張鴻民臺灣大學：食品科技研究所白利文Pai, Li-WenLi-WenPai2007-11-272018-06-292007-11-272018-06-292004http://ntur.lib.ntu.edu.tw//handle/246246/56266惡性腫瘤為為台灣十大死因之首，且其死亡率逐年增加，因此許多學者都致力於減緩或抑制癌細胞的增生。樹突狀細胞為公認最具潛力的抗原呈獻細胞 (antigen presenting cell, APC)，它能捕捉及處理外來的抗原，並將獨特的抗原胜肽 (peptide) 藉由主要組織相容複體 (major histocompability complex, MHC) 呈獻給T細胞，並活化T細胞進而啟動適應性免疫反應 (adaptive immune response)，因樹突狀細胞具有這些功能性，因此以樹突狀細胞為主的免疫療法在近幾年越來越受到重視。在這個實驗中，將探討小鼠骨髓樹突狀細胞活化的混合淋巴細胞於秀珍菇水萃物存在下對小鼠CT26細胞 (小鼠大腸腺癌細胞株) 的影響。由小鼠骨髓細胞所衍生而來的樹突狀細胞經過LPS (lipopolysaccharide) 或CT26 lysate (藉由反覆冷凍解凍製備) 刺激一天後，與小鼠脾臟中的混合淋巴細胞以1比10的比例共培養兩天，之後再將活化後的混合淋巴細胞與CT26細胞 (1比20) 在有無添加秀珍菇水粗萃物的情況下共培養兩天，藉以觀察對CT26細胞的影響。由實驗結果可知，樹突狀細胞會因吞噬CT26 lysate而成熟，且會使共培養的混合淋巴細胞活化及增生，而活化後的混合淋巴細胞即具有顯著抑制CT26細胞的功效，且其抑制率可達80%左右，而活化後的混合淋巴細胞於秀珍菇冷水粗萃物的存在下，可進一步的將對CT26細胞的抑制率提高到90%左右。不過在秀珍菇水萃物方面，我們發現熱水粗萃物可以小幅增生CT26細胞與活化後的混合淋巴細胞，而冷水萃物雖能抑制CT26細胞及增生脾臟中的混合淋巴細胞，但卻也會抑制活化後的混合淋巴細胞繼續增生，推測其原因可能是不同粗萃物中所含成分不同，而呈現不同的抑制作用。Malignant tumor is ranked top among the ten major causes of death with an increasing mortality year by year. Dendritic cell (DC) is a potent antigen presenting cell (APC) and is capable of uptake and processing foreign antigens, such as bacteria and tumor cells, and presents specific peptides of antigen to major histocompability complex (MHC). As a consequence, activation of T cells occurs and leads to strong adaptive immune response. Such characteristics of DC enable it to be focused recently on cancer therapy. In this study, to study the growth inhibition of activated mixed lymphocytes in presence with various levels of cold water extracts from PO on CT26 cells. Mouse DCs were cultured from bone marrow and then activated and matured by incubation for 1 day with LPS or CT26 cell lysates, which were prepared by repeated freezing and thawing. The mature DCs were then co-cultured with mixed lymphocytes from mouse splenocytes at a ratio of 1/10 to enhance the proliferation and specific cytotoxicity of mixed lymphocytes (containing T-cells). Subsequently, the activated mixed lymphocytes were co-cultured with CT26 cells (at a ratio of 1/20), in the presence or absence of PO water extracts, to observe the inhibition on CT26 cells. Results showed that mature DCs were effective in proliferating and enhancing the cytotoxic specificity of mixed lymphocytes. PO cold water extracts exhibited CT26 cell inhibition on growth, while stimulating the proliferation of mixed lymphocytes, as detected by a MTT assay. However, PO cold water extracts showed anti-proliferation on activated mixed lymphocytes by about 20 %. The activated mixed lymphocytes displayed remarkable inhibition of CT26 cells by about 80 %; however, in the presence (200 - 600 μg/mL) of PO cold water extracts, they showed much stronger inhibition on CT26 