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研究生:簡靖純
研究生(外文):Chien, Ching-Chun
論文名稱:探討熱休克蛋白27在具化療抗性的肺癌幹細胞中所扮演之角色
論文名稱(外文):The role of heat shock protein 27 in drug-resistant lung cancer stem cells
指導教授:聶鑫聶鑫引用關係林曜祥林曜祥引用關係
指導教授(外文):Nieh, ShinLin, Yaoh-Shiang
口試委員:聶鑫林曜祥陳素鳳
口試委員(外文):Nieh, ShinLin, Yaoh-ShiangChen, Su-Feng
口試日期:101.5.25
學位類別:碩士
校院名稱:國防醫學院
系所名稱:微生物及免疫學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:53
中文關鍵詞:抗藥性
外文關鍵詞:drug resistant
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肺癌在全世界始終是癌症死亡的第一名,其中非小細胞肺癌就佔了八成五。肺腺癌是目前國人肺癌的重要細胞類型,五年的存活率為12~15%,值得注意的是,目前最令人憂心的女性肺腺癌,幾乎均是不吸菸的族群。目前臨床常用順鉑治療肺癌,但臨床上還是常發現癌症復發的情況,這有可能是因為癌細胞會產生對順鉑的抗藥性有關。
癌症幹細胞在產生抗藥性的過程中扮演重要的角色,但是目前的研究對於癌症幹細胞、順鉑的抗藥性與癌症的復發,這三者的關係並不是很清楚。在這篇研究我們嘗試由具化療抗性之細胞分離出具有癌症幹細胞特性之細胞,做為我們實驗的細胞模式,進而協助未來肺癌治療標的之設定。從細胞存活率分析發現,此種細胞對順鉑具有較高的抗藥性,另外,在此細胞也發現p38MAPK會活化熱休克蛋白27之現象。所以當我們抑制熱休克蛋白27時,會減低對順鉑的抗藥性,並會誘導細胞走向凋亡路徑。
綜合以上結果,我們發現p38MAPK-Hsp27路徑,在此細胞獲得抗藥性之過程中可能扮演重要的角色,此結果顯示未來這可能可以做為治療肺癌的參考。
Lung cancer is one of the leading causes of cancer-related death worldwide. Non-small cell lung carcinoma (NSCLC) is an aggressive type of lung cancer, accounting for the majority of the lung cancer. Cancer recurrence followed by relapse and metastasis is an obstacle in clinical treatment, often as a result of acquiring the resistance to cisplatin-based chemotherapy that is a common treatment for lung cancer patients. It has been known that cancer stem cells (CSCs) serve as a key factor for the development of drug resistance in tumors. However, the correlation among CSCs, cisplatin-resistance and tumor recurrence remains elusive. In the present study, we isolated sphere cells from cisplatin-resistant cells (DRSP) to serve as our cell model. In MTT assay, DRSP cells displayed greater resistant ability to cisplatin. Moreover, phosphorylation of heat shock protein (HSP) 27 via activation of p38 MAPK signaling was observed in DRSP cells. Knockdown of HSP27 reduced the cisplatin resistance and induced apoptosis of DRSP cells. Taken together, our results demonstrate the crucial role of p38 MAPK-HSP27 axis renders in CSCs-induced cisplatin resistance. We suggest that p38 MAPK-HSP27 axis is worthy of attention as a potential therapeutic target in lung cancer.
目錄

目錄 I
圖目錄 Ⅳ
中文摘要 Ⅴ
英文摘要 VI
第一章 緒論 1
第一節 肺癌 1
第二節 惡性肋膜積水 2
第三節 熱休克蛋白 3
第四節 癌症幹細胞 5
第五節 抗藥性 6
第六節 研究目的 7
第一節 實驗材料 8
壹. 肺癌細胞株介紹 8
貳. 實驗藥物 8
參. 抗體 8
第二節 實驗方法 9
壹. 培養肺癌細胞株NCI-H23 9
貳. 分離和培養惡性胸水中癌症幹細胞 12
參. 西方點墨法 (Western blot) 12
肆. 反轉錄聚合酶連鎖反應 (RT-PCR) 18
伍. 細胞存活率分析 (MTT assay) 20
陸. 流式細胞分析 (Flow cytometry) 21
柒. 免疫螢光染色 (Immunofluorescence) 22
捌. 短段髮夾核糖核酸轉殖 (siRNA transfecion) 22
第三章 結果 24
第一節 建立具化療抗性的肺癌幹細胞之研究模式 24
壹.以非貼附式的培養誘發肺癌細胞形成球體幹細胞 24
貳.利用cisplatin篩選出具化療抗性之肺癌細胞 25
參.鑑定誘導成球狀細胞之幹細胞特性 26
肆.鑑定誘導成球狀細胞之抗藥性特性 26
第二節 熱休克蛋白27 在DR-SP中扮演之角色 27
壹. 鑑定H23與DR-SP幹細胞及抗藥性蛋白表現 27
貳. 觀察熱休克蛋白27活化的現象 28
參. 觀察凋亡路徑活化的現象 28
肆. 觀察p38MAPK-Hsp27路徑對凋亡的影響 28
第四章 討論 30
第五章 結論 34
第六章 參考文獻 35
第七章 圖與表 40

圖目錄
圖一. 40
圖二. 41
圖三. 42
圖四. 43
圖五. 44
圖六. 45
圖七. 46
圖八. 47
圖九. 48
圖十. 49
圖十一. 50
圖十二. 51
圖十三. 52
圖十四. 53

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