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研究生:曾炫凱
研究生(外文):Hsuan-Kai Tseng
論文名稱:利用癌組織異體移植體外培養與轉移性腫瘤動物模式:探討桑黃多醣萃取物輔助化學治療之分子作用機制
論文名稱(外文):The molecular mechanism of adjuvant action of polysaccharide extract of Phellinus linteus is synergistic with chemotherapies in xenografted tumor histoculture and metastatic animal model
指導教授:褚俊傑褚俊傑引用關係
指導教授(外文):Jiunn-Jye Chuu
學位類別:碩士
校院名稱:南台科技大學
系所名稱:生物科技系
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:中文
論文頁數:90
中文關鍵詞:桑黃 抗氧化 肺癌 黑色素細胞癌 巨噬細胞 轉移性 輔助化療 動物模式
外文關鍵詞:Phellinus linteusantioxidantlung cancermelanomamacrophagemetastasisadjuvant chemotherapyanimal model
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桑黃的學名為Phellinus linteus,是寄生於桑樹上的一種多年生蕈類屬於刺革菌科真菌,在韓國與日本已作為醫治癌症的藥物。近年研究發現,桑黃具有免疫調節與抗腫瘤等藥理活性功效,同時具有無副作用等優點,目前已成功開發為正式的健康食品與具抗癌劑功效 之醫藥品來販售。然而野生桑黃生長緩慢且數量有限,故本研究乃利用菌絲體發酵技術,來研究桑黃多醣萃取物與合併抗癌藥物cisplatin及etoposide 之組合物,來評估其對原發性與轉移性肺癌之輔助癌症化學治療與降低化療副作用之分子作用機制。
首先,我們評估桑黃多醣萃取液對肺癌細胞與黑色素癌細胞等細胞毒殺能力,接著透過接種(implation)肺癌細胞(LL2)與黑色素癌細胞(B16F10)在實驗動物的荷瘤模式(xenografted model),將這些已增生之癌組織(mass)進行體外活體培養技術,來進一步探討在癌組織與巨噬細胞共同培養下,桑黃多醣萃取液如何透過免疫調節作用來產生其抗腫瘤增生之能力與輔助癌症化學治療之藥理作用機制。最後,以直接靜脈注射黑色素癌細胞所誘導轉移性腫瘤小鼠模式,來測試桑黃多醣萃取液是否能輔助抗癌藥物抑制肺癌的增生作用與延長在腫瘤動物的存活時間等抗癌能力。
研究結果顯示,桑黃菌絲體樣品經由酚硫酸呈色法與自由基清除試驗中發現,利用熱水萃取暨酒精沉澱取得的多醣粗抽物(crude extract)具有較好的抗氧化活性。我們挑選了試驗組中粗多醣(crude polysaccharides)含量較高的桑黃組,來進行計畫中的各項抗腫瘤活性評估與輔助化療試驗。我們發現桑黃多醣萃取物合併抗癌藥物(cisplatin與etoposide)對老鼠肺癌細胞LL2,人類肺癌細胞A549肺癌細胞與具轉移性老鼠黑色素癌細胞(B16F10)的毒殺作用有顯著的輔助效果,在黑色素癌細胞轉移試驗中也有減緩細胞遷徙(migration)的情形。此外,桑黃多醣萃取物與巨噬細胞(RAW 264.7)共同培養下,對於皮下荷瘤癌組織與巨噬細胞(RAW 264.7)共同培養(ex vivo histoculture)下,也有顯著抑制腫瘤組織增生的表現。ELISA分析試驗中,可發現桑黃多醣萃取液可分別在24小時後之培養基與活體腫瘤動物模式中,提高細胞激素TNF-與降低M-CSF的表現量。同時,在腫瘤轉移動物模式下,我們可由有餵食桑黃多糖萃取物的老鼠血中白血球與血小板會有明顯提升,來判斷桑黃多醣萃取物能降低抗癌藥的副作用。特別是餵食桑黃多醣萃取物對轉移性肺癌組織的腫瘤發生率(腫瘤重量/體重)與結節數也有明顯之降低且顯著提高轉移性肺癌動物之存活率。由以上結果顯示,我們可以推斷桑黃多醣萃取物本身即具有減緩腫瘤轉移的藥理活性,尤其是對於癌症輔助治療與降低相關副作用具有良好的改善成效,值得臨床上針對轉移性癌症(如肺癌)在化學治療的開發運用上,提供一項新的輔助治療標的。
The Phellinus linteus (Hymenochaetaceae), found on mulberry trees growing on wild mulberry trees, is a well-known fungus which belonged to one of perennial mushrooms. In South Korea and Japan, it have been served as a pharmaceuticals and health food for preventing cancer. Recent studies also have shown that Phellinus linteus has the effects of anti-tumor and immunomodulation without any side-effect. However, the wild Phellinus linteus is growing slowly and show serious productive limit, thus we try to develope a proprietary production technology based on submerged fermentation in this study to evaluate the molecular mechanism of adjuvant action of polysaccharide extract of Phellinus linteus (PLPE) and/or combined with anticancer drugs (eg. cisplatin and etoposide) in original and metastatic tumors by the models of xenografted tumor histoculture and metastatic animal.
