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Effects of Polysaccharide-rich Fraction from Fu-Ling [Poria cocos (Schew.) Wolf] on the Immunomodulation and CT26 Cell-Implanted Balb/c Mice
No full text本研究以茯苓乾燥子實體之酒精沉澱粗多醣為樣品進行動物實驗，探討Balb/c鼠之非特異性免疫、特異性免疫反應以及對皮下轉殖CT26腫瘤之作用。
在非特異性免疫實驗部分，將不同濃度茯苓粗多醣餵食Balb/c鼠33天後，發現其生長狀況良好，且器官相對重量不受影響。犧牲動物取脾臟細胞以PHA刺激後，發現餵食茯苓粗多醣具有促進脾臟細胞增生的能力，自然殺手細胞的活性方面，雖與控制組雖無顯著差異，但有較高之趨勢比例。脾臟細胞表面抗原分析得知，實驗組中T細胞、Th細胞、Tc細胞及B細胞和控制組相比無顯著差異，代表餵食茯苓粗多醣不會影響脾臟細胞中各淋巴細胞的消長，可維持平衡穩定狀態。
特異性免疫實驗方面，以腹腔注射ovalbumin (OVA)對Balb/c鼠致敏，得知小鼠餵食茯苓粗多醣不會影響其生長狀況。特異性抗體分析後，顯示餵食茯苓粗多醣對小鼠血清中抗OVA專一性抗體IgG和IgM含量有下降情形，餵食100 mg/kg bw茯苓粗多醣脾臟中CD8(+) Tc細胞比例下降，濃度200 mg/kg bw則會提高CD4(+) Th細胞和CD8(+) Tc細胞比例。
於Balb/c鼠皮下轉殖CT26腫瘤後，發現餵食400 mg/kg bw的茯苓粗多醣組和控制組相比，其腫瘤抑制率為32.1%。分析血清中抗體IgG含量，得知餵食茯苓粗多醣有助於明顯(p < 0.05)提升IgG含量，及促進經PHA刺激後之脾臟細胞增生作用。脾臟細胞經表面抗原分析，轉殖腫瘤會使淋巴細胞比例降低，而餵食茯苓粗多醣可提高小鼠脾臟中Th細胞、Tc細胞及B細胞比例。Polysaccharide-rich fraction (PRF) from dried fruiting body of Fu-Ling [Poria cocos (Schew.) Wolf] was prepared from cool-water extracts by ethanol precipitation to study its immunomodulatory and anti-tumor effects in vivo.
In non-specific immune response, oral administration of PRF for 33 days did not affect the growth and relative organ weight of Balb/c mice. Relative proliferation ratio of PHA-stimulated spleonocytes in mice was significantly increased, while natural killer activity, splenocyte surface markers of T, Th, Tc, and B cells were similar to the corresponding result of the control group.
Mice were immunized with ovalbumin (OVA) to conduct the specific immune response experiment. It was found that oral administration of PRF induced the reduction of anti-OVA IgG and IgM levels. Oral administration of 100 mg/kg bw PRF significantly reduced the percentage of CD8(+) Tc splenocytes, while higher dose of 200 mg/kg bw significantly increased CD4(+) Th and CD8(+) Tc percentage.
In anti-tumor test of CT26-implanted Balb/c mice, about 32.1% of tumor was suppressed at dose of 400 mg/kg bw PRF. The content of serum IgG and proliferation of PHA-stimulated splenocytes for mice orally administrated with PRF were significantly higher than that of control group. Ratio of lymphocyte of CT26-implanted Balb/c mice decreased in splenocytes, but oral administration of PRF increased the cell numbers of Th, Tc, and B cells.中文摘要 - 9 -
