- 學位論文
葉酸缺乏促進癌幹細胞發展之探討
Folic deficiency Enhance Stemness Expression in Colon cancer cells
摘要
由於國人飲食習慣的西化，飲食越來越趨向高脂肪低纖維，導致大腸直腸癌在癌症主要死亡率逐年增加，多數研究認為大腸直腸癌罹患率攀升和飲食習慣中葉酸攝取量有很大關係。因為葉酸在哺乳動物體內無法自行合成，只能仰賴從飲食獲得，加上飲食西化的因素造成葉酸攝取缺乏，導致國人大腸直腸癌病人數逐年增加，已成為全民健康的一項重大問題。另一方面越來越多的研究認為人類的癌症可以被當作是一種幹細胞的疾病，在大腸直腸癌中研究中已經發現大腸直腸癌幹細胞的存在，而且被認為可能是導致腫瘤形成與惡性化的主因之一，使得癌症難以治癒。因此本研究的目的在於探討葉酸缺乏的情形之下是否可能導致癌幹細胞形成，期從癌幹細胞的特性誘發著手，透過當前分析癌幹細胞方法學來瞭解此一問題。一開始先看葉酸缺乏的環境下，大腸直腸癌細胞株的改變，結果顯示葉酸缺乏的環境下，C26細胞生長曲線相較於正常培養狀況要來的緩慢，但細胞並沒有死亡的情形﹔同時利用顯微鏡觀察細胞，發現葉酸缺乏下細胞型態也隨作用時間逐漸改變。細胞毒性試驗發現，在葉酸缺乏時細胞對於化療用藥Doxorubincin呈現抗藥性。由此結果可推測在葉酸缺乏的環境下，可能會引起腸癌細胞表現出癌幹細胞的特性。進一步利用流式細胞儀分析癌細胞的表面標記CD133和CD44表現狀況，結果發現當葉酸缺乏時，此二個腸癌細胞表面標記的表現量皆高於正常培養條件下。另一方面本研究也透過PT-PCR的方法分析與癌幹細胞相關之基因，如SOX2, Nanog, C-myc的表現，同樣發現這些基因表現皆呈現葉酸缺乏環境下高於正常培養的條件。本研究也利用癌幹細胞具較高致瘤性(tumorigenicity)方法測試，結果也發現腸癌細胞經葉酸缺乏環境馴化後，以較少量的細胞數就能誘發小鼠身上的腫瘤生成。綜合上述研究結果，吾人推測葉酸的缺乏，的確可誘導癌幹細胞族群生成與增加。
並列摘要
Due to the westernization of diet, such as high fat and low fiber, mortality of colorectal carcinoma in Taiwan are higher than past. Because folic acid can’t be synthesized in mammals and majority only rely on food uptake, westernization diet would result in low folic acid intake. Several researches suggested that the risks of colorectal cancer are highly correlated with folic acid uptake in daily life. Base on such observation, incidence of colorectal cancer are getting higher and higher annually. In the past decades, cancer stem cells (CSCs) were thought to be the major population leading to tumor formation, drug resistant, and recurrence. Therefore, we hypothesized that folic acid deficiency may create a microenvironment which lead to cancer stem cells formation. Through different type of analyzing methods, enrichment of colorectal cancer stem cells population after folic acid deficiency was evaluated. As results showed in growth rate study, comparing to normal condition, folic acid deficiency would decrease doubling time of colon cancer cell line (C26) without affecting its’ survival. By Liu staining and microscopy observation, cell morphology changed gradually while increasing incubation time under folic acid deficiency. Cytotoxicity test also revealed that folic acid deficiency environment would induce drug resistant to doxorubincin treatment. Further characterization of commonly used CSCs’ surface markers, CD133 and CD44, by flow cytometry showed both CD133- and CD44-containing population increase under folate deficiency condition. In addition, genes related to stemness properties have been analyzed after C26 was growth at folic acid deficiency environment. SOX2, Nanog, and c-Myc genes were highly induced. Finally, tumorigenicity of C26 after incubation at folic-acid deficiency had been evaluated. One thousand of C26 cells, which had been cultivated under folic acid deficiency, could induce tumor formation after 2 weeks of subcutanusly inoculated at mice, while need 1x105 cells for control group. All these data indicated that folic acid deficiency might create a micro-environment for cancer stem cell formation.