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研究生:張佳惠
研究生(外文):Jia-Huei Chang
論文名稱:愛玉瘦果中多胜肽區分之抗腫瘤及免疫調節作用
論文名稱(外文):Anti-tumor and Immunomodulation of Polypeptide Fraction from Jelly-fig (Ficus awkeotsang Makino) Achenes
指導教授:張鴻民張鴻民引用關係
學位類別:博士
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:209
中文關鍵詞:誘導分化細胞凋亡愛玉瘦果多胜肽區分單核細胞條件培養液
外文關鍵詞:differentiation inductionapoptosisMNC-CNsJelly-figPolypeptide Fraction
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本研究以愛玉子多胜肽區分(polypeptide fraction, PF)於體外細胞模式(in vitro)下探討PF抑制白血病細胞(U937)生長並促使其分化及誘導細胞凋亡之能力,並以動物模式(in vivo)探討愛玉子PF於生物體內經由免疫調節方式達到抗CT26腫瘤的效果。將濃度為100 μg/mL之愛玉子PF刺激人類單核球細胞一天所製備之單核細胞條件培養液(mononuclear cell conditioned media, MNC-CMs),可有效抑制U937細胞生長(94%),且以NBT還原陽性率測試、NSE陽性率、型態學觀察及細胞表面抗原檢測下,皆可顯示U937細胞株可被誘導分化為正常的單核或巨噬細胞。而此單核細胞條件培養液中亦可測得高量的IL-1β(4446.5±58.5 pg/mL)、TNF-α(1324.8±90.6 pg/mL)、GM-CSF(305.9±13.2 pg/m)及IFN-γ(1557.7±16.2 pg/mL)等細胞激素。將愛玉子PF(100 μg/mL)與U937共同培養,其生長抑制率可達90%以上,且會使U937細胞週期停滯,而於DNA片段化、Annexin-V檢測可知PF可誘使細胞凋亡之發生,具有濃度及時間效應;而由流式細胞儀檢測可知,PF亦會影響粒腺體膜電位且活化caspase-3。故可得知,由PF所製備之單核細胞條件培養液可誘導白血病細胞株分化,而將PF與白血病細胞株共同培養則可誘導細胞凋亡,兩者皆可抑制白血病細胞生長。
另外為了研究愛玉子PF於生物體內經由免疫調節方式達到抗腫瘤的效果,在Balb/c小鼠皮下移植大腸癌細胞(CT26)形成腫瘤,分別於施打腫瘤同時(處理組)或待腫瘤長出後(治療組)及腫瘤移植前(預防組)餵食愛玉子PF。結果發現,處理組之小鼠餵食愛玉子PF劑量為150 mg/kg bw/day時能明顯抑制腫瘤生長(35.9%),同時能促進移植腫瘤小鼠之脾臟細胞增生及提高其脾臟細胞內TH、TC及巨噬細胞比例。而治療組當投予小鼠每公斤體重100 mg劑量之愛玉子PF可有效抑制腫瘤生長,但其促進脾臟細胞增生能力則不明顯。而預防組之結果顯示,當小鼠先給予劑量為100及200 mg/kg bw/day之愛玉子PF後,其腫瘤抑制率可高達58.1及62.8%,若配合5-FU之使用,抑制率更高達81%左右,但愛玉子PF無法改善因5-FU所引起的脾臟腫大及脾臟增生能力降低之現象。顯示愛玉子PF於細胞模式及動物模式下皆具有抑制腫瘤生長及具有免疫調節能力。
Polypeptide fraction (PF) from jelly-fig (Ficus awkeotsang Makino) achenes was prepared to determine its effect on proliferation, differentiation, and apoptosis of human leukemic cells (U937) in vitro and immunomodulation and antitumor of CT26 in vivo. First, human peripheral blood mononuclear cells were stimulated by PF (100 μg/mL) for 1 day to prepared conditioned media (PF-MNC-CM-1). With which high growth inhibition (94%) on U937 cells and nitroblue tetrazolium (NBT) positive percentage, nonspecific esterase (NSE) positive percentage and surface antigen expressions of differentiated cells were observed. By morphological examination, U937 cells were confirmed to be differentiated into mature monocytes or/and macrophages. Through an ELISA, we found that MNC-CM-1 (stimulated by 100 μg/mL PF) contained high levels of IL-1β (4446.5±58.5 pg/mL), TNF-?(1324.8±90.6 pg/mL), GM-CSF (305.9±13.2 pg/mL), and IFN-γ (1557.7±16.2 pg/mL). The results of DNA ladder and Annexin V test showed that PF induced apoptosis of U937 cells when co-cultured. The PF-induced apoptosis was dose- and time-dependent. PF caused a dose-dependent influence on mitochondria transmembrane potential (MTP, Δψ) and caspase-3 activation when detected by a flow cytometry. PF displayed strong cell cycle arrest on various phases, depending on the dose and incubation time. Taken together, these results demonstrated that PF potently inhibited the proliferation of human leukemia U937 cell lines via differentiation induction (MNC-CM method) and apoptosis (co-culturation).
