臺灣博碩士論文加值系統

　　巴西蘑菇原產於巴西，巴西蘑菇最常用來抑制癌症也被用來改善許多疾病，例如:糖尿病、高膽固醇、軟骨症。巴西蘑菇含有豐富的多醣體，這些多醣體被發表可增進免疫功能進而殺死癌細胞。雖然巴西蘑菇的好處很多，但它的生物特性及成分還沒有被完整的研究清楚。我們的研究就是去探討巴西蘑菇的抗氧化性，我們利用肺癌上皮細胞A549，骨癌上皮細胞Saos-2、老鼠纖維母細胞NIH3T3 和大鼠的心肌細胞H9c2 當作研究材料。這些細胞加入不同濃度的巴西蘑菇萃取液，再利用MTT 方法觀察細胞存活率。結果發現加入巴西蘑菇萃取液高達10 mg/ml 也不會影響細胞存活率，顯示說巴西蘑菇在10mg/ml 濃度下不具有細胞毒性。另外當細胞遭受到氧化性傷害時巴西蘑菇也顯示出保護作用。為了研究巴西蘑菇對細胞分子層面作用機制，我們利用NIH3T3 細胞加入巴西蘑菇萃取液並用過氧化氫(H2O2)來製造細胞的氧化性傷害，接著用流式細胞儀偵測反應性氧化物質(ROS)的數量。結果發現加入巴西蘑菇萃取液可減少ROS 的數量。再來我們利用彗星分析(comet assay)和南西方墨點法(south-western immunoblot)觀察到巴西蘑菇萃取液可減少H2O2 造成的氧化性DNA，即是8-oxoguanine 的量減少。因此我們的結果發現巴西蘑菇更進一步，本實驗利用硫酸銨鹽析沉澱法找到降低巴西蘑菇中降低ROS 的分餾物，但是我們目前尚未找到巴西蘑菇影響ROS 的分子機制，這將是未來主要的研究方向。

　　The Agaricus blazei Murill (ABM) mushroom is native to south-eastern Brazil and has been known for its medicinal properties. The mushroom is used to combat diabetes, hypercholesterolemia and osteoporosis. The protein-polysaccharide complex extracted from A. blazei Murill have previously been shown to have antitumor activity and stimulate immunity benefits. Nevertheless, scientific knowledge about its biological properties is still insufficient. In the present study, the antioxidant effects of A. blazei have been studied. By the analysis of MTT assay, the mouse fibroblast (NIH3T3), lung carcinoma epithelial cell (A549), osteosarcoma epithelial cell (Saos-2) and rat heart myocardium (H9c2) were treated with A. blazei. The results indicate the concentration (≦10 mg/ml) of A. blazei is no genotoxic. The aqueous extract dose (≦4 mg/ml) of A. blazei is able to protect from oxidative damage in all cells except, but not in Saos-2 cell. To investigate the molecular affect of A.blazei in NIH3T3 cell. We found the addition of A. blazei to cells reduces the amounts of reaction oxygen species (ROS), as demonstrated by flow cytometry. By comet assay and south-western immunoblot, we also found the DNA damage caused by hydrogen peroxide was decreased with treatment of A. blazei. The results suggest that extracts of Agaricus blazei Murill present antioxidant activity. We find out the fraction of A. blazei which can reduce ROS by salting out with ammonium sulfate. But we have not identified the mechanism which the level of ROS is reduced by A. blazei. It remains future investigation to elucidate the detail mechanism.

中文摘要------------------------------------------------------------------- I
英文摘要------------------------------------------------------------------ III
誌謝------------------------------------------------------------------------IV
目錄-------------------------------------------------------------------------V
表目錄---------------------------------------------------------------------VIII
圖目錄-----------------------------------------------------------------------IX
簡寫表-----------------------------------------------------------------------XI

第一章緒論
1.1 前言--------------------------------------------------------------------- 1
1.2 飲食與癌症--------------------------------------------------------------- 3
1.3 巴西蘑菇----------------------------------------------------------------- 5
1.4 巴西蘑菇的作用----------------------------------------------------------- 6
1.5 自由基與癌症的發生------------------------------------------------------- 8
1.6 研究動機---------------------------------------------------------------- 12

第二章實驗材料與方法
2.1 巴西蘑菇萃取------------------------------------------------------------ 13
2.2 細胞培養---------------------------------------------------------------- 13
2.3 細胞存活率試驗(MTT assay)----------------------------------------------- 14
2.4 流式細胞儀分析細胞反應性氧化物質(ROS)----------------------------------- 15
2.5 單細胞電泳法(Single-cell electrophoresis；Comet assay)------------------ 16
2.6 萃取細胞之DNA----------------------------------------------------------- 18
2.7 西南方墨點法(south-western blot)---------------------------------------- 19
2.8 p53 轉活化分析(p53 transactivation assay)------------------------------- 20
2.8.1抽取質體--------------------------------------------------------------- 21
2.8.2 以冷光儀偵測luciferase 的活性----------------------------------------- 22
2.9 純化巴西蘑菇成分-------------------------------------------------------- 22
2.91 熱水及凝膠過濾管柱萃取法----------------------------------------------- 22
2.92 鹽析沉澱法------------------------------------------------------------- 23
2.93 醣類測定--------------------------------------------------------------- 24
2.94 蛋白質測定------------------------------------------------------------- 25
2.10 統計方法--------------------------------------------------------------- 26

第三章結果
3.1 巴西蘑菇不具有細胞毒性-------------------------------------------------- 27
3.2 巴西蘑菇對氧化性傷害具有保護作用---------------------------------------- 27
3.3 巴西蘑菇萃取液可減少氧化性物質(ROS)的數量------------------------------- 29
3.4 巴西蘑菇萃取液可減少氧化性DNA 傷害-------------------------------------- 30
3.5 巴西蘑菇不影響p53 transactivation--------------------------------------- 32
3.6 巴西蘑菇內的成分影響降低氧化性物質(ROS)--------------------------------- 32
3.6.1 巴西蘑菇的多醣體成分-------------------------------------------------- 33
3.6.2 巴西蘑菇的蛋白質成分-------------------------------------------------- 33

第四章討論------------------------------------------------------------------ 36

參考文獻-------------------------------------------------------------------- 40

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