臺灣博碩士論文加值系統

絞股藍皂苷是絞股藍萃取物中重要的組成，且有多種不同的藥理性，包括有抗腫瘤的增生及腫瘤的形成。最近研究顯示，在許多人類的癌細胞株中，絞股藍皂苷能誘發細胞週期停滯及引發細胞凋亡。然而在人類口腔癌細胞株的研究中，並沒有關於絞股藍皂苷對於DNA損傷，DNA修復的相關基因表現，細胞凋亡及預防細胞侵襲的相關報導。
本實驗的目的，為研究絞股藍皂苷對於人類口腔癌細胞株SAS其DNA損傷的影響，DNA修復的相關基因表現，細胞凋亡及細胞移動或侵襲的影響。
流式細胞儀被使用來檢測及量化SAS細胞的存活率，彗星試驗的結果顯示在絞股藍皂苷不同濃度的作用下，DNA損傷(彗星拖尾)的程度有濃度的依賴性。又在Real-time PCR的檢測下，當絞股藍皂苷的濃度為180 μg/ml，作用於SAS細胞株24小時後，將使14-3-3σ、DNA-PK、p53、ATM、ATR、BRCA1 的mRNA表現量減少。
流式細胞儀分析結果也顯示絞股藍皂苷誘導SAS細胞株細胞週期停滯在S期，且在DAPI實驗及DNA凝膠電泳的檢測下證明細胞凋亡的存在。又絞股藍皂苷誘導細胞中 ROS 、 Ca2+、 ER stress的表現增加，同時降低粒腺體膜電位的表現，引發Cytochrome c 的釋放。再由西方墨點法分析結果顯示絞股藍皂苷經由減少Bcl-2的表現，增強 Bax的表現量，進而降低粒腺體膜電位，造成Cytochrome c 的釋放，接著活化下游 Caspase-9 ,-3的活性，或藉由誘導 Endo G 從粒腺體的釋放，而導致細胞凋亡。
除了PI3K之外，絞股藍皂苷在降低SOS、Ras、uPA、FAK、Akt 、NF-κB、COX-2、 ERK1/2 、 MMP-9、 MMP-2的蛋白表現量方面，有時間的依賴性。另外絞股藍皂苷能減少MMP-2, MMP-7, MMP-9的 mRNA表現，但在FAK與Rho 其mRNA並無明顯變化。
綜合上述數據顯示， 絞股藍皂苷對人類口腔癌細胞株SAS能造成 DNA 損傷，使細胞週期停滯，同時抑制DNA 的修復，誘導細胞凋亡，並且抑制細胞的轉移和細胞侵襲的能力。

Gypenosides (Gyp) is the major component of Gynostemma pentaphyllum Makino extracts and is known to have diverse pharmacologic activities including anti-proliferation and anti-cancer effects. Recently, Gyp has been shown to induce cell cycle arrest and apoptosis in many human cancer cell lines. However, there is no available information to address the effects of Gyp on DNA damage, DNA repair associated gene expression, apoptosis and in preventing invasion in human oral cancer cells.
The purpose of this study was to investigate the effects of Gyp treatment on DNA damage, DNA repair gene expression, apoptosis and the migration and invasion in human oral cancer SAS cells.
Flow cytometry analysis was used to examine and quantitate the percentage of viable SAS cells. The results from Comet assay indicated that the incubation of SAS cells with various doses of Gyp led to a longer DNA migration smear (Comet tail) when compared with control and these effects are in a dose-dependent manner. The results from real time PCR analysis indicated that 180 μg/ml of Gyp for 24 h treatment in SAS cells led to decrease 14-3-3 proteins sigma (14-3-3σ), DNA-dependent serine/threonine protein kinase (DNA-PK), p53, ataxia telangiectasia mutated (ATM), ataxia-telangiectasia and Rad3-related (ATR) and breast cancer gene 1 (BRCA1) mRNA expression.
Flow cytometry analysis also indicated that Gyp induced cell cycle arrest (S phase arrest) and apoptosis in SAS cells.
Gyp induced production of ROS and Ca2+ and ER stress and decreased the levels of ΔΨm, causing Cytochrome c release. Western blotting results demonstrated that Gyp decreased Bcl-2 and increased Bax levels, decreased the levels of ΔΨm, caused Cytochrome c release, activations of Caspase-9 and -3, or induced Endo G release from mitochondria leading to apoptosis.
Gyp decreased SOS, Ras, urokinase-type plasminogen activator (uPA), focal adhesion kinase (FAK), RAC-alpha serine/threonine-protein kinase (Akt) , nuclear factor kappa B (NF-κB), cyclooxygenase-2 (COX-2), extracellular signal-regulated kinase 1/2 (ERK1/2) and matrix metalloproteinase -9, -2 (MMP-9, MMP-2) in a time-dependent manner except phosphatidylinositol 3-kinase (PI3K) levels. In addition, Gyp decreased the mRNA levels of MMP-2, MMP-7 and MMP-9 but not FAK and Rho in SAS cells.
These observations may offer information for the molecular mechanisms leading to cell apoptosis by Gyp in SAS cells. Taken together, Gyp induced DNA damage, inhibited the DNA repair, arrested the cell cycle, induced apoptosis and inhibited the metastatic and invasive capacity of oral cancer cells in vitro.

