臺灣博碩士論文加值系統

衛福部國民健康署統計資料顯示，大腸癌是台灣癌症發生人數最多的癌症，每年約有1萬多人得到結腸直腸癌，其中約五分之一的病患為第四期。癌細胞的侵襲及轉移往往是癌症病患致死且導致預後不良的主要原因之一，而其侵入則與細胞間質的分解、細胞的貼附能力及移動性的改變有關。有研究發現，舞菇的子實體及菌絲體均含有豐富的營養成分，其對糖尿病、高血壓、高血脂等有明顯的治療效果，其中舞菇含有的多醣體在腸胃中具有提升免疫力和抗腫瘤效果。本實驗使用灰、白兩種舞菇水萃取物處理具有侵襲能力的人類結腸直腸癌細胞株LoVo，分別先進行24、48小時MTT assay，並從中找到安全劑量，以wound healing assay及transwell migration等實驗評估舞菇水萃取物抑制結腸直腸癌細胞轉移的能力。實驗結果顯示，LoVo細胞在經過不同劑量下的灰、白舞菇水粗萃物並培養24、48小時後，隨著萃取物的濃度增加，LoVo細胞的存活率明顯下降，也得知兩種舞菇水萃取物的安全濃度為0.8 mg/mL以下。在wound healing assay及transwell migration方面，兩種舞菇萃取物分別處理48小時後皆呈現劑量效應，而且兩種舞菇水萃取物顯著抑制細胞爬行距離以及transwell migration程度。經由western blot分析，也發現灰、白舞菇水萃取物可以減少誘發EMT過程之相關因子N-cadherin、β-catenin、Vimentin、Snail及Slug的蛋白質表現量。由以上實驗結果可以得知，低濃度之灰、白舞菇水萃取物能有效抑制LoVo轉移的功能，可能具有輔助治療大腸癌轉移之能力。

According to statistics from the Health promotion Administration, Ministry of Health and Welfare, colorectal cancer is the most commonly occurring cancer in Taiwan. About 10,000 people get colorectal cancer each year, and about one in five patients are in the fourth phase. Invasion and metastasis of cancer cells are often one of the major causes of cancer death and lead to poor prognosis. Invasion is related to the breakdown of cell stroma, cell attachment and mobility changes. Studies have found that fruiting bodies of Grifola frondosa and mycelium are rich in nutrients, which have significant effects on diabetes, hypertension, hyperlipidemia, etc., in which the polysaccharide contained in Grifola frondosa has the immunity in the stomach and anti-tumor effect. In this experiment, grey and white Grifola frondosa water extracts with invasive human colorectal cancer cell line LoVo, respectively, 24,48 hours MTT assay and find a safe dose to wound healing assay and transwell migration two an experiment was conducted to evaluate the ability of Grifola frondosa water extracts to inhibit the metastasis of colorectal cancer cells. The experimental results showed that after LoVo cells were cultured for 24,48 hours under different doses of grey and white Grifola frondosa water extracts, the survival rate of LoVo cells decreased significantly with the increase of the extract content, The Grifola frondosa water extracts safe concentration of 0.8 mg / mL or less. In the wound healing assay and transwell migration, the two kinds of Grifola frondosa water extracts were treated for 48 hours, respectively, showing dose-effect, and two kinds of Grifola frondosa water extracts significantly inhibited cell creep distance and transwell migration degree. Western blot analysis also showed that Grifola frondosa water extracts can reduce the protein expression of N-cadherin, β-catenin, Vimentin, Snail and Slug, which are involved in the process of EMT. From the above experimental results we can see that the low concentration of grey and white Grifola frondosa water extracts rough extract effectively inhibit the function of LoVo metastasis may have the ability to assist the treatment of metastatic colorectal cancer.