To study the antitumor effect through immunomodulation, Balb/c mice were orally administrated with crude PF (50-200 mg/kg bw/day) simultaneously (treatment group), 24 days before (prevention group) or 1 week after (curing group) implantation of CT26 tumor. Results of treatment group showed that crude PF inhibited 35.9% tumor growth and enhanced ratios of TH, TC, and macrophage in splenocytes, in comparison with control, at a dose of 150 mg /kg bw/day. In curing group, administration of 100 mg/kg bw reduced tumor markedly while showed unapparent effect in splenocytes proliferation. Of note, in prevention group, PF displayed remarkable (58.1 and 62.8%) tumor growth inhibition when mice were administrated with 100 and 200 mg/kg bw, respectively, revealing the effectiveness of PF as an alternative cancer therapy. However, PF was ineffective in reducing spleen swelling and in improving the proliferation reduction of splenocytes induced by the tumor implantation. In combination with 5-FU, antitumor effect can be as high as 81%. Above findings suggested that PF is potent in reducing tumor cell growth and in immunomodulation, in vitro and in vivo.
摘 要……………………………………………………………….I
Abstract………………………………………………………………….III
第一章 文獻整理……………………………………………………..1
第一節 愛玉子………………………………………………..1
1-1-1 愛玉子果膠酯酶抑制劑. …………………………………..2
第二節 白血病………………………………………………..3
1-2-1 白血病之定義及分類………………………………………3
1-2-2 白血病之流行病學與病理因素. …………………………..5
1-2-3 白血病之治療方法…………………………………………5
第三節 人類白血病細胞株U937及誘導分化之特性……………..6
1-3-1 U937細胞株之建立………………………………………..6
1-3-2 U937細胞株之特性………………………………………..6
1-3-3 評估U937細胞株分化之方法…………………………….8
第四節 以人類周邊血液單核球細胞(peripheral blood mononuclear cells, PBMNC)探討食品對腫瘤免疫之研究…………10
1-4-1 以PBMNC探討間接誘導U937分化之研究……………10
1-4-2 PBMNC之應用…………………………………………...11
第五節 細胞週期(cell cycle)…………………………………….11
1-5-1 細胞週期調控因子………………………………………..12
第六節 細胞凋亡作用(apoptosis)……………………………….14
1-6-1 細胞凋亡的特徵…………………………………………..14
1-6-2 細胞凋亡之調控機轉……………………………………..15
第六節 以移植腫瘤小鼠之動物模式探討食品對免疫調節抗腫瘤之研究…………………………………………………...19
1-6-1 小鼠大腸癌細胞株(CT26)之簡介…………………….19
1-6-2 氟尿嘧啶(5-Fluorouracil, 5-FU)之抗癌效果………….20
第二章 實驗架構……………………………………………………..23
第三章 愛玉子PF抑制人類白血病細胞增殖與誘導其分化及細胞凋亡作用. …………………………………………………………33
第一節 摘要…………………………………………………33
第二節 前言…………………………………………………34
第三節 實驗材料…………………………………………….35
3-3-1 愛玉來源. …………………………………………………35
3-3-2 實驗細胞……………………………………………………35
3-3-3實驗藥品及試劑…………………………………………….35
3-3-4 實驗器材…………………………………………………..39
3-3-5 儀器設備. …………………………………………………40
第四節 實驗方法…………………………………………….41
3-4-1 愛玉子多�@肽區分之製備………………………………..41
3-3-2 愛玉子PEI活性測定法…………………………………..41
3-4-3 愛玉子多�@肽區分物之初步區分………………………..44
3-4-4 愛玉子PF成分之分析……………………………………44
3-4-5 U937細胞之培養、冷凍及解凍………………………….46
3-4-6 測試樣品之前處理………………………………………..47
3-4-7 細胞計數法:錐藍染液排出法…………………………..47
3-4-8 人類單核細胞條件培養液(PBMNC-CM)之製備…….48
3-4-9 愛玉子PF對抑制U937生長之活性測試……………….48
3-4-10 NBT還原試驗………………………………………...…51
3-4-11 NSE染色測試……………………………………………51
3-4-12 U937細胞形態學變化之分析……………………………..52
3-4-13 細胞表面抗原CD11b、CD14及CD68之測定……….53
3-4-14 單核細胞條件培養液中IL-1b、TNF-a、IFN-g及GM-CSF含量之測定……………………………………………...53
3-4-15 細胞週期檢測……………………………………………54
