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研究生:張俊傑
研究生(外文):CHANG CHUN-CHIEH
論文名稱:上皮生長因子抑制劑引發c-Myc過度表現之老鼠乳癌細胞株抗藥性機制的研究
論文名稱(外文):Epidermal growth factor receptor inhibitor (EGFRI)-induced drug resistance in c-Myc-overexpressing mouse mammary carcinoma cell line
指導教授:王正康
學位類別:碩士
校院名稱:國防醫學院
系所名稱:生物化學研究所
學門:生命科學學門
學類:生物化學學類
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:77
外文關鍵詞:c-Myc EGFRI drug resistancE
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一般認為癌症病人抗藥性的產生,大部分是因為多重抗藥蛋白(MDR)過度表現而使化療藥物被排出所導致,可是利用MDR產生抗藥性的機制卻不能解釋癌症病人對於放射治療後仍會產生的抵抗性,所以推測應該是有其他的因子參與其抵抗機制!而在流行病學的統計結果顯示,c-Myc過量表現的癌症病患中,在化療一段時間後,病人體中便有抗藥性的情況出現,進而對預後產生不良的影響。從我們實驗室之前的研究結果觀察到: c-Myc過度表現的基因轉殖鼠癌化之乳腺上皮細胞Myc83,若生長在含有PD153035 (上皮細胞生長因子接受器酪氨酸基酶抑制劑 )的培養液中,則會有大量的細胞進行細胞凋亡,所以我們利用PD153035篩選出具有抗藥能力的Myc83細胞株 ( 稱為PD ),藉此模擬c-Myc過度表現的癌細胞產生抗藥性的情形,希望能找到和抗藥性產生相關的機制。為了使細胞在篩選過程中,能對於藥物傷害做出完全的反應,所以除了維持加藥48小時,更繼續增加篩選次數,以取得到更強的抗藥性細胞株,利用細胞凋亡測試証實經由PD153035篩選出來的抗藥性細胞所產生的抗藥性對普遍癌症的化學療法都具有抵抗作用。此外配合生長型態及生長速度的觀察,發現抗藥性細胞的生長狀況似乎有隨著藥物篩選次數的增加而有所改變。從過去的研究中推測隨著抗藥性增加,在個別繼代培養後p53有下降的趨勢,因此我們將不同抗藥性細胞個別做繼代培養後,觀察到p53的表現的確有下降的現象,但隨著繼代培養的代數增加,p53有回升的情形,由於推測抗藥性的產生可能和p53表現下降有關,所以我們將不同抗藥性細胞之間,個別繼代培養出來取p53表現量較低的代數,重新做加藥的實驗,結果觀察到抗藥性細胞的抗藥性能力的確隨著篩選次數增加而增加,另外這些不同抗藥性細胞在個別繼代培養過程中,亦伴隨有1. p53第十五位置絲胺酸磷酸化程度有先降後升,2. p38 MAPK活化的程度上升以及3. 細胞存活路徑Erk1/2有活化增加的現象,所以推測抗藥性的產生可能和這些有變化的蛋白質有關。
In general, acquired drug resistance is frequently observed in cancer patients with the treatment of chemotherapy. Chemotherapy-induced drug resistance is driven by MDR, which could be irrelevant for radiation resistance. Therefore﹑we hyphothesize there should be some other factor(s) contribute to drug resistance. According to Epidemiology, overexpression of c-Myc is associated with poor prognosis. Thus we utilized Myc83,a c-Myc-overexpressing mouse mammary carcinoma cell line, to study the possible relationship between c-Myc and drug resistance. Myc83 cells were treated with PD153035 to develop drug resistance cells ( PD cell line ).To obtain stronger drug-resistant cell clones , we prolong the time of treatment from previous 24 hours to 48 hours and avoid the possibility that the drug resistantance is caused by the delay of apoptosis. After a serial of treatments, we have selected different independent drug resistant cell clones by repeating treatment of PD153035. We named these drug resistant cells:PD8、PD14 and PD22,respectively.It is consisitant with MDR related papers that these resistant cells are also resistant to IR and 5-FU.To learn the drug resistance mechanism, we examined the morphology and proliferation rate and found dramatic change along with the increment of the PD153035 treatment.Oru previous results have shoen that PD153035 can trigger the down-regulation of p53, there we wanted to observe the level of p53 and subcultured the drug resistant cells and lets the cell fully respond to the impact of PD153035. Interestingly we find the level of p53 is decreased after individual drug resistant cell line subculture. However, p53 level is reversed in the late passage. Since p53 is critical for cancer therapy, we believe that there could be some relationships between drug-resistantance and the downregulation of p53. we also found the phosphorylation of p53 (serine-15) is decreased in early passage,but increased in late passage. when we checlced some signaling pathways, we also found the phosphorylation of p38 MAPK, and Akt, and Erk1/2 are increased. Therefore p53, p38 MAPK, Akt and Erk1/2 could be important in c-Myc-induved drug resistant mechanism. Our results have revealed some possible factors in c-Myc induced drug resistant mechanism, however further experiments need to be performed.
