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研究生:簡佳怡
研究生(外文):Chia-Yi Chein
論文名稱:鴻喜菇蛋白質對小鼠大腸癌CT26細胞生長抑制機制及對Balb/c鼠之該腫瘤轉移抑制效果
論文名稱(外文):Inhibitory Effect of proteins from Hypsizigus marmoreus on CT26 Colon Adenocarcinoma Cells and Metastasis in Balb/c Mice
指導教授:張鴻民張鴻民引用關係
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:食品科技研究所
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2005
畢業學年度:93
語文別:中文
論文頁數:110
中文關鍵詞:鴻喜菇小鼠大腸癌細胞肺部轉移
外文關鍵詞:Hypsizigus marmoreusCT26 Colon Adenocarcinoma Cellspulmonary metastasis
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鴻喜菇為低熱量、低脂肪且蛋白質含量高的食用菇,在日本培育多年,長期以來被視為高級食材且不斷推廣,目前是我國國內新興栽培的食用菇之ㄧ。文獻指出鴻喜菇具有抗腫瘤及抗真菌等之功效。本實驗中,將探討鴻喜菇40~80%硫酸銨沉澱物對小鼠大腸癌CT26腫瘤細胞株生長抑制及抗轉移之效果。細胞實驗中,利用鴻喜菇40~80%硫酸銨沉澱物與CT26細胞共同培養不同時間,以MTT法觀察其生長抑制率,並探討其對CT26細胞週期之變化。結果顯示,鴻喜菇40~80%硫酸銨沉澱物具較高的直接生長抑制效果,在濃度為600 μg/mL的生長抑制率可達67.90%以上,再經由流式細胞儀分析可知,鴻喜菇40~80%硫酸銨沉澱物可將CT26細胞週期停滯於G2/M期,並且使其走向細胞凋亡。在動物實驗中,於Balb/c鼠尾靜脈注射CT26腫瘤細胞株,建立腫瘤轉移肺臟之模式,並餵食不同劑量之鴻喜菇40~80%硫酸銨沉澱物,以探討其抗腫瘤轉移之效果。結果顯示,有餵食鴻喜菇40~80%硫酸銨沉澱物之小鼠,其腫瘤轉移至肺臟之小節結數,會隨濃度增加抑制效果愈明顯(P<0.05)。另外,於顯微鏡下觀察小鼠之肺臟及腎臟病理組織切片,於肺臟組織切片圖中,可看到腫瘤轉移後使組織間質增厚產生病變之情形;而腎臟組織切片圖顯示組織並未有病變。
Hypsizigus marmoreus (HM), one of popular dietary mushrooms, contains high protein, low calorie, and low fat and has long been consumed in Japan. Several researches indicate that HM has antitumor and antifungal activities. In the present study, HM was used to study its antitumor activity in vitro and in vivo. First, 40~80% ammonium sulfate precipitates of cold-salt water extracts from HM were prepared and incubated with mouse colon CT26 cells to observe the changes in growth inhibition (%) for 24, 48 and 72 hr by MTT assay, followed by the cell cycle arrest by a Flow Cytometry. Results showed that remarkable growth inhibition, higher than 68 %, was observed when CT26 cells were incubated with 600 μg/mL of 40~80 % ammonium sulfate precipitates of cold-salt water extracts from HM. In addition, cell cycle arrest of CT26 cells at G2/M phase and apoptosis were induced. In vivo, pulmonary metastasis of CT26 tumor on male Balb/c mice orally administrated for 13 days with doses (200, 400 and 600 mg/kg bw/day) of ammonium sulfate precipitates of cold-salt water extracts from HM and injected (iv) with 1×105 CT26 cells via a tail vein was established to observe the results. Metastasic lung nodules in experimental group fed with 200, 400 or 600 mg/kg bw/day decreased in a dose-dependent manner, revealing the significant (p<0.05) anti-metastatic effect of HM proteins. Histopathologic results in lung was consistent with the finding in vivo. Insignificant changes in renal and immunohistograph were observed in mice at those three dosages.