First, the cytotoxity of PLPE in several cancer cells, lung cancer cell (LL2, A549) and melanoma cell (B16F10) was estimated in vitro. Continuously, the lung cancer cells and melanoma cells were implanted onto the ICR nude mice, then they were taken off the tumors, while the proliferating solid mass grew up to be exceed 1 cm around the tumor. In the presence of macrophages, the tissue samples were treated with the PLPE in the co-culture system of xenografted tissues and macrophages, to explore the anti-proliferation and the potential benefits of adjuvant chemotherapy via the activation of immune response. Finally, we evaluate the synergistic effects of PLPE with chemotherapies for the inhibition of tumor proliferation and the improvement of animal survival in metastatic animal model using the direct-Injection model of melanoma cell by tail vein.
The phenol-sulfuric acid method and antioxidant test showed that PLPE from the use of hot-water extraction and alcohol precipitation had better antioxidant activity than others. It was found that PLPE was significantly synergistic with anti-cancer drugs (cisplatin, etoposide) in the cytoxicity of cancer cells and cell migration assay in vitro. In the cocultured histoculture system containing macrophage cells (RAW 264.7), PLPE also inhibited the growth of LL2, not B16F10, however, the combination of PLPE and anti-cancer drugs had a remarked growth inhibition of B16F10, through decreasing the expression of p-AKT. Using the ELISA assay, the stimulation of PLPE resulted in the elevated TNF-level and M-CSF level decreased in the medium 24h later. We also found that PLPE can diminish the tumor weight in body weight ratio, reduce incidences of tumor nodules and elevate the survival of BALB/c mice with increasing the numbers of white blood cells and platelets in metastasis studies. Taken together, we believed that PLPE itself could alleviate the invasive tumor growth and PLPE also potentiate the effectiveness of chemotherapies treatment by a better side-effect profile in metastastic animal model.
摘要 i
目次 iii
圖及表目錄 v
縮 寫 表 vi
<第一章、緒論及文獻探討> 1
1. 桑黃簡介 1
1.1 桑黃的產地與基源 1
1.2 桑黃的分類與型態 2
1.3 桑黃的多醣 4
1.4 桑黃之生物活性應用及研究 10
2. 桑黃與癌症 13
2.1 癌症簡介 13
2.2 腫瘤轉移 13
2.3 黑色素瘤 15
2.4 癌症治療 16
2.5 抗癌藥物副作用 17
<第二章、研究動機及目的> 18
<第三章、實驗材料及方法> 19
3.1 材料 19
3.2 儀器設備 20
3.3 桑黃熱水萃取物 21
3.4 桑黃乙醇萃取物 21
3.5 桑黃多糖酒精沉澱萃取法 21
3.6 DPPH自由基抑制試驗 21
3.7 凝膠滲透層析(Gel Permeation Chromatography, GPC) 22
3.8 酚硫酸呈色法 23
3.9 細胞培養及繼代 23
3.10 抗癌藥物配製 23
3.11 細胞毒性測試-MTT Assay 23
3.12 細胞轉移試驗 24
3.13 西方墨點法 25
3.16 皮下靜脈注射誘導腫瘤轉移模式 31
3.17 腫瘤組織細胞毒性測試-MTT Assay 31
3.18 組織培養液中之細胞激素測定(M-CSF、IL-1與TNF-) 32
3.19 腫瘤轉移實驗動物之藥物給予 33
3.20 腫瘤轉移實驗動物之血中生化值 33
3.21 腫瘤轉移實驗動物之肺腫瘤結節計數 33
3.22 腫瘤轉移實驗動物之肺腫瘤/體重之比率 33
3.23 腫瘤轉移實驗動物之生存曲線 33
3.24 數據處理與分析 33
<第四章、結果> 35
4.1 DPPH自由基抑制試驗 35
4.2 膠體層析法(GPC)-分子量分佈分析 35
4.3 酚硫酸呈色法-多醣含量分析 35
4.4 桑黃多糖萃取物輔助抗癌藥物-細胞毒性測試 35
4.5 B16F10細胞創傷癒合實驗 36
4.6 異體癌組織體外之培養-細胞毒性測試 36
4.7 異體癌組織體外之培養-ELISA assay 37
4.8 抗癌藥物副作用抑制效果 38
4.9 腫瘤轉移動物模式實驗 38
<第六章、結論> 43
<第七章、實驗結果> 45
<第八章、參考文獻> 74
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