3-4-16 細胞凋亡-DNA片段化之檢測………………………….55
3-4-17 粒腺體膜電位之檢測……………………………………55
3-4-18 早期細胞凋亡之檢測……………………………………56
3-4-19 Caspase-3之檢測………………………………………..56
3-4-20 細胞總蛋白質之萃取……………………………………57
3-4-21 SDS-PAGE電泳分析……………………………………57
3-4-22 西方墨點法(Western blotting)……………………….58
3-4-23 原態電泳檢定法…………………………………………58
3-4-24 統計方法…………………………………………………59
第五節 結果與討論………………………………………………...61
3-5-1 愛玉子PF之基本性質……………………………………61
3-5-2 間接作用模式下愛玉子PF對U937細胞生長之影響…..65
愛玉子PF之間接作用濃度效應………………………………..65
3-5-3 間接作用模式下愛玉子PF誘導U937細胞分化之探討 69
間接作用NBT還原試驗濃度效應……………………………..69
愛玉PF對U937間接作用後細胞之NSE陽性率……………..73
U937細胞之形態學變化………………………………………..76
細胞表面抗原CD11b、CD14及CD68之測定………………..79
單核細胞條件培養液中細胞激素含量之變化…………………86
3-5-4 直接作用模式下愛玉子PF對U937細胞生長之影響…..97
愛玉子PF之直接作用濃度效應………………………………..97
3-5-5 直接作用模式下愛玉子PF對U937細胞週期之影響…100
愛玉子PF對U937細胞週期之影響………………………….100
探討愛玉子PF對U937細胞週期之影響是否為可逆性……..105
3-5-6 直接作用模式下愛玉子PF誘導U937細胞凋亡之探討……………………………………………………….108
DNA片段化之檢測……………………………………………108
早期細胞凋亡之檢測…………………………………………..110
3-5-7 細胞凋亡途徑之探討……………………………………113
愛玉PF對U937細胞粒腺體膜電位之影響………………….113
以流式細胞儀檢測細胞凋亡相關蛋白質Caspase-3………….116
愛玉子PF對U937細胞凋亡作用之相關蛋白質探討……….118
3-5-8 愛玉子PF不同區分物對U937細胞之影響……………124
愛玉子PF不同區分對U937細胞直接及間接抑制率之比較 124
愛玉子PF不同區分間接及直接作用NBT還原試驗………..126
3-5-9 愛玉子PF經酒精沈澱之區分物對U937細胞之影響…126
愛玉子PF經酒精沈澱之區分物對U937細胞直接抑制率之比較………………………………………………………………..128
第六節 結論………………………………………………………...131
第四章 愛玉子PF抑制腫瘤生長…………………………………..132
第一節 摘要……………………………………………………….132
第二節 前言……………………………………………………….134
第三節 實驗材料………………………………………………….135
4-3-1 愛玉子來源………………………………………………135
4-3-2 實驗動物…………………………………………………135
4-3-3 實驗細胞…………………………………………………135
4-3-4 實驗藥品及試劑…………………………………………135
4-3-5 實驗器材…………………………………………………137
4-3-6 儀器……………………………………………………....138
第四節 實驗方法…………………………………………...139
4-4-1 愛玉子PF之製備………………………………………..139
4-4-2 CT26細胞之冷凍、解凍及培養………………………..139
4-4-3 細胞計數法:錐藍染液排出法…………………………140
4-4-4 PF對抑制CT26細胞生長之活性測試………………….140
4-4-5 動物飼養…………………………………………………141
4-4-6 管餵或腹腔注射樣品配置………………………………141
4-4-7 移植腫瘤…………………………………………………142
4-4-8 脾臟細胞之收集與樣品之取得…………………………143
4-4-9 脾臟細胞增生能力測定…………………………………145
4-4-10 脾臟細胞之分析………………………………………..145
4-4-11 統計方法………………………………………………..146
第五節 結果與討論…………………………………………147
4-5-1 愛玉子PF對CT26細胞生長之影響……………………147
4-5-2 愛玉子PF對未移植腫瘤組小鼠之體重、脾臟細胞之影響……………………………………………………….147
未移植腫瘤組之體重變化……………………………………..147
愛玉子PF對未移植腫瘤組Balb/c小鼠脾臟之影響…………150
愛玉子PF對未移植腫瘤組Balb/c小鼠脾臟細胞增生能力之影響………………………………………………………………..150
4-5-3 愛玉子PF對處理組小鼠之體重、脾臟細胞及免疫力之影響……………………………………………………….154
處理組之體重變化……………………………………………..154
處理組腫瘤大小及重量的測定………………………………..154
愛玉子PF對處理組Balb/c小鼠脾臟之影響…………………162
愛玉子PF對處理組Balb/c小鼠脾臟細胞增生能力之影響…164
愛玉子PF對處理組Balb/c小鼠脾臟細胞之影響……………166
4-5-4 愛玉子PF對治療組小鼠之體重、脾臟細胞及免疫力之影響……………………………………………………….171
治療組之體重變化……………………………………………..171
治療組腫瘤大小、重量及腫瘤抑制率的測定………………..171
愛玉子PF對治療組Balb/c小鼠脾臟之影響…………………174
愛玉子PF對治療組Balb/c小鼠脾臟細胞增生能力之影響…178
愛玉子PF對治療組Balb/c小鼠脾臟細胞之影響……………178
4-5-5 愛玉子PF對預防組小鼠之體重、脾臟細胞及免疫力之影響……………………………………………………….182
預防組之體重變化……………………………………………..182
預防組腫瘤大小、重量及腫瘤抑制率的測定………………..184
愛玉子PF對預防組Balb/c小鼠脾臟之影響…………………187
愛玉子PF對預防組Balb/c小鼠脾臟細胞增生能力之影響…190
第六節 結論. ………………………………………………192
第五章 參考文獻……………………………………………………194
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