張鴻民臺灣大學：食品科技研究所王韻寧Wang, Yun-NingYun-NingWang2007-11-272018-06-292007-11-272018-06-292004http://ntur.lib.ntu.edu.tw//handle/246246/56265自古以來，黑豆在傳統中醫藥學即被認為是防老抗衰、醫食俱優的產品。黑豆的研究在近幾年來蓬勃發展，本實驗欲探討黑豆浸泡液之免疫調節幼臚庛~瘤抑制的效果。將黑豆浸泡於室溫蒸餾水中過夜，取浸泡液刺激人類單核球細胞一天，去除細胞後取其條件培養液加入人類白血病細胞株U937培養五天。而後計數細胞觀察其生長抑制情形，在濃度400 μg/mL可抑制86.8% U937生長；進一步以75%酒精萃取黑豆浸泡液後，將黑豆浸泡液酒精沉澱物200 μg/mL之條件培養液刺激U937，得81.5%抑制率；黑豆浸泡液酒精沉澱物經Saphadex G-15餾分出638 Da（區分一）之物質，在濃度200 μg/mL時，其條件培養液對U937抑制率為84.2%，在濃度100μg/mL仍有75.5%抑制率。以NBT還原試驗觀察經條件培養液刺激後的U937是否能分化為成熟的巨噬細胞，黑豆浸泡液濃度400 μg/mL時，NBT還原率為62.6%，濃度200 μg/mL為34.3%；黑豆酒精沈澱物濃度400 μg/mL 時NBT還原率為59.1%，濃度200 μg/mL為34.2%；黑豆浸泡液酒精沉澱物區分一濃度400 μg/mL時NBT還原率為67.3%，濃度200 μg/mL時為55.8%。黑豆浸泡液可能為有效的免疫促進物。 為探討黑豆浸泡液的抗腫瘤效果，利用皮下轉殖小鼠大腸癌細胞株CT26 1×106個於Balb/c小鼠，觀察實質固態瘤 (solid tumors) 的形成變化並觀察小鼠的體重變化，24天後犧牲觀察脾臟細胞增殖、B/T淋巴細胞及巨噬細胞增長情形、脾臟細胞培養液的細胞激素含量，以推論是否藉由提升小鼠的自身免疫，達到抑制腫瘤生長的效果。餵食黑豆浸泡液 400 mg/Kg bw/day，可抑制40.5%腫瘤生長，且經由PHA誘導後脾臟細胞明顯增生，及促進其分泌IL-2與TNF-ㄐA因此黑豆浸泡液為有效提身體內免疫系統及抑制腫瘤生長的物質。Black soybean (BSB) has been consumed as food and been used as medicinal herb for hundreds of years in oriental countries. Some reports indicated that BSB has chemotherapeutic functions and showed immunomodulating effects in vivo. In an effort to understand the antiproliferative and differentiating effects on U937 cells, the water-soluble portion (WSP) of intact BSB prepared at room temperature was used to stimulate peripheral blood mononuclear cells (MNC) for one day to prepare conditioned medium (WSP-MNC-CM). With which, U937 cells were incubated for 5 days and the growth inhibition and differentiation-inducing effect were studied. It was found that at the level of 400 μg WSP/mL, 86.8 % growth inhibition and 62.6 % differentiation was observed. Precipitates from WSP of BSB by 75% alcohol (AP-WSP) were added to stimulate MNC and show 81.5 % growth inhibition and 34.2 % differentiation at a level of 200 μg/mL. Fraction 1 (F-1-AP-WSP) on Sephadex G-15 was determined to be about 638 Da, which showed 84.2% growth inhibition and 55.8 % differentiation at a level of 200 μg/mL. In NBT test, WSP at a level of 400 and 200μg/mL, U937 exhibited 62.6 and 34.4 % positive percentage, while AP-WSP at a level 400 and 200 μg/mL displayed 59.1 and 34.2% positive percentage. Moreover, F-1-AP-WSP showed stronger differentiation-inducing effect, revealing the apparent immunity-enhancing effect. Of in vivo test, Balb/c mice were orally administrated with WSP and the growth inhibition of implanted (s.c.) CT26 cells were checked. Of note, about 40.5% of tumor growth was inhibited at the dosage of 400 mg/kg bw/day . Also, proliferation of spleen cells and increased IL-2 and TNF-ωecretion were observed in the group of mice orally administrated WSP-BSB of with 400 mg/kg bw/day. In conclusion, water-soluble portion of intack black soybean would have immunomodulating and antitumor activities.謝誌 …………………………………………………………………1 中文摘要 ……………………………………………………………2 英文摘要 ……………………………………………………………3 目錄 …………………………………………………………………5 圖表次 ………………………………………………………………10 第一章 文獻回顧 1.1 黑豆………………………………………………………….12 1. 黑豆簡介…………………………………………………12 2. 