目錄
第一章 前言 1
第二章 文獻探討 6
第一節 中醫與腫瘤的關係 6
1-1.中醫腫瘤的病因 6
1-2.中醫腫瘤的病機 7
1-3.中醫腫瘤的治法 7 1-4.中藥抗腫瘤的有效成分 9 1-5.中藥抗腫瘤的作用機轉 10
1-6.中醫治療腫瘤優勢 11
1-7.中藥治療腫瘤細胞的特性(多靶點效應) 11
第二節 絞股藍 13
2-1.天然青草藥與中藥之區別，用語等疑義 13
2-2.絞股藍為可同時提供食品使用之中藥材 13
2-3.絞股藍始載於明代朱橚著的《救荒本草》 13
2-4.別名與形態 13
2-5.種類與分佈 14
2-6.性味歸經 14
2-7.功能、主治 14
2-8.絞股藍化學成分 15
2-9.絞股藍的近代藥理研究 17
2-10.絞股藍的近代臨床研究 23
第三節 DNA 損傷(DNA damage) 24
第四節 細胞週期檢查點(cell cycle checkpoint) 25
4-1. G1/S期檢查點 25
4-2. S期檢查點 25
4-3. G2/M期檢查點 26
第五節 DNA的修復 27
第六節 細胞週期(Cell cycle) 31
6-1.細胞週期調控分子可分為三大類 31
6-2.細胞週期檢查點 32
第七節 細胞凋亡(apoptosis) 33
7-1.細胞凋亡 33
7-2.檢測細胞凋亡的方法(Annexin V-FITC/PI) 33
7-3.細胞凋亡的鑑別方法有 33
7-4.細胞凋亡路徑 34
第八節 細胞轉移 40
第三章 材料與方法 42
第一節 實驗材料 42
1-1.細胞株來源 42
1-2.藥物來源 42
1-3.藥品試劑 43
1-4.設備與器材 46
1-5.實驗動物來源 47
第二節 實驗方法 47
2-1.絞股藍與絞股藍皂苷濃度配製 47
2-2.細胞冷凍保存 48
2-3.冷凍細胞的活化 48
2-4.人類口腔癌細胞株(SAS cell lines)的培養 49
2-5.絞股藍作用於SAS細胞株細胞存活率及細胞凋亡研究 49
2-6.絞股藍皂苷誘導SAS細胞株細胞凋亡及轉移抑制研究 51
第三節 分析方法 72
3-1.數據分析 72
第四章 研究結果 73
第一節 絞股藍與絞股藍皂苷對SAS細胞株存活率的影響 73
1-1.存活率(viability) 的影響 73
第二節 絞股藍與絞股藍皂苷對SAS細胞株細胞凋亡的影響 75
2-1.細胞形態學(morphology)的影響 75
2-2.細胞凋亡的影響 76
第三節 絞股藍皂苷對細胞株SAS造成DNA損傷的影響 78
3-1.存活率(viability) 的影響 78
3-2.利用彗星試驗(comet assay)觀察細胞DNA damage現象 79
第四節 絞股藍皂苷對細胞株SAS造成細胞週期停滯的影響 81
4-1.存活率(viability) 的影響 81
4-2.細胞週期 (cell cycle)的影響 82
4-3.細胞週期停滯相關蛋白的影響 83
第五節 絞股藍皂苷對SAS修復抑制的影響 85
5-1.利用Real-time PCR 探討DNA修復相關基因的表現 85
第六節 絞股藍皂苷對SAS細胞凋亡的影響 89
6-1.細胞形態學(morphology)的影響 89
6-2.細胞凋亡(apoptosis)比例的影響 90
6-3.利用DNA凝膠電泳，檢測DNA 碎片的形成 91
6-4.利用DAPI staining觀察細胞凋亡的形態變化 92
6-5.絞股藍皂苷對SAS細胞株 ROS的影響 94
6-6.絞股藍皂苷對SAS細胞株 Ca2+的影響 96
6-7.絞股藍皂苷對SAS細胞株粒腺體膜電位的影響 98
6-8.絞股藍皂苷與NAC對SAS 存活率的影響 100
a. NAC對ΔΨm的影響 100
b. NAC對Ca2+的影響 102
c. NAC對細胞存活率及凋亡比率的影響 104
6-9.絞股藍皂苷與BAPTA對SAS 存活率的影響 105
a. BAPTA對ΔΨm的影響 105
b. BAPTA對存活率及細胞凋亡比率的影響 107
6-10.絞股藍皂苷對SAS Caspase活性的影響 108
a. Caspase-3活性影響 108
b. Caspase-8活性影響 110
c. Caspase-9活性影響 112
6-11. Caspase inhibitor 對細胞存活率及凋亡比率的影響 114
a. Z-DEVD-FMK 114

b. Z-IETD-FMK 116

c. Z-LEHD-FMK 118

d. Z-VAD-FMK 120
6-12.絞股藍皂苷對 SAS 凋亡路徑相關蛋白的影響 122
a.粒腺體路徑 122
b.內質網路徑 125
c.死亡接受器路徑 126
d.生長因數路徑 128
6-13.利用免疫螢光法及共軛焦顯微鏡探討 130
a. Cytochrome c 130
b. Endo G 130
c. AIF 131
d. GADD153 131
6-14.利用Real-time PCR 探討細胞凋亡相關基因的表現 136
第七節 細胞轉移相關實驗 140
7-1.絞股藍皂苷對SAS細胞傷口癒合分析試驗的影響 140
7-2.絞股藍皂苷對SAS 細胞移動分析試驗的影響 142
7-3.絞股藍皂苷對SAS 細胞侵入分析試驗實驗的影響 144
7-4.利用Real-time PCR 探討細胞轉移相關基因的表現 146
7-5.絞股藍皂苷對SAS細胞移動和侵入抑制相關蛋白影響 149
第八節 絞股藍皂苷對SAS 體內動物實驗 152
第五章 討論 159
第六章 結論 179
參考文獻 184
英文摘要 200
謝辭 202

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