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系統識別號 U0026-0812200914004122
論文名稱(中文) 桑黃抗癌效果之研究
論文名稱(英文) Study on the Anti-Tumor Effects of Phellinus Linteus
校院名稱 成功大學
系所名稱(中) 醫學檢驗生物技術學系碩博士班
系所名稱(英) Department of Medical Laboratory Science and Biotechnology
學年度 95
學期 2
出版年 96
研究生(中文) 陳光渝
研究生(英文) Kuang-yu Chen
電子信箱 t3694101@mail.ncku.edu.tw
學號 t3694101
學位類別 碩士
語文別 中文
論文頁數 69頁
口試委員 口試委員-張權發
口試委員-林尊湄
指導教授-黃暉升
中文關鍵字 小鼠路易士肺癌  桑黃萃取物  多醣體 
英文關鍵字 Phellinus linteus extraction  polysaccharides  Louis lung carcinoma 
學科別分類
中文摘要 桑黃菇是一種傳統中草藥,被發現有顯著的抗腫瘤功能。本著強而有力的療效,桑黃在韓國成了癌症輔助治療的核可藥物。其多醣體被發現有多種免疫調節功能。例如,一項腫瘤小鼠的體內實驗顯示,桑黃分離出的醣蛋白,可以讓助手型T淋巴細胞分化趨向第一型的免疫型式,並且抑制MCA-102癌細胞的生長。不幸地,桑黃子實體在野外生長緩慢。因此,以液態培養出桑黃菌絲體,再濃縮其中有效的分離物,會是較為迅速的研究方法。所以,我們想知道此種液態培養的桑黃,是否能造成如前人所說的腫瘤抑制及免疫調節反應。桑黃樣本被分為菌絲體水煮液(PLw)、水煮液中的多醣體(PLPS)及桑黃發酵過的培養液(PLm)。一開始先以C57BL/6j小鼠做28日藥物毒性測試,最高劑量組給予1000毫克/公斤體重.日之下,可看出桑黃的安全性。在C57BL/6j鼠接種小鼠肺癌細胞LL/2腫瘤模型中,PLm+PLw及PLw有抑制腫瘤效果,但PLPS則否。PLm+PLw在200及400毫克/公斤體重.日的服用下,35天後對腫瘤體積抑制率分別為38﹪及36﹪;PLw在200及400毫克/公斤體重.日的服用下,36天後對腫瘤體積抑制率分別為61﹪及67﹪。然而,體外實驗顯示了PLm及PLw對LL/2及人類肺癌細胞A549的活性抑制效果,以及Sub-G1值的提升。脾臟中細胞的CD4/CD8比值,及血清中IL-12值沒有明顯改變。以PLPS刺激小鼠巨噬細胞RAW 264.7可以提升其吞噬能力。因此,我們的結論認為液態培養的桑黃在體外有抑制癌細胞及免疫調節功能,但是在動物模型的效果則需要進一步釐清。
英文摘要 Phellinus linteus (PL) is a traditional Chinese herbal medicine and found to have remarkably tumor-inhibiting functions. With powerful therapeutic effects, PL becomes an approved medicine in Korea for the co-treatment of cancer. Its polysaccharides (PS) are found to have immune-modulate functions. An in vivo mice tumor model shows that the proteoglycan isolated from PL may lead type-I helper T cell-dominant immune state and inhibit the growth of MCA-102 cells. Unfortunately, the PL fruit body grows slowly on the wild field. Therefore, liquid culture of the PL mycelia and then concentration of the functional fractions is a more rapid method to study on its effects. Thus, we want to know whether the liquid cultured PL causes tumor-inhibiting and immune-modulating reactions as the previous studies show. PL samples were separated into water extractions (PLw), polysaccharides in PLw (PLPS), and PL broth medium (PLm). A 28-days drug toxicity test was performed in the beginning by treating C57BL/6j mouse with the highest dosage of 1000 mg/kg.day and showed the safety of PL. PLm+PLw and PLw have tumor-inhibiting functions to LL/2 tumor bearing C57BL/6j male mice, but PLPS doesn’t. The tumor inhibition rates came to 38﹪ and 36﹪ under 35 days treatment of 200 and 400 mg/kg.day of PLm+PLw; 61﹪ and 67﹪ under 36 days treatment of 200 and 400 mg/kg.day of PLw. However, the in vitro assay shows the inhibitory effects of PLm and PLw to LL/2 and human lung carcinoma A549 and the up-regulation of their Sub-G1 values. The ratios of CD4/CD8 of spleenocytes and the level of IL-12 in serum had no obvious change. Stimulation of RAW 264.7 cells by PLPS may enhance the abilities of its phagocytosis. Therefore, we conclude liquid culture system of PL has tumor-inhibiting and immune-modulating functions in vitro, but the effects to the animal model should be further elucidated.
論文目次 考試合格證明 II
中文摘要 III
英文摘要 IV
致謝 VI
目錄 VII
表目錄 X
圖目錄 XI
縮寫表 XIII

主文
第一章 研究背景……………………………………1
第一節 癌症與抗癌療程…………………………………………………………1
第二節 細胞凋亡…………………………………………………………………2
電三節 免疫系統與癌症…………………………………………………………3
第四節 桑黃背景…………………………………………………………………4
第五節 桑黃之生物活性…………………………………………………………5
第二章 研究目的……………………………………7
第三章 實驗材料…………………………………8
第一節 細胞株……………………………………………………………………8
第二節 試劑………………………………………………………………………9
第三節 桑黃分離物 ……………………………………………………………11
第一段 液態醱酵培養 ……………………………………………………11
第二段 桑黃樣本分離 ……………………………………………………11
第四章 實驗方法 ………………………13
第一節 活體實驗 ………………………………………………………………13
第一段 28日短期體內安全性實驗 ………………………………………13
第二段 癌細胞誘發皮下原位腫瘤模式 …………………………………14
第三段 統計方法 …………………………………………………………15
第二節 離體實驗 ………………………………………………………………15
第一段 細胞培養 …………………………………………………………15
第二段 離體細胞毒性 ……………………………………………………17
第三段 醣體定量 …………………………………………………………18
第四段 脾臟細胞分離 ……………………………………………………19
第五段 脾臟細胞分型 ……………………………………………………20
第六段 巨噬細胞吞噬細菌能力測定 ……………………………………21
第七段 巨噬細胞吞噬螢光乳膠微粒能力測定 …………………………22
第八段 酵素連結免疫吸附分析法 ………………………………………22
第九段 細胞週期分析 ……………………………………………………23
第十段 統計方法.…………………………………………………………24
第五章 實驗結果 ……………………25
第一節 液態培養的桑黃對正常小鼠沒有短期毒性 …………………………25
第二節 PLm+PLw及PLw在體內實驗中能抑制LL/2腫瘤 ……………………25
第三節 PLm及PLw在體外都能抑制LL/2………………………………………25
第四節 PLm+PLw,PLw,PLPS都沒有影響T淋巴細胞分化…………………26
第五節 所有的桑黃分離物都沒有提升12-介白素(IL-12)…………………26
第六節 PLPS在體外可以活化小鼠巨噬細胞 …………………………………27
第七節 PLw在體外提高LL/2及A549的Sub-G1值 …………………………27
第六章 討論 …………………………………28
參考文獻………………………………………35
圖表
附錄……………………………………………………68
作者簡歷……………………………………………69
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