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研究生:韓享祐
研究生(外文):Hsiang-Yu Han
論文名稱:大腸癌細胞與癌幹細胞之抗藥性及抗細胞凋亡特性之研究
論文名稱(外文):Investigation of the disparity of chemoresistance and anti-apoptosis between colon cancer cells and colon cancer stem-like cells
指導教授:謝銘鈞謝銘鈞引用關係
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:醫學工程學研究所
學門:工程學門
學類:綜合工程學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:英文
論文頁數:38
中文關鍵詞:大腸直腸癌癌症幹細胞抗藥性抗細胞凋亡流式細胞分選儀
外文關鍵詞:cancer stem-like cellreal-time PCRdrug resistanceanti-apoptosissphere culturecell sorting
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傳統治療癌症的策略為大量毒殺癌細胞。然而對於癌症後期的病患,這樣的方式並無法有效治癒。目前,越來越多的證據顯示只有少數特定的腫瘤細胞能形成新的癌組織。這些細胞與正常幹細胞所表現的性質有許多相似之處,例如具有自我更新、分化與不斷的增生的能力,因此被稱為癌症幹細胞。
癌症幹細胞因為具有較強的抗藥及抗細胞凋亡機制,不易被化療藥物殺死,而成為腫瘤復發的源頭。因此,進一步研究癌症幹細胞的抗藥及抗細胞凋亡機制有其必要性。在本篇論文中,我們選擇大腸直腸癌幹細胞做相關研究。
首先,利用流式細胞分選儀,將大腸直腸癌病患腫瘤組織分離出來原代培養細胞,以及大腸直腸癌症細胞株(HT-29)中帶有癌症幹細胞表面抗原的細胞篩選出來。
在細胞毒性測試中,我們驗證了癌症幹細胞對目前臨床化療藥物有比較強的抗藥性。接著比較非癌症幹細胞與癌症幹細胞兩者抗藥性及抗細胞凋亡基因的相對表現量。其中在大腸直腸癌症株幹細胞中,抗藥性基因ABCB1及ABCC3有相較於大腸直腸癌細胞8-21倍的表現。至於在抗細胞凋亡基因BCL-XL及NAIP,在人類大腸直腸癌症幹細胞有相較於大腸直腸癌細胞9-12倍高的相對基因表現。
在原代培養細胞中,癌症幹細胞的抗藥性基因ABCB1的表現量約是非癌症幹細胞52倍。至於抗細胞凋亡基因BCL-XL、NAIP、livin及Survivin,在大腸直腸癌幹細胞約高於大腸直腸癌細胞3-58倍的相對基因表現。由以上研究結果可推論,抑制這些高表現量基因,將可降低大腸直腸癌症幹細胞的抗細胞凋亡能力和對化療藥物的抗性,再結合臨床的標靶治療,或許是更有潛力的大腸直腸癌治療模式。
In recent years, the hypothesis of cancer stem-like cells (CSCs) received striking attention. This hypothesis indicated CSCs possess stem cell properties, the ability of self-renew and differentiate, and proliferate infinitely. Besides, they also have characteristics of chemotherapy-resistance and anti-apoptosis. CSCs are more difficult to eradicate and easy to cause cancer relapse because of these properties. Therefore, the mechanisms for CSCs which can escape from death after therapy merits further study.
Here, colorectal cancer cells (CRCs) were studied by investigating the characteristics of drug-resistance and anti-apoptosis within colon CSCs and non-CSCs. First, colon CSCs and non-CSCs were separated by FACS. The cytotoxicity assay showed that CSCs were more resistant to clinic chemotherapeutic drugs than non-CSCs. Moreover, the result of real-time PCR indicated that the gene expression of CSCs on certain drug resistant and anti-apoptosis was at a higher level than non-CSCs. Therefore, these findings provided practical information to design more effective therapeutic strategy for eradicating CRC.
中文摘要 Ι
英文摘要 Π
1.Introduction 1
2.Materials and methods 5
2.1 Materials 6
2.2Flow Cytometry Activated Cell Sorting ( FACSAria cell sorter) with CD133 mAb and CD44 mAb Staining 8
2.3 Sphere culture assay 9
2.4 Soft Agar Assay for Colony formation 10
2.5 Immunocytochemistry and confocal microscopy 10
2.6 Evaluation of cytotoxicity (MTT and WST-1 assay) 11
2.7 real-time PCR 12
2.8 Statistical analysis 15
3. Results 16
3.1 Flow Cytometry Activated Cell Sorting ( FACSAria cell sorter) 16
3.2 The stem cell culture condition 16
3.3 The colony formation assay 16
3.4 The proliferation ability determined by PCNA immunofluorescence staining 17
3.5 The sensitivity to chemotherapy drugs 17
3.6 real-time PCR 18
4. Discussion 19
5. Conclusion 23
6. References 24
7. Appendix 31
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