臺灣博碩士論文加值系統

惡性腦瘤，如嬰孩兒常見之兒童神經母細胞瘤(Neuroblastoma)和成人之多型性神經膠質母細胞瘤(Glioblastoma multiforme, GBM)，目前仍屬人類最具致命性的腫瘤疾病。近年來，免疫治療被認為繼手術切除、放射線治療、化學治療之後的第四種治療方式，因此有關腫瘤免疫治療的研究在最近幾年中漸受到重視。文獻指出在惡性腦瘤病人體內會出現免疫系統功能不良(dysfunction)的情形。本實驗的目的為利用體外培養的方式，探討腦腫瘤細胞對免疫抑制情形。利用蛋白質染劑CFDA-SE標記正常人周邊T淋巴球細胞、或多型性神經膠原母細胞瘤病人體內周邊Ｔ淋巴球細胞，分別與神經膠原母細胞瘤細胞株(Neuroblastoma cell line, TE671)、或病人自體腫瘤細胞進行體外淋巴球細胞與腫瘤細胞混合培養(Mixed Lymphocyte and tumor cell co-culture)。T淋巴球與不同比率腫瘤細胞混合培養數天後，使用流式細胞儀進行CFSE和表面抗原分子(CD4、CD8、CD25、CD45RO)的螢光偵測，並採用修飾後公式D0 / Dn=2x (其中D0為第0天CFSE平均值、Dn第n天CFSE平均值、x為分裂次數) 去計算T淋巴球生長增殖的情形。結果顯示，不論是正常人或腦瘤病人周邊T淋巴球細胞，和腫瘤細胞混合培養之下，CD8+ T淋巴球細胞增殖情形會受到抑制；而部分CD4+ T淋巴球細胞之增殖情形為受到刺激而增長。另外，GBM病人的周邊血液T淋巴球群，和高比率的自體腫瘤細胞混養之下，CD4+CD25+middle T淋巴球的增殖會提升，而CD4+ CD45RO+ T淋巴球細胞增長則受抑制。綜合以上的結果，我們認為惡性腦瘤細胞會直接刺激調控性T淋巴球增殖，卻使毒殺型或記憶型T淋巴球之生長情況減緩。以此推論腫瘤細胞可能藉由「刺激調節性T淋巴球的增殖」此機制以逃避免疫系統的監
視。

Malignant tumors, including childhood neuroblastoma and adult glioblastoma multiforme (GBM), are among the most fatal human cancer diseases. In search for novel cancer treatment measures, immuno-therapeutic strategies have recently attracted considerable interest. However, patients of malignant brain tumors may show immune dysfunction. The goal of this study is to investigate the immuno-suppressive effects of brain tumor cells in vitro. Peripheral blood T lymphocytes of healthy donors or GBM patients were labeled with 5-(and-6) carboxyfluorescein diacetate succinyl ester (CFDA-SE) and co-cultivated with irradiated neuroblastoma TE671 cells or autologous GBM tumor cells. At various time of co-culture, flow cytometric analysis measurement was performed to measure CFSE fluorescence intensities of individual lymphocytes. Using the equation D0/Dn=2x, where D0 and Dn are the mean CFSE fluorescence intensities at day 0 and day n, respectively, and x is the cell division number, we found that both neuroblastoma TE671 and patient GBM cells, at high tumor cell/lymphocyte ratios, could inhibit the proliferation of CD8 lymphocytes but not CD4 lymphocytes. In the presence of autologous co-cultivated tumor cells in vitro, CD8+, CD4+CD25+high, and CD4+ CD45RO+ T cells showed slower rates of cell growth, while growth of CD4+CD25middle+ T cells was increased. Our results suggest that CD4+CD25+middle regulatory T cells were stimulated by the co-cultivated tumor cells and might in turn suppress the autologous CD8+ T and CD4+CD45RO+ T cells, allowing tumor cells to evade immune surveillance by immune T lymphocytes.

中文摘要...……………………………………….……………………..Ⅰ
Abstract………………………………………………………………...Ⅱ
致謝……………………………………………………………………..Ⅲ
前言………………………………………………………………………1
背景介紹（Introduction）…………….……………………………….3
實驗設計………………………………………………………………..11
實驗材料和實驗方法（Material and Methods）……………………12
結果（Results）………………………………………………………..24
討論（Discussion）……………………………………………………29
圖表……………………………………………………………………..35
參考文獻………………………………………………………………..52
附錄..……………………………………………………………………56

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