臺灣博碩士論文加值系統

近年來，大腸直腸癌(colorectal cancer)的發生率及復發率逐年增加，發生年齡年年下降，死亡人數也逐年高升，是目前台灣發生人數最多的癌症。根據腫瘤幹細胞假說，腫瘤的形成、轉移及復發，是由一小群特殊的癌細胞所造成，這些所謂的腫瘤幹細胞同時也具有幹細胞的特性，包括高度表現幹性基因。因此本研究假設，若將幹性基因外送至癌細胞株，也許可使癌細胞惡性程度增加。本實驗使用慢病毒 (lenti virus)將兩個幹性基因，Oct4或Nanog，過度表現在SW480及HT29兩株大腸癌細胞中，建立六株細胞株HT29-pCDH、HT29-Oct4、HT29-Nanog、SW480-pCDH、SW480-Oct4、以及SW480-Nanog，來觀測in vitro中細胞行為的變化以及in vivo腫瘤的生長和癌細胞在活體動物中細胞遷移之情況。
目前in vitro實驗結果顯示Oct4或Nanog過度表現會改變細胞形態。Oct4過度表現可增加細胞貼附的能力。Nanog則會增加SW480及HT29細胞株sphere及colony formation的能力，Oct4的過度表現則兩株細胞之colony formation的能力提升，但對於HT29之sphere formaiton能力沒有影響。同時Nanog過度表現可誘發其他stemness genes之蛋白質表現。Oct4基因過度表現對於HT29大腸癌細胞會提高IL-8的mRNA level，而Nanog基因過度表現則使SW480大腸癌細胞提高IL-6、IL-8、以及IL-32的mRNA表現量。然而Oct4過度表現對於細胞內許多訊息傳導路徑，包括β-Catenin、MAPK、及PI3K/AKT pathways並沒有顯著影響，而Nanog之過度表現在兩株細胞中卻會有相反的效應。小鼠異體移植實驗結果顯示，Oct4及Nanog過度表現之HT29細胞，在in vivo環境中明顯具有較強之tumorigenicity及轉移能力。
總而言之，Oct4及Nanog的過度表現，可增加HT29及SW480之部分腫瘤幹細胞特性，特別是活體腫瘤生長能力。然而此兩個基因對細胞內分子層級之影響，仍有待更進一步研究。

In recent years, increasing incidence and recurrence rate of colorectal cancers have been observed and ages of patience diagnosis with colorectal cancers are decreasing and the mortalities of patience with colorectal cancer are increasing. Besides, colorectal cancer is so far the most prevalence type of cancer in Taiwan. Hypothesis has that cancer may rise from a small population of cancer cells which possess stem cell like properties and these cancer stem-like/initiating cells involves many aspects of tumorigenesis: tumor formation, metastasis, and recurrences. Based on these assumptions, we ask whether it is possible to establish inducible cancer stem-like/initiating cells by transferring two stemness genes: Oct4 and Nanog in SW480 and HT29 which are both colorectal cancer cell lines. We totally established six colorectal cancer cell lines: HT29-pCDH, HT29-Oct4, HT29-Nanog, SW480-pCDH, SW480-Oct4, and SW480-Nanog as model systems for further studies for in vitro cell biological based studies and in vivo animal models for studying tumor growth and metastasis.
In vitro experiment results support that overexpression of Oct4 or Nanog promotes cell morphology and overexpressing of Oct4 increases cellular adhesion abilities of colorectal cells, while Nanog overexpression, on the other hand, increases the self-renewal abilities in both SW480 and HT29 cells and furthermore, overexpression of Nanog induces expressions of other stemness genes. Among those cytokines we had examined, overexpression of Oct4 increases the expression levels of IL-8 transcripts in HT29 cells, and Nanog overexpression triggers the expression levels of IL-6, IL-8, and IL-32 transcripts in SW480 cells. However, there is little or no effect of Oct4 overexpression on β-Catenin, MAPK, and PI3K/AKT signaling pathways. Surprisingly, overexpression of Nanog among HT29 and SW480 cells shows completely opposite effects. The animal model experiments have shown that overexpression of Oct4 or Nanong promotes stronger tumorigenicity effects in HT29 cell.
Namely, several tumorigenicity properties of HT29 and SW480 cells are increased in overexpression of Oct4 or Nanog, the effects are even more pronounced in in vivo experiments and metastasis. However, the detailed mechanisms downstream of Oct4 and Nanog still need further investigations.

中 文 摘 要 - 1 -
Abstract - 3 -
目 錄 - 5 -
圖 目 錄 - 7 -
緒 言 - 9 -
一、大腸直腸癌 - 10 -
二、癌症幹細胞 - 13 -
三、腫瘤幹細胞的基因調控 - 16 -
研 究 目 的 - 18 -
研 究 材 料 及 方 法 - 20 -
一、細胞培養(Cell culture) - 21 -
二、細胞活性分析(MTT assay) - 21 -
三、Cell cycle的檢定 - 22 -
四、細胞免疫染色(Immunofluorescence ) - 23 -
五、西方點墨法(Western blotting) - 24 -
六、Sphere formation assay - 26 -
七、蘇木紫-伊紅染色 (Hematoxylin ＆ Eosin stain) - 27 -
八、免疫組織化學染色（Immunohistochemical staining） - 27 -
九、質體製備 (Plasmid construct) - 29 -
十、慢病毒的製備與感染 (Lentivirus production and infection) - 30 -
十一、RNA的萃取 - 31 -
十二、反轉錄聚合酶鏈反應（Reverse-transcriptase polymerase chain reaction, RT-PCR） - 32 -
十三、即時定量聚合酶鏈反應（Real-time polymerase chain reaction ） - 32 -
十四、流式細胞儀 - 33 -
十五、非固著依賴性細胞生長 (Soft agar assay) - 33 -
十六、細胞貼附實驗 (Adhesive capacity assay) - 34 -
十七、活體腫瘤生長試驗 (In vivo tumorigenicity assays) - 35 -
結 果 - 36 -
討 論 - 52 -
結 論 - 57 -
參 考 文 獻 - 60 -
圖 表 - 67 -
附 錄 - 90 -

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