藥物效力減少;第二種機制是因為細胞中tubulin產生改變。本實驗利用microarray和Real-time PCR分析,發現A549 Taxotere resistant cell line的ATP-binding cassette (ABC) transporter isotypes mRNA表現量均有上升的現象,而ABCB1是上升最多的,與TBAs抗藥性機制中,ABCB1大量表現相吻合,且再加入抑制劑Verapamil後,細胞的存活率明顯下降許多,而在A549 Vincristine resistant cell line中,ABC transporter isotypes mRNA表現量比親代A549細胞無明顯差異,且tubulin也無產生明顯改變,但是對Taxotere還是具有抵抗性,所以推測還有其它抗藥機制的存在,未來希望可以找尋其不同的抗藥機制,看是否有抑制劑可合併化療藥物使用,使抗藥性細胞對藥物敏感性增加。


Lung cancer is the leading cause of cancer deaths worldwide. Non-small cell lung cancer (NSCLC) accounts for about 85 %, while SCLC accounts for the rest 15 %. The tubulin-binding agents (TBAs) are used extensively in the treatment of cancer. However, the prognosis for patients remains dismal due to the emergence of drug-resistant tumors which significantly limits the clinical utility of these drugs in the treatment of NSCLC. Therefore, we study the TBAs resistant mechanisms on the TBAs-resistant cell lines. We established A549 taxotere resistant cell lines and vincristine resistant cell lines and analyzed their drug resistance by MTT assay. The obtained results from A549 taxotere- resistant cell lines showed 700-fold resistance against taxotere and >400-fold resistance against vincristine when compared with the parental A549 cell.. The A549 vincristine-resistant cell line showed 200-fold resistance against taxotere and 250-fold resistance against vincristine .
There are two drug-resistant mechanisms associated with TBAs. One is the tumor cells expressed multi-drug resistant (MDR; ABCB1) phenotype. The phosphoglycoproteins on the cell membrane are the pumps for exporting drugs. Taxanes and Vinca alkaloids are the ligands of the pumps. So the drug could be exported out of cells and reduced the drugs efficiency by the pumps. The other mechanism is the tubulin gene alteration. We found ATP-binding cassette (ABC) transporter isotypes, especially ABCB1 expression were upregulated in A549 taxotere-resistant cells analyzed by microarray and real-time PCR. Then we found that A549 taxotere-resistant cell survival rate was obviously decreased after treating with the ABCB1 inhibitor Verapamil. Interestingly, the ABC transporter isotypes mRNA and tubulin expression of A549 vincristine-resistant cell line was not different when compared to A549. cells. Since the A549 vincristine-resistant cell lines still resisted to taxotere treatment, therefore, we presume that other drug-resistant mechanism may exist in the vincristine-resistant cells. In the future, we want to search those drug-resistant mechanisms in addition to ABC, and find out inhibitors to cooperate with chemotherapy drugs to induce sensitivity of drug-resistant cancer cells.


目錄 頁次
壹、 中文摘要 1

貳、 英文摘要 2

参、 縮寫表 4

肆、 緒論

一、 肺癌 5

二、化療藥物Tubulin-Binding Agents (TBAs) 6

三、 ATP-binding cassette (ABC) transporter的結構及功能 8

四、 Microtubule的結構及功能 8

五、 Tubulin-Binding Agents (TBAs)的抗藥機制 9

伍、 研究動機 11

陸、 實驗材料
12
一、 藥品試劑
12
二、 細胞株
12
三、 儀器

柒、 實驗方法
14
一、 抗藥細胞株篩選
15
二、 細胞培養與分盤
16
三、 細胞毒性測試(MTT assay)

四、 細胞RNA的萃取 16

五、 cDNA的合成與Real time PCR 17

六、 西方點墨法(Western blot) 19

七、 免疫螢光染色 21

捌、 實驗結果

一、 利用MTT assay偵測抗藥株與親代A549細胞株對
TBAs的IC50 ( 24 h ) 22

二、 利用MTT assay偵測抗藥株與親代A549細胞株對
TBAs的IC50 ( 48 h ) 22

三、 利用microarray檢測抗藥株與親代A549細胞株基因的
變化 22

四、 在Taxotere抗藥株中,ABC transporter isotypes mRNA
會大量表現 23

五、 在Vincristine抗藥株中,ABC transporter isotypes mRNA
表現量是不同於Taxotere抗藥株 23

六、 在Taxotere抗藥株中,ABCB1蛋白會大量表現 23

七、 加入ABCB1抑制劑(verapamil) 後,Taxotere抗藥株對
化療藥物敏感性增加 24

八、 比較tubulin在抗藥株與親代A549細胞株中的相對表
現量 24

玖、 討論 25

拾、 圖表說明

圖一、 利用MTT assay偵測抗藥株與親代A549細胞株對
TBAs的IC50 (24h) 28

圖二、 利用MTT assay偵測抗藥株與親代A549細胞株對
TBAs的IC50 (48h) 29

圖三、利用microarray檢測抗藥株與親代A549細胞株基因
的變化 30

圖四、 比較A549、A549 Taxotere resistant cell line和A549
Vincristine resistant cell line中ABC轉運蛋白表現量
mRNA的差異 33

圖五、 在Taxotere抗藥株中,ABCB1蛋白會大量表現 35

圖六、 加入ABCB1抑制劑(verapamil)後,Taxotere抗藥株
對化療藥物敏感性增加 36

圖七、 比較tubulin在抗藥株與親代A549細胞株中的相對
表現量 38

拾壹、 附圖表

附表一、 非小細胞肺癌分組分期 39

附表二、 引子序列表 40

附表三、 SDS-page配製法 41

附圖一、 Mitosis 42

附圖二、 Structures of Taxotere and Vincristine 43

附圖三、 Structures of ABC transporters 44

附圖四、 Structures of MTT and its formazan product. 45

拾貳、 參考文獻 46


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