表目錄.................................IV
圖目錄..................................V
縮寫檢索表.............................VII
摘要.................................VIII
Abstract................................X
第一章 緒論............................1
1.1 大腸直腸癌.......................1
1.1.1 大腸直腸癌之流行病學..............1
1.1.2 大腸直腸癌臨床表徵及病理上變化.....2
1.2 舞菇.............................6
1.2.1 舞菇介紹 ........................6
1.2.2 舞菇成份 ........................7
1.2.3 生理機能.........................8
1.3 細胞的轉移與侵襲.................11
1.3.1 上皮－間質轉化作用...............13
第二章 研究目的........................17
第三章 實驗材料與來源...................19
3.1 細胞來源........................19
3.2 藥品試劑........................19
3.3 儀器設備與耗材...................22
第四章 實驗方法........................24
4.1 製備舞菇之水萃取物...............24
4.2 人類大腸癌細胞株(LoVo)之培養方法..24
4.2.1 活化細胞........................24
4.2.2 細胞繼代........................24
4.2.3 冷凍細胞........................26
4.2.4 細胞計數........................26
4.3 Total phenol 測定...............27
4.4 細胞存活率分析(Cell Viability Assay).....28
4.5 細胞傷口癒合試驗(Wound Healing Assay).....29
4.6 細胞轉移試驗（Migration）........30
4.7 西方墨點法（Western blot）.......31
第五章 統計方法 ........................39
第六章 實驗結果.........................40
6.1 總多酚含量測定...................40
6.2 灰、白舞菇水萃取物對LoVo細胞株存活抑制率的影響......41
6.3 灰、白舞菇水萃取物對LoVo細胞株移行能力之影響........43
6.4 灰、白舞菇水萃取物對LoVo細胞株轉移能力之影響........47
6.5 灰、白舞菇水萃取物對LoVo細胞株之轉移相關蛋白的影響...49
第七章 討論............................55
第八章 結論............................59
第九章 參考文獻.........................60
表目錄
表一、大腸直腸癌AJCC第七版TNM分期表........4
表二、SDS-PAGE gel配方...................35
表三、灰及白舞菇水萃取物分別之總多酚含量....40
圖目錄
圖一、（a）灰舞菇（b）白舞菇之外觀..........6
圖二、細胞侵襲與轉移的過程.................13
圖三、MTT及Formazan之結構式...............29
圖四、transwell migrationg示意圖..........31
圖五、western blot transfer holding示意圖........37
圖六、LoVo細胞株經過不同濃度之灰舞菇水萃取物（0.1、0.2、0.4、0.8、1.2、1.6、2及2.5 mg/mL）處理後分別培養24及48小時之存活抑制率........41
圖七、LoVo細胞株經過不同濃度之白舞菇水萃取物（0.1、0.2、0.4、0.8、1.2、1.6、2及2.5 mg/mL）處理後分別培養24及48小時之存活抑制率........42
圖八、利用倒立式相位差顯微鏡（100X）觀察LoVo細胞株經過不同濃度之（a）灰、（b）白舞菇水萃取物（0、0.4及0.8 mg/mL）處理後，分別培養24及48小時之細胞移動情形........44
圖九、LoVo細胞經由不同濃度之（a）灰、（b）白舞菇水萃取物（0、0.4及0.8 mg/mL）處理後，分別培養24及48小時之移動距離..................45
圖十、LoVo細胞株經由crystal violet solution染色後，利用倒立式顯微鏡（200X）觀察其經過不同濃度之（a）灰、（b）白舞菇水萃取物（0、0.2、0.4及0.8 mg/mL）處理後，並培養48小時之轉移情形.................47
圖十一、觀察LoVo細胞株經過不同濃度之灰、白舞菇水萃取物（0、0.2、0.4及0.8 mg/mL）處理後，並培養48小時之轉移情形............48
圖十二、不同濃度之灰、白舞菇水萃取物對LoVo細胞株對β-catenin蛋白表現量之影響 ....50
圖十三、不同濃度之灰、白舞菇水萃取物對LoVo細胞株對N-cadherin蛋白表現量之影響.....51
圖十四、不同濃度之灰、白舞菇水萃取物對LoVo細胞株對Snail蛋白表現量之影響..........52
圖十五、不同濃度之灰、白舞菇水萃取物對LoVo細胞株對Slug蛋白表現量之影響...........53
圖十六、不同濃度之灰、白舞菇水萃取物對LoVo細胞株對vimentin蛋白表現量之影響.......54

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