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研究生:楊士杰
論文名稱:巴西蘑菇中4-Hydroxy-17-methylincisterol調控人類周邊血單核細胞增生、基因表現、細胞激素產生與細胞週期之進行
論文名稱(外文):Regulation of Cell Proliferation, Gene Expression, Production of Cytokines and Cell Cycle Progression in Human Peripheral Blood Mononuclear Cells by 4-Hydroxy-17-methylincisterol Isolated from Agaricus blazei
指導教授:郭育綺郭育綺引用關係
指導教授(外文):Yuh-Chi Kuo
學位類別:碩士
校院名稱:輔仁大學
系所名稱:生命科學系碩士班
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:127
中文關鍵詞:人類周邊血單核細胞植物凝集素
外文關鍵詞:Human Peripheral Blood Mononuclear CellsPBMCsPhytohemagglutininPHA
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巴西蘑菇 (Agaricus blazei Murill) 又別名巴西洋菇、姬松茸,近年來由於其降血糖、抗發炎作用、降膽固醇、抗病毒、抗癌等功效陸續被報導出來,而成為各國民間熱門的養生與保健的食品。本研究乃根據生物活性測定,欲探討由巴西蘑菇所純化之有效成分 4-Hydroxy-17-methylincisterol (4-HM) 對人類周邊血單核細胞 (Human Peripheral Blood Mononuclear Cells;PBMCs) 的細胞增生、細胞激素基因表現、細胞週期進行、轉錄因子之活化及相關訊息傳導路徑之影響。實驗結果顯示:(1) 利用 3H-thymidine 吸收檢測法,發現 4-HM 可抑制由植物凝集素 (Phytohemagglutinin;PHA) 所引起之人類周邊血單核細胞增生,其抑制作用百分之五十之濃度 (IC50) 為3.16 microM;(2) 利用Propidium Iodide (PI) 染色與流式細胞分析儀 (Flow cytometer) 偵測4-HM 對於PBMCs細胞週期進行之影響,結果發現4-HM可以阻斷PHA所引起的細胞週期進行,使細胞停留在 G0/G1 phase;(3) 由西方墨點法 (Western Blotting) 分析結果發現,4-HM 可以抑制 PHA 所引起之細胞週期蛋白 (Cyclins) 的表現例如 Cyclin D3 與 Cyclin E;(4) 利用免疫酵素連結測定法 (Enzyme-linked immunosorbent assay;ELISA) 發現 4-HM 可以降低PHA所引起的第二介白質 (Interleukin-2;IL-2)、第四介白質 (Interleukin-4;IL-4)、第十介白質 (Interleukin-10;IL-10)、甲種腫瘤壞死因子 (Tumor necrosis factor-alpha;TNF-alpha) 與丙種干擾素 (Interferon-gamma;IFN-gamma) 等細胞激素 (Cytokines) 之產生;(5) 利用反轉錄聚合酶連鎖反應 (Reverse transcriptase-polymerase chain reaction;RT-PCR) 與即時定量聚合酶連鎖反應 (Real-time PCR) 分析顯示4-HM 可以減少 IL-2、IL-4、IL-10、TNF-alpha 與 IFN-gamma 等細胞激素之 mRNA 在 PBMCs 中的表現量;(6) 利用免疫螢光染色 (Immunofluorescent staining) 及共軛焦顯微鏡 (Confocal microscope) 觀察,顯示4-HM可以阻斷 PHA 所引起的 Nuclear factor kappa B (NF-kappaB) 與 Nuclear factor of activated T cells (NF-AT) 的活化;(7) 利用螢光顯示劑 Fluo-3/AM 與螢光光譜儀 (Fluorescence spectrophotometer) 分析,顯示 4-HM 可以降低 PHA 所引起的細胞內鈣離子濃度之增加。綜合以上實驗結果,我們認為巴西蘑菇中含有免疫調控因子,它可藉著調控人類周邊血單核細胞中轉錄因子 NF-kappaB 與 NF-AT 之活化與細胞內鈣離子濃度而降低 IL-2、IL-4與 IFN-gamma等細胞激素與細胞週期蛋白之表現,進而抑制人類周邊血單核細胞細胞週期進行與增生。
Agaricus blazei Murill is useful edible and health care food for its antiinflammation, antiviral, and antitumor activities. This study was designed to analyze the effects of 4-hydroxy-17-methylincisterol (4-HM;C21H32O3; M.W. 332) from A. blazei on immune responses of peripheral blood mononuclear cells (PBMCs). The regulation of 4-HM on cell proliferation, gene expression, production of cytokines, cell cycle progression and cellular signal in PBMCs induced by phytohemagglutinin (PHA) was investigated. The cell proliferation was determined by tritiated thymidine uptake. Results showed that 4-HM suppressed PBMC proliferation at about 0 — 48 hr following stimulation with PHA. Cell cycle analysis indicated that 4-HM arrested the cell cycle progression of activated PBMC from the G1 transition to the S phase. To localize the point in the cell cycle where arrest occurred, a set of key regulatory events leading to the G1/S boundary, including gene expression of interleukin-2 (IL-2), IL-4, and interferon-gamma (IFN-gamma), production of cyclins, and activation of NF-κB and NF-AT was examined. 4-HM suppressed, in activated PBMC, the production and mRNA expression of IL-2, IL-4, IL-10, TNF-alpha and IFN-gamma in a concentration-dependent manner. The levels of Cyclin D3 and Cyclin E protein in activated PBMCs were reduced by 4-HM. The 4-HM impaired activation of NF-AT and NF-B in PBMCs induced with PHA. Thus, the suppressant effects of 4-HM on proliferation of PBMCs activated by PHA appeared to be mediated, at least in part, through reduction of NF-AT and NF-kappaB activation, inhibition of cytokines and cyclins production, and arresting cell cycle progression in the cells.
縮簡表………………………………2
中文摘要……………………………...4
英文摘要…………………………...…5
壹、序論……………………………………………………………...…………………6
貳、材料與方法………………………………………………..……………………11
參、實驗結果…………………………………………………………………………24
肆、討論……………………………………………………………………………...…40
伍、參考文獻…………………………………………………………………………48
表……………………………………………………………………………….………...57
圖……………………………………………………………………………………..…..60
附錄…………………………………………………………………………..……..…..95
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