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研究生:呂俊杰
研究生(外文):Jun-Jay Lu
論文名稱:Canavailiagladiata地方品種抗癌免疫活性之研究
論文名稱(外文):The anticancer immunity of a local variety of Canavailia gladiata
指導教授:李益謙李益謙引用關係
指導教授(外文):Eric I. C. Li
學位類別:碩士
校院名稱:國立成功大學
系所名稱:藥理學研究所
學門:醫藥衛生學門
學類:藥學學類
論文種類:學術論文
論文出版年:2003
畢業學年度:91
語文別:中文
論文頁數:98
中文關鍵詞:自然殺手細胞腫瘤免疫
外文關鍵詞:tumor immuneNK cell
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我們實驗室從坊間宣稱具有抗癌作用的豆科植物中,收集到地方品種Canavailia galadiata種子,初步萃取有效成分IC-1,並在體內實驗已經證實對腫瘤具有明顯的抑制效果。而從體外實驗,也已得知IC-1可以透過直接毒殺腫瘤和抑制腫瘤血管生成的機轉,來達成腫瘤抑制效果。然而從IC-1於體外也可活化脾臟免疫細胞增生的實驗結果,我們認為IC-1中可能含有類似Con A的凝集素物質,在腫瘤治療上除了直接毒殺和抑制血管新生外,可能還可以經由透過活化體內免疫來對抗腫瘤細胞。因此我針對IC-1的腫瘤免疫調節機轉來進行一序列研究,並選擇一株在C3H/HeN小鼠經致癌物(carcinogen)誘導產生的膀胱癌細胞株;MBT-2,做為抗腫瘤的動物研究模式。首先利用3H-thymidine incorporation的方法,證實IC-1不管在體外及體內都可以使正常Balb/cj小鼠的免疫細胞增生。進一步由流式細胞儀分析(FACScan)實驗,得知IC-1可以明顯使得B細胞、毒殺性(cytotoxic)T細胞及自然殺手(natural killer)細胞的數目增加,並且可以使分泌IL-2及IFN-□的細胞比例上升。這些細胞及分子在人體免疫系統對抗腫瘤中扮演了非常重要的角色。另外在對膀胱癌細胞株MBT-2的實驗中,初步發現IC-1不管在體外及體內實驗,都可以明顯的抑制MBT-2腫瘤生長,有效的殺死癌細胞。由3H-thymidine incorporation的方法,得知IC-1可以回復cisplatin所誘導的免疫抑制現象。進一步由腫瘤免疫組織染色法,發現經IC-1處理過的腫瘤有些微毒殺性T細胞(Cytotoxic T cell)及明顯自然殺手細胞(NK cell)浸潤的現象。最後由免疫細胞活性評估實驗,發現經IC-1治療後只有自然殺手細胞的活性有明顯提升的情形,推測IC-1的抗癌免疫作用主要是經由自然殺手細胞活化的路徑產生。
From a local variety of Canavailia galadiata our lab has isolated a protein fraction, termed IC-1, that possesses some interesting anticancer activities. The anticancer immunity of the protein fraction is investigated and reported here. Using fresh splenocytes isolated from Balb/cj mouse and the 3H-thymidine incorporation method, I found that IC-1 can stimulate the proliferation of these splenocytes. The same finding also occurs in vivo when IC-1 was injected intraperitoneally into Balb/cj mouse and the splenocytes isolated and tested in vitro. The proliferated cell population was found by the fluorescence activated cell sorter (FACS) scanning to be CD8 cytotoxic T cells and natural killer cells and, to a lesser extent, B cells. At the same time, IC-1 stimulates expressions of cytokine IL-2 and INF-□□in mouse, regardless whether the animal bears tumor cells or not. IC-1 also shows a strong in vitro cytotoxicity toward a murine bladder cancer cell line MBT-2 in a dose-dependent manner. The cytotoxicity is also reflected in vivo when the same cells were transplanted subcutaneously into C3H/HeN mouse and IC-1 injected intraperitoneally into the testing animals. The extract reduces the tumor size by 90% as compared to the controls in a 20-day experimental period. The tumor infiltrating lymphocytes (TIL) are found by the immuo- histochemistry method to be CD8 cytotoxic T cells and natural killer cells, but only natural killer cells reveal cytolytic activity. The rusult suggests that natural killer cells play an important role in the tumor reduction mechanism of IC-1. The same animal model was used to investigate the combination therapy of cisplatin and IC-1 on bladder cancer. Cisplatin treatment alone seriously impairs the immune system as manifested by the marked reduction of spleen size and of the numbers of CD8 T, CD19 B and NK cells in the spleen. The reduction, however, can be reversed almost to normal size or values when IC-1 is used concurrently with cisplatin. The results suggest a possible clinical usage of IC-1 as, at least, a supplementary material for cancer chemotherapy when immune suppression drugs are used.
目錄.......................................................I
圖目錄...................................................III
表目錄....................................................IV
中文摘要...................................................V
英文摘要..................................................VI
縮寫表..................................................VIII
第一章 研究背景...........................................1
第二章 研究目的..........................................13
第三章 實驗材料
第一節 細胞株.......................................16
第二節 實驗動物.....................................16
第三節 試劑.........................................16
第四節 抗體.........................................18
第五節 Kit..........................................18
第六節 儀器.........................................19
第四章 實驗方法
第一節 植物蛋白質萃取與純化.........................20
第二節 植物蛋白質的定量.............................20
第三節 SDS-PAGE蛋白質電泳...........................21
第四節 細胞培養.....................................23
第五節 離體細胞毒殺測定.............................25
第六節 小鼠脾臟細胞分離.............................25
第七節 小鼠脾臟細胞增生測定.........................27
第八節 流體細胞分析儀測定細胞表面抗原..............28
第九節 流體細胞分析儀測定細胞激素表現..............28
第十節 腫瘤冷凍組織免疫染色........................30
第十一節 自然殺手細胞活性試驗........................33
第十二節 毒殺性T細胞活性試驗.........................34
第十三節 活體動物腫瘤實驗............................35
第十四節 統計分析....................................36
第五章 實驗結果.........................................37
第六章 討論.............................................47
參考文獻.................................................54
圖表.....................................................64
附錄.....................................................93
作者簡歷.................................................98
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