目錄
中文摘要…………………………………………………………………i
英文摘要…………………………………………………………………ii
第一章 前言…………………………………………………..………..1
第一節 食用菇與免疫抗癌之相關性………..……………………...1
1-1-1.1癌症……………………………..………………………….1
1-1-1.2 食品與免疫之作用……………..…………………………1
1-1-1.3免疫反應………………………..………………………….2
第二節 食用菇之簡介……………………..………………………..5
1-1-2.1 食用菇……………………………..………………………5
1-1-2.2 鴻喜菇……………………………..………………………8
壹、以鴻喜菇對CT26細胞生長抑制並探討其細胞週期之調控…...12
第三節 細胞週期之調控…………………….……………………..12
貳、以尾靜脈注射CT26之動物模式建立以探討鴻喜菇抗腫瘤轉移之能力………………………………………………………………..15
第一節 小鼠大腸癌細胞CT26之介紹……………………………15
1-2-1.1 CT26細胞株之特性……………………………………15
1-2-1.2 CT26之生瘤性…………………………………………15
第二節 癌細胞之轉移………………………………………………16
1-2-2.1 轉移………………………………………………………16
1-2-2.2 基質金屬蛋白酶…………………………………………18
1-2-2.2.1 活化…………………………………………………19
1-2-2.2.2 抑制…………………………………………………20
1-2-2.3 基質金屬蛋白酶在癌症方面之研究……………………20
1-2-2.4 組織觀察…………………………………………………22
1-2-2.4.1 組織切片……………………………………………22
1-2-2.4.2 組織切片之製作……………………………………22
第二章 鴻喜菇對小鼠大腸癌CT26細胞株生長抑制機制………...24
第一節 前言…………………………………………………………24
第二節 實驗材料……………………………………………………25
2-2.1 實驗樣品來源………………………………………………25
2-2.2 實驗細胞株…………………………………………………25
2-2.3 實驗藥品及試劑……………………………………………25
2-2.4 實驗器材……………………………………………………26
2-2.5 儀器設備……………………………………………………26
第三節 實驗方法……………………………………………………28
2-3.1 菇類鹽溶液粗萃硫酸銨沉澱物之製備……………………28
2-3.2 菇類活性物質測試…………………………………………28
2-3.3 CT26細胞株之培養………………………………………35
2-3.4 CT26生長抑制率測定……………………………………36
2-3.5 形態學上的觀察……………………………………………38
2-3.6 細胞週期生長抑制之分析測定……………………………38
2-3.7 統計分析……………………………………………………39
第四節 結果與討論………………………………………………...40
2-4.1 鴻喜菇40~80%硫酸銨沉澱物活性成分分析…………….40
2-4.2 鴻喜菇40~80%硫酸銨沉澱物電泳檢定………………….41
2-4.3 鴻喜菇40~80%硫酸銨沉澱物對CT26細胞之影響….….41
2-4.3.1 鴻喜菇40~80%硫酸銨沉澱物對CT26間接生長抑制率………………………………………………………41
2-4.3.2 鴻喜菇40~80%硫酸銨沉澱物對CT26不同時間直接生長抑制率…………………………………………........42
2-4.4 細胞週期之分析……………………………………………43
第三章 鴻喜菇經由活體之調節抑制腫瘤轉移之研究……..………56
第一節 前言………………………………………………………….56
第二節 實驗材料…………………………………………………….57
3-2.1 實驗樣品來源………………………………………………57
3-2.2 實驗動物…………………………………………………...57
3-2.3 實驗細胞株…………………………………………………57
3-2.4 實驗藥品及試劑……………………………………………57
3-2.5 實驗器材……………………………………………………58
3-2.6 儀器設備 ……………………………………………………59
第三節 實驗方法…………………………………………………....61
3-3.1 菇類鹽溶液粗萃硫酸銨沉澱物之製備………………..…61
3-3.2 CT26細胞株之冷凍、解凍及培養………………….…..61
3-3.3 動物飼養……………………………………………….…62
3-3.4 動物實驗……………………………………………….…62
3-3.5 基質金屬蛋白酶之Gelatin Zymography分析…………....67
3-3.6 統計分析……………………………………………….…..69
第四節 結果與討論…………………………………………….…..70
3-4.1 小鼠體重變化………………………………………….…..70
3-4.2 小鼠白血球數目變化………………………………….…..71
3-4.3 鴻喜菇40~80%硫酸銨沉澱物對小鼠脾臟之影響…….…72
3-4.4 鴻喜菇40~80%硫酸銨沉澱物對小鼠脾臟細胞增生之能力……………………………………………………….…..73
3-4.5 鴻喜菇40~80%硫酸銨沉澱物抑制小鼠肺臟轉移之能力…………………………………………………………...73
3-4.6 小鼠肝腎毒性測定…………………………………….…..74
3-4.7 組織病理切片………………………………………….…..75
3-4.8 小鼠脾臟細胞之條件培養液細胞激素之測定…………...76
3-4.9 鴻喜菇40~80%硫酸銨沉澱物對基質金屬蛋白酶之抑制效果…………………………………………………………...78
第四章 參考文獻…………………………………………………….94
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