英文摘要 - 10 -
第一章 前言 - 11 -
第二章 文獻整理 - 12 -
一、茯苓簡介 - 12 -
二、茯苓主要化學成分 - 12 -
三、茯苓之研究 - 14 -
(一)免疫調節作用 - 14 -
(二)抗腫瘤活性 - 16 -
(三)抗發炎活性 - 18 -
(四)抗氧化活性 - 19 -
(五)其他活性 - 20 -
四、免疫系統 - 20 -
(一)先天性免疫反應 - 20 -
1. 身體性的屏障 - 20 -
2. 生理性屏障 - 21 -
3. 吞噬性屏障 - 21 -
4. 發炎屏障 - 21 -
(二)後天性免疫反應 - 21 -
1. B淋巴細胞 - 22 -
2. T淋巴細胞 - 22 -
3. 抗原呈獻細胞 - 23 -
(三)自然殺手細胞 - 23 -
(四)細胞激素 - 24 -
(五)免疫球蛋白 - 24 -
1. 免疫球蛋白G - 26 -
2. 免疫球蛋白M - 26 -
3. 免疫球蛋白A - 26 -
4. 免疫球蛋白E - 26 -
5. 免疫球蛋白D - 27 -
五、健康食品之免疫調節功能評估 - 27 -
六、實驗架構 - 28 -
第三章 材料與方法 - 29 -
一、茯苓粗多醣萃取 - 29 -
(一)茯苓粗多醣萃取流程 - 29 -
(二)實驗材料 - 30 -
1. 原料 - 30 -
2. 實驗藥品與試劑 - 30 -
3. 實驗器材與儀器 - 30 -
(三)萃取方法和步驟 - 31 -
1. 樣品製備 - 31 -
2. 樣品蛋白質含量測定 - 31 -
3. 樣品總醣含量測定 - 32 -
二、茯苓粗多醣萃取物對Balb/c鼠體內非特異性免疫反應之影響 - 33 -
(一)實驗流程 - 33 -
(二)實驗材料 - 34 -
1. 樣品來源 - 34 -
2. 動物飼料 - 34 -
3. 實驗動物 - 34 -
4. 實驗器材和設備 - 34 -
(三)實驗步驟和方法 - 36 -
1. 動物飼養 - 36 -
2. 實驗組別 - 36 -
3. 血樣的收集 - 37 -
4. 臟器的收集 - 37 -
5. 脾臟細胞的收集 - 37 -
6. 自然殺手細胞活性分析 - 38 -
7. 製備脾臟細胞培養液 - 38 -
8. 脾臟細胞增生能力測定 - 39 -
9. 血清中非特異性抗體的測定 - 40 -
10. 脾臟細胞表面標記分析 - 41 -
三、茯苓粗多醣萃取物對Balb/c鼠體內特異性免疫反應之影響 - 42 -
(一)實驗流程 - 42 -
(二)實驗材料 - 43 -
1. 樣品來源 - 43 -
2. 動物飼料 - 43 -
3. 實驗動物 - 43 -
4. 實驗器材和設備 - 43 -
(三)實驗步驟和方法 - 45 -
1. 動物飼養 - 45 -
2. 實驗組別 - 45 -
3. 致敏操作 - 46 -
4. 血清中特異性抗體的測定 - 47 -
四、茯苓粗多醣萃取物對Balb/c鼠轉殖CT26腫瘤之作用 - 48 -
(一)實驗流程 - 48 -
(二)實驗材料 - 49 -
1. 樣品來源 - 49 -
2. 實驗細胞株 - 49 -
3. 實驗動物 - 49 -
4. 實驗器材與儀器 - 49 -
(三)實驗步驟和方法 - 51 -
1. CT26細胞之冷凍保存 - 51 -
2. CT26細胞之解凍 - 51 -
3. CT26細胞之培養 - 51 -
4. 實驗組別 - 52 -
5. 轉殖腫瘤 - 53 -
6. 細胞激素分泌量之分析 - 53 -
7. 統計分析 - 54 -
第四章 結果與討論 - 56 -
一、茯苓粗多醣萃取 - 56 -
茯苓粗多醣之蛋白質及總醣含量 - 56 -
二、茯苓粗多醣萃取物對Balb/c鼠體內非特異性免疫反應之影響 - 56 -
(一)體重變化 - 56 -
(二)器官相對重量 - 56 -
(三)血清中IgG含量 - 57 -
(四)脾臟細胞增生能力 - 57 -
(五)自然殺手細胞活性 - 58 -
(六)脾臟細胞表面抗原分析 - 58 -
三、茯苓粗多醣萃取物對Balb/c鼠體內特異性免疫反應之影響 - 60 -
(一)體重變化 - 60 -
(二)器官相對重量 - 61 -
(三)茯苓粗多醣對OVA致敏之Balb/c鼠脾臟細胞表面抗原分析 - 61 -
(四)茯苓粗多醣對OVA致敏之Balb/c鼠抗OVA專一性抗體IgG和IgM之影響 - 62 -
四、茯苓粗多醣萃取物對Balb/c鼠轉殖CT26腫瘤之作用 - 63 -
(一)體重變化 - 63 -
(二)器官相對重量 - 64 -
(三)腫瘤生長大小 - 64 -
(四)茯苓粗多醣對轉殖CT26腫瘤之Balb/c鼠血清中抗體IgG含量之影響 - 64 -
(五)茯苓粗多醣對轉殖CT26腫瘤之Balb/c鼠脾臟細胞增生能力之影響 - 65 -
(六)茯苓粗多醣對轉殖CT26腫瘤之Balb/c鼠脾臟細胞表面抗原分析... - 66 -
(七)茯苓粗多醣對轉殖CT26腫瘤之Balb/c鼠脾臟細胞條件培養液中TNF-α含量之影響 - 67 -
第五章 總結 - 69 -
參考文獻 - 102
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