cells by about 90 %, revealing the activation of DCs and activated mixed lymophocytes by PO cold water extracts. On the other hands, PO hot water extracts proliferated both CT26 cells and activated mixed lymphocytes. Although PO cold water extracts showed adverse results on CT26 cells and mixed lymphocytes, they were effective in inhibiting CT26 cell growth by increased cytotoxity of T cells as a result of the co-cultivation with PO extracts. Reasons for proliferating or anti-proliferating the activated mixed lymphocytes could be due to the difference in compositions between PO cold and hot water extracts.摘要………………………………………………………………………I Abstract……………………………………………………………..II 目錄…………………………………………………………………..IV 圖次…………………………………………………………….....VII 表次………………………………………………………………...VIII 第一章 前言……………………………………………………………1 第一節 樹突狀細胞……………………………………….1 1.1.1 樹突狀細胞的起源與生活史………..1 1.1.2 樹突狀細胞主要亞群………………..2 1.1.3 樹突狀細胞的表現型………………..5 1.1.4 樹突狀細胞的活化與成熟…………..6 1.1.5 樹突狀細胞的功能性………………..7 1.1.6 樹突狀細胞反應訊號………………..7 1.1.7 樹突狀細胞的移動…………………..8 1.1.8 樹突狀細胞與免疫療法……………..9 第二節 癌症……………………………………………………...10 第三節 秀珍菇…………………………………………………...12 第四節 研究動機……………………………………………...…13 第二章 實驗方法與材料…………………………………………..14 第一節 藥品與試劑……………………………………………...14 第二節 溶液……………………………………………………...15 第三節 器材……………………………………………………...16 第四節 儀器……………………………………………………...17 第五節 小鼠……………………………………………………...18 第六節 CT26小鼠 (BALB/cJ) 大腸癌細胞…………………...18 第七節 腫瘤細胞抗原的製備…………………………………...19 第八節 骨髓樹突狀細胞之培養………………………………...19 第九節 型態學上的觀察………………………………………...20 第十節 利用流式細胞儀分析樹突狀細胞……………………...20 第十一節 脾臟混合淋巴細胞之製備…………………………...20 第十二節 利用流式細胞儀分析脾臟混合淋巴細胞…………...21 第十三節 利用MTT比色法檢測樹突狀細胞對混合淋巴細胞的活化與增生……………………………..…………….21 第十四節 經過樹突狀細胞活化後的混合淋巴細胞對CT26的抑制……………………………………………………...22 第十五節 秀珍菇來源與冷、熱水萃物之製備………….………23 第十六節 秀珍菇中蛋白質與總糖含量之測定………………...23 第十七節 利用MTT比色法測定秀珍菇之冷、熱水萃物對CT26的影響………………………………………………...24 第十八節 利用MTT比色法測定秀珍菇冷、熱水萃物對脾臟混合淋巴細胞的影響…………………………………...25 第十九節 經過樹突狀細胞活化後的混合淋巴細胞於秀珍菇冷水萃物的存在下對CT26的影響……………………25 第二十節 利用MTT比色法測定秀珍菇冷水萃物對經過樹突狀細胞活化後的脾臟混合淋巴細胞的影響…………...26 第二十一節 統計分析……………………………………...26 第三章 結果討論…………………………………………………..27 第一節 骨髓衍生的樹突狀細胞的細胞型態與表面抗原分析...27 第二節 利用Histopaque-1083分離出的脾臟中混合淋巴細胞之表面抗原分析…………………………………………...28 第三節 經不同外來物刺激的樹突狀細胞對脾臟混合淋巴細胞的影響…………………………………………………...29 第四節 混合淋巴細胞與樹突狀細胞共培養兩天後對CT26的抑制率………………………………………………...30 第五節 秀珍菇水萃物蛋白質與總糖含量……………………...30 第六節 利用MTT比色法檢測秀珍菇水萃物對CT26生長的影響……………………………………………………...…30 第七節 利用MTT比色法檢測秀珍菇水粗萃物對脾臟中混合淋巴細胞的影響…………………………………………...31 第八節 對CT26專一性活化後的混合淋巴細胞於秀珍菇冷水萃物存在下對CT26的影響……………………………….31 第九節 利用MTT比色法測定秀珍菇水萃物對專一性活化後的脾臟中混合淋巴細胞的影響…………………………...32 第四章 結論………………………………………………………..34 第五章 參考文獻…………………………………………………..511725270 bytesapplication/pdfen-US混合淋巴細胞秀珍菇樹突狀細胞大腸癌細胞Dendritic Cellmixed lymphocytesPleurotus ostreatusCT26[SDGs]SDG3thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/56266/1/ntu-93-R91641022-1.pdf