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研究生:劉姿君
研究生(外文):Tzu-Chun Liu
論文名稱:自CT-26小鼠大腸癌細胞株分離幹細胞群及其特性分析
論文名稱(外文):Characterization of the Stem Cell-like Population from CT-26 Mouse Colon Cancer Cells
指導教授:蘇瑀蘇瑀引用關係
指導教授(外文):Yeu Su
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生物藥學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:53
中文關鍵詞:CT-26小鼠大腸癌細胞株幹細胞
外文關鍵詞:CT-26 Mouse Colon Cancer CellsStem Cell
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大腸癌為一高轉移性的惡性腫瘤,目前對其形成的原因仍有許多未明之處。大腸癌幹細胞(Colon cancer stem cell, Co-CSC)已陸續自病人腫瘤組織和一些已建立的癌細胞株中被發現;該類細胞被認為是腫瘤形成、惡性轉移及抗藥性產生的元兇,因此了解大腸癌幹細胞與非幹細胞間的差異,將有助於發展更有效之治療方法。因此在本研究中,我以無血清但含有N-2, EGF和bFGF的培養液培養CT-26小鼠大腸癌細胞,懸浮的CT-26細胞球體(spheres)約在一星期後出現。將初級細胞球體打散並種植於六公分培養盤,約一星期後發現有次級細胞球體形成,以同樣方法將次級細胞球體打散並種植於六公分培養盤亦可於一星期後形成三級細胞球體,顯示CT-26細胞球體的確具有自我更新之能力。利用含有10% 小牛血清的RPMI培養基培養CT-26初級細胞球體,發現其能分化成近似原始以及多核細胞。利用即時定量RT-PCR 分析一些可能是Co-CSC的標記基因在CT-26細胞球體內的表現,發現CD44、CD29及Msi-1在細胞球體的表現高於原始的CT-26細胞,而西方點墨分析結果也顯示CD44蛋白在細胞球體內的表現高於原始細胞。利用皮下注射方式評估球體細胞腫瘤形成能力,結果發現注射少量細胞時,球體細胞即可形成腫瘤,腫瘤大小與原始細胞處理組無明顯差異,但其腫瘤形成率較原始細胞低。根據microarray的結果,我進一步利用即時定量RT-PCR分析CT-26原始細胞和細胞球體之基因表現差異,發現connective tissue growth factor (CTGF) mRNA在CT-26細胞球體內表現遠低於原始的CT-26細胞。雖然曾有研究指出在易轉移之大腸癌細胞株內CTGF表現較低,但我卻發現CT-26球體細胞的體外移動及侵犯能力較原始細胞差;在活體試驗中也發現CT-26球體細胞的轉移能力遜於其原始細胞。利用即時定量RT-PCR分析CT-26細胞球體內一些與epithelial-mesenchymal-transition (EMT) 有關的基因,發現N-cadherin及Slug在細胞球體的表現低於原始細胞,而fibronectin 和vimentin則無顯著差異。未來將進一步釐清CTGF在CT-26細胞球體的形成及其分化過程中扮演的角色。
Colorectal carcinoma (CRC) is a deadly malignancy due mainly to its metastasis. Recent discovery of colon cancer stem cells (Co-CSCs) that are responsible for tumor initiation, metastasis and relapse after therapy from the primary tumor tissues of CRC patients as well as several established tumor cell lines should be helpful in developing more effective therapies for this disease. Hence, distinguishing the differences between Co-CSC and their regular counterparts became an important task in cancer research. To enrich Co-CSCs from CT-26 mouse colon cancer cells, they were cultured in a serum-free RPMI medium containing N-2 supplement, EGF and bFGF. Floating spheres appeared one week after cells being cultured in this condition. We found that single spheroid cells could form secondary spheres which in turn could form tertiary spheres in self-renewal assays. After being incubated in 10 % FBS containing-RPMI, floating spheres became attached and differentiated into parental-like as well as multinucleated cells. Real-time RT-PCR was then performed to analyze the expression of several potential stem cell marker genes. We found that mRNA levels of CD44, CD29 and Msi-1 in CT-26 spheres were increased. We subcutaneously injected spheroid cells to the mice and tumors formed three weeks post-injection. However, the tumorigenecity of spheroid cells were lower than that of the parental cells. Interestingly, the tumorigenecity of spheroid cells increased 20% when injecting less cell number. To further elucidate the differences between CT-26 cells and spheres derived from them, microarray analysis was carried out. Among genes identified, the one encoding connective tissue growth factor (CTGF) appears to be most intriguing since its downregulation has been found in invasive colon cancer cells. Indeed, the mRNA levels of CTGF in CT-26 spheres were lower than those in the parental cells. We then examined several genes related to epithelial-mesenchymal-transition (EMT) since EMT usually accompany with metastasis. However, real-time RT-PCR analysis shown that except fibronectin and vimentin, the mRNA levels of N-cadherin and Slug in CT-26 spheres were lower than those in the parental cells. Indeed, both in vitro invasion and in vivo metastasis abilities of CT-26 spheroid cells were markedly lower than those of the parental cells, suggesting that downregulation of this growth factor in CT-26 spheres does not promote their invasion. Hence, the roles of CTGF in the formation and maintenance of CT-26 spheres are under investigation.
目錄

目錄 1
圖表目錄 2
中文摘要 3
英文摘要 4
緒論 5
實驗設計 10
實驗材料與方法 11
實驗結果 19
討論 24
參考文獻 30
圖表 36
行政院衛生署國民健康局2006年癌症登記報告

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