黑豆營養成分……………………………………………12 3. 黑豆之藥理地位…………………………………………13 1.2 豆類研究…………………………………………………….13 1. 大豆異黃酮……………………………………………….14 2. 皂素……………………………………………...………..15 3. 胰蛋白酶抑制劑……………………………….….……..16 4. 大豆凝集素………………………………………..……..16 5. 其他……………………………………………..………..16 1.3 黑豆研究…………………………………………….………17 1.4免疫系統……………………………………………………..17 1. T淋巴球…………………………………………….…….20 2. B淋巴球………………………………………….……….21 3. 自然殺手細胞……………………………………..……...21 4. 單核球與巨噬細胞…………………………………..…...21 5. 顆粒性白血球…………………………………..………...22 1.5 細胞激素…………………………………………….………22 1. 干擾素……………………………………………………..23 2. 間白素……………………………………………………..23 3. 腫瘤壞死因子……………………………………………..23 4. 其他………………………………………………………...24 1.6 癌症發生與免疫系統…………………………………………24 1.7 白血病…………………………………………………………25 1.8 白血病的治療…………………………………………………25 1.9人類白血病細胞株 U937……………………………………..26 1.0 小鼠大腸腺癌細胞株 CT26………………………………….28 1.11 流式細胞技術………………………………………………..28 1.12 實驗動機與設計……………………………………………..29 第二章 材料與方法 2.1 黑豆萃取………………………………………………………31 1. 黑豆萃取流程……………………………………………...31 2. 實驗材料…………………………………………………..32 (1) 實驗原料來源………………………………………..32 (2) 實驗試藥……………………………………………...32 (3)實驗器材與儀器……………………………………….32 3. 萃取方法…………………………………………………...33 (1) 黑豆粗萃取………………………………………….33 (2) 膠體管柱層析…………………………………….....34 (3) 測定各階段樣品蛋白質與總醣量………………….34 2.2 以細胞模式檢驗黑豆之有效成分及免疫調節能力………...37 1. 實驗流程…………………………………………………...37 2. 實驗材料………………………………………………...…38 (1) 樣品來源……………………………………………..38 (2) 實驗細胞……………………………….……………..38 (3) 實驗試藥………………………………….…………..38 (4) 實驗器材與儀器…………………………….………..39 2.3 實驗方法與步驟………………………………………………40 (1) 細胞解凍………………………………….…………..40 (2) 細胞冷凍…………………………………….…..……41 (3) 細胞存活率……………………………………..…….41 (4) 樣品對人類單核球細胞 (U937) 抑制作用 (直接模式)……………………………………..……..42 (5) 人類單核球細胞之分離……………………...……....42 (6) 條件培養液之製備……………………………....……43 (7) 樣品條件培養液對 U937 的抑制作用 (間接模式)…………………………………..………..43 (8) U937 分化試驗……………………………..………...44 (9) U937 形態變化分析………………………..………..44 (10) 統計分析……………………………………...…….45 2.3 以動物模式探討黑豆浸泡液抗腫瘤活性………………..….46 1. 實驗流程……………………………………………...……46 2. 實驗材料………………………………………………..…47 (1) 樣品來源…………………………………………..…47 (2) 實驗細胞………………………………………….…47 (3) 實驗動物………………………………………….….47 (4) 實驗試藥……………………………………….…….47 (5) 實驗器材與儀器……………………………….…….49 3.實驗步驟與方法……………………………………………50 (1) 小鼠飼養…………………………………………….50 (2) CT26 之培養………………………………………...51 (3) 轉殖腫瘤…………………………………………….51 (4) 餵食樣品…………………………………………….52 (5) 量測體重與腫瘤…………………………………….52 (6) 製備小鼠脾臟細胞………………………………….52 (7) 製備小鼠脾臟細胞條件培養液…………………….53 (8) 脾臟細胞增生試驗………………………………….53 (9) 脾臟細胞表面抗原分析……………………………..53 (10) 細胞激素測定………………………………………54 (11) 統計分析…………………………………………....54 第三章 結果討論 3.1 黑豆萃取………………………………………………………55 1. 黑豆與黃豆之浸泡液與水萃物其 蛋白質含量及總醣含量………..………………………...55 2. 黑豆浸泡液酒精沉澱之管柱層析………………………..55 3.2 以細胞模式檢驗黑豆之有效成分及免疫調節能力 1. 樣品對人類白血病 U937 細胞直接抑制作用…………..56 2. 樣品刺激人類單核球細胞之條件培養液對 U937 細胞抑制作用(間接模式)…………………………56 3. U937誘導分化試驗………………………………………...57 4. 以管柱層析預測黑豆浸泡液酒精沉澱物………………...57 餾分物分子量 5. 黑豆皮與黑豆仁浸泡液之條件培養液 對U937細胞間接抑制作用…………………………..…60 3.3 以動物模式探討黑豆浸泡液抗腫瘤活性 1. 體重變化與腫瘤生長大小………………………………...62 2. 脾臟細胞增生比率………………………………………..63 3. 脾臟細胞表面抗原分析…………………………………..64 4. 脾臟細胞條件培養液中IL-2 及TNF-α含量…………..65 結論…………………………………………………………………….68 圖表…………………………………………………………………….70 參考文獻……………………………………………………………….91en-US腫瘤抑制Balb/c小鼠黑豆浸泡液單核細胞條件培養液人類白血病細胞(U937)U937 cellblack soybeanBalb/c miceantitumorthesis