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研究生:黃美淑
研究生(外文):HUANG, MEI-SHU
論文名稱:樟芝水萃取液抑制口腔腫瘤細胞增生、轉移之研究
論文名稱(外文):A bioactivity study of niu-chang-chih extract on oral carcinoma cell lines
指導教授:陳健祺
指導教授(外文):Chen, Jian-Chyi
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:101
畢業學年度:100
語文別:中文
論文頁數:78
中文關鍵詞:樟芝轉移細胞凋亡MMP-9Baxpro-caspase 9Bcl-2口腔鱗狀上皮細胞癌
外文關鍵詞:Antrodia camphoratemetastasisapoptosisoral squamous cell carcinoma
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口腔癌為全世界第六常見的惡性腫瘤。在台灣,自1991 年起,也是前十大癌症死亡原因之一。近年來口腔癌的發生率及死亡率有逐漸增加的趨勢,嚼食檳榔也被證實為造成口腔黏膜下纖維化及口腔癌的主要致病因子之一。雖然目前治療口腔癌的方法有好幾種,但口腔癌之五年存活率在過去十幾年來並沒有明顯改善。所以尋找更有效治療口腔癌的策略是重要的。
樟芝(Antrodia camphorata)是台灣稀有且極為昂貴的藥用真菌。過去的研究發現,樟芝對人體肝癌具有促進抗氧化、抗發炎的效果,但目前仍沒任何資料證明樟芝可作為治療口腔癌。因此,這個研究的目的是要探討從樟芝萃取液抑制口腔腫瘤細胞株生長及促進它發生細胞凋亡的分子機制。
首先利用MTT assay測試樟芝水萃取液抑制檳榔刺激人類口腔腫瘤細胞株(OECM-1)增生的試驗,再以流式細胞儀來探討樟芝萃取液在抑制口腔腫瘤細胞株的作用機制,例如細胞凋亡。接著進一步使用西方點墨法研究樟芝萃取液是否能有效活化各個與細胞週期調控及細胞凋亡有關之指標分子表現(Bax、pro-caspase 9、Bcl-2)。此外,樟芝萃取液抑制口腔癌細胞株的轉移試驗及抑制口腔細胞株纖維化的生物活性也被探討與研究。在real-time PCR結果,樟芝萃取液處理OECM-1細胞會使MMP-9之mRNA相較於沒有處理而有高度表現。而migration assay實驗顯示樟芝亦可抑制OECM-1細胞的移動。
Oral cancer is the sixth most common cancer worldwide. In Taiwan, oral cancer has been one of the top 10 leading causes of death from cancer since 1991. In the past decades, incidence and mortality rate of oral squamous cell carcinoma (OSCC) increased remarkably, areca quid chewing is demonstrated to be one of the major risk factors leading to oral submucous fibrosis, and oral cancer. Although there are many treatment options for oral cancer, the 5-year survival rate of OSCC has not been improved significantly.
Developing more effective strategies against oral tumors is a very important object. Although previous studies have shown that Antrodia camphorata possesses anti-oxidative and anti-inflammatory effects on human hepatocellular carcinomas, no documented effects of
A.camphorata in treatment of oral cancers have been described.
Therefore, the purpose of this study was to investigate the medicinal properties of A.camphorata that can be used to treat oral cancers.
The medical effects of A.camphorata on OSCC have been evaluated by MTT assay and the growth-inhibition mechanism, such as apoptosis, was evaluated by Flow cytometer during this study. We further analyzed whether the A. camphorata can regulate the cell cycle-and apoptosis-related molecules by western blot. Furthermore,
the effect of A. camphorata on MMP-9 expression was also studied by gelatin zymograph assay. MMP-9 mRNA expression was upregulated after treatment with A.camphorata in OECM-1cell as compared with the untreated group.
The migration assay supported that A. camphorata that inhibited the migration of OECM-1 cells.
Conclusively, the demonstrated medicinal properties of Antrodia camphorate that can be used to treat oral cancers, or used as supplements to augment its therapeutic effects.
中文摘要 i
英文摘要 iii
第一章 緒論
一、口腔癌
1.1 口腔癌的簡介 1
1.2 口腔癌之危險因子 2
1.3 口腔鱗狀細胞癌之病理分類 3
1.4 口腔癌之預後 4
1.5 口腔癌的治療 4
二、細胞凋亡 5
1.2.1 細胞凋亡的型態與特徵 5
1.2.2 細胞凋亡的訊息傳導路徑 7
1.2.3 基質金屬蛋白酶 13
三、樟芝(Antrodia camphorata)的簡介 16
1.4.1 樟芝的分類與特徵 16
1.4.2 樟芝之生物學特性 17
1.4.3 樟芝之生物活性與藥理療效 17
第二章 研究動機 20
第三章 實驗架構 21
第四章 材料方法 22
一、實驗細胞與藥品 22
4.1 實驗細胞株 22
4.2 樟芝水萃取法 22
4.3 實驗藥品 22
4.3.1 購自SIGMA (USA) 22
4.3.2 購自波仕特公司 23
4.3.3 購自德國MERCK 23
4.3.4 購自美國Amersham 23
4.3.5 購自Bio‐Rad 23
4.3.6 抗體使用一覽表 24
4.3.7 購自Gibco 24
4.3.8 引子 24
4.4 細胞培養 (Cell culture) 24
二、實驗方法 25
4.5 細胞存活分析 (MTT cell viability assay) 25
4.6 細胞週期測定Propidium iodide(PI)染色 26
4.7 利用倒立式位相差顯微鏡評估細胞型態 27
4.8 西方墨點法(Western blot) 27
4.9 Gelatin Zymography Assay 30
4.10 反轉錄聚合酵素連鎖反應 33
4.10 移行實驗 34
4.11 統計分析及實驗操作方法 35
第五章 實驗結果 36
5.1 樟芝萃取液抑制腫瘤細胞增生試驗 36
5.2 利用倒立式位相差顯微鏡觀察細胞型態 36
5.3 流式細胞儀測定樟芝萃取液對OECM-1 細胞的影響 37
5.4.1 樟芝萃取液經由內在凋亡路徑誘發口腔腫瘤細胞凋亡 37
5.4.2 樟芝萃取液誘導內生性細胞凋亡(intrinsic apoptotic
pathway)機制 38
5.5 樟芝萃取液對口腔癌細胞株OECM-1 移行性之影響 41
5.6 樟芝萃取液對matrix metalloproteinase-9 表現影響之探討 39
5.7 樟芝萃取液會抑制OECM-1 細胞之MMP-9 的mRNA 表現 39
第六章 討論 40
第七章 結論 44
第八章 未來工作 45
第九章 參考文獻 46
第九章 參考文獻
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