目錄
圖目錄 IV
縮寫表 VI
中文摘要 VIII
英文摘要 X
第一章 緒論 1
第一節 癌症 (惡性腫瘤的形成) 1
第二節 原致癌基因 (proto-oncogene)、致癌基因 (oncogene) 與抑癌基
因(Tumor suppressor gene) 3
第三節 原致癌基因:c-Myc 5
第四節 抑癌基因:p53 7
第五節 細胞凋亡 (apoptosis) 10
第六節 Caspases 13
第七節 Mitogen-activated protein kinase (MAPK)超級家族 14
第八節 上皮生長因子接受器 (epidermal growth factor receptor)所調控
之細胞存活訊息傳遞路徑 15

第九節 癌細胞的抗藥性 17
第二章 實驗材料與方法 19
第一節 實驗材料 19
第二節 實驗方法 25
第三章 實驗結果 35
第一節 PD153035藥物對c-Myc高度表現得老鼠乳癌細胞Myc83有大
量細胞凋亡的現象 35
第二節 利用Myc83細胞株篩選出具有抗藥性之細胞株 35
第三節 延長加藥時間篩選出的抗藥性細胞,觀察其生長型態及生長
速度 37
第四節 延長加藥時間篩選出的抗藥性細胞,觀察個別在繼代培養的
過程中,p53的表現情況 38
第五節 觀察抗藥性細胞間,個別繼代培養所得之p53表現量較低的細胞株重新加入PD153035、放射線及傳統化療藥物之後,觀察細胞凋亡的情形 39
第六節 觀察繼代培養之抗藥細胞在加入PD153035之後p53、pp38 MAPK以及p38MAPK的表現量 40
第七節 p38 MAPK蛋白質的移除對於抗藥性機制的影響 41
第八節 觀察c-Myc表現的移除,對於抗藥性機制的影響 41
第九節 觀察抗藥性細胞在繼代培養的過程以及加入PD153035、放射線
及傳統化療藥物之後的抗藥細胞株中細胞存活訊號傳遞路徑的
變化 44
第四章 討論 45
第一節 藉由增加藥物篩選次數成功由c-Myc過度表現之乳癌上皮
細胞得到對藥物具有更加抗藥性的細胞株 45
第二節 藉由增加篩選次數所得到的抗藥性細胞,其生長型態及生長
速度有逐漸改變的現象 46
第三節 藉由PD153035所篩選出的抗藥性細胞在繼代培養的過程
中都有觀察到P53蛋白下降的現象 48
第四節 將不同抗藥性細胞,個別繼代培養的過程中P53表現較低
的代數做加藥實驗,觀察細胞中蛋白質的變化仍具有抗藥性
程度上的差異 49
第五節 不同抗藥性細胞株,在個別繼代培養的過程中,PI3K/Akt及
Erk1/2訊號傳遞路徑可能參與抗藥性機制的發生對於降低細胞
中p38α MAPK及c-Myc表現的結果 50
第六節 對於降低細胞中p38α MAPK及c-Myc表現的結果 51
第五章 結論 52
第六章 參考文獻 54
圖 59
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