臺灣博碩士論文加值系統

根據行政院衛生署十幾年來的統計，男女性罹患胃癌的比例仍居於十大癌症排名中，且因初期症狀不明顯，確診時大多已無法手術切除。而對於無法手術切除之胃癌患者則多以化學治療為主，但由於副作用大所以臨床應用有限。因此，許多天然物對抗癌症之研究便紛紛興起。牛樟芝 ( Taiwanofungus camphorates ) 為台灣特有之珍貴真菌，僅生長於台灣獨有的牛樟樹中。在台灣已是眾所皆知的傳統藥物，而近十年來許多相關研究證實其具有相當多之藥理活性，如抗氧化及抗腫瘤等。因此，本實驗以胃癌為研究對象，篩選能抑制人類胃癌細胞株 AGS 生長之特定牛樟芝子實體純化物，探討其作用機制，並與市售抗癌藥物結合，測試對 AGS 與人類乳腺細胞 H184B5F5/M10 之存活率影響。細胞存活率測試結果顯示，特定牛樟芝子實體純化物 K4-2 能有效抑制 AGS 生長，IC50為 8.3 μg/ml。以不同濃度之 K4-2 (1.25、2.5、5、10、20 μg/ml) 處理 AGS，可觀察到細胞形態改變、細胞週期 sub-G1 phase 增加、細胞膜雙層磷脂質內側之磷脂質絲氨酸外翻、DNA 片段化產生、Pan-caspase 活性增加以及粒線體膜電位去極化，證實 K4-2 會誘導 AGS 細胞凋亡。經 RT-PCR 及 western blotting 結果證實 K4-2 會促使 TRAIL 與 DR4、DR5 結合，進而促使下游 caspase -3,-7,-8,-9及促進凋亡因子 Bax 與 Bad之表現量增加並使抑制凋亡因子 Bcl-2 及 Bcl-xL 之表現量降低。此外，經 K4-2 處理後 IGF1 、IGF1R 與 p-AKT 的表現量下降，証實 K4-2 會同時經由 TRAIL/DR4、DR5、mitochondria membrance potential 與IGF1R- mediated PI3K/AKT 訊息傳遞路徑調控以抑制 AGS 細胞生長並誘導凋亡。而與市售抗癌藥物結合測試的結果顯示，結合處理對 AGS之抑制生長效果比單獨處理抗癌藥物明顯。其中，對 AGS 於相同抑制效果下，結合處理之 cisplatin、 doxorubicin、 etoposide 和 5-FU 濃度較單獨使用時低且不會增加對 H184B5F5/M10 之毒殺性，顯示未來能進一步發展成胃癌治療之化療增敏劑。

Gastric cancer remains one of the top ten’s malignancies that leading causes of cancer death in Taiwan. Symptoms are clinically silent in early-stages, most of patients diagnosed with gastric cancer have unresectable locally advanced. Chemotherapy is a major therapeutic approach in treatment of advanced gastric cancer, but its clinical applications are limited because of severe side effects. Recently, increasing researches have been focused on the application of natural products in cancer therapy. Taiwanofungus camphorata is a traditional medicinal mushroom which grows only on the inner cavity of the endemic species Cinnamomum kanegirae of Taiwan. The fruiting bodies of T. camphorata is well known in Taiwan as a traditional medicines, and it has been recognized to exhibit antioxidant and anticancer effects. This study using human gastric carcinoma cell line, AGS measured the cell viability after treated with the specific compound purified from Taiwanofungus camphorata fruiting body which called K4-2 and explored the molecular mechanism. Measured the cell viability after treated with K4-2 combined with common anti-tumor agents which used to treat gastric cancer on AGS cell and human mammary epithelial cell, H184B5F5/M10 as control test. Cell viability assay showed that K4-2 treatment significantly inhibited growth of AGS cell and the effective does ( IC50 ) were 8.3 μg/ml. AGS cells treated with K4-2 ( 1.25、2.5、5、10、20 μg/ml ) were induced apoptosis, following by cellular morphology changed, phosphatidylserine exposure, sub-G1 phase increased, DNA molecules were fragment, pan-caspase activity increased and mitochondrial membrane potential depolarization. The results of RT-PCR and western blotting showed that K4-2 treatment significantly increased the level of caspase-3,-7,-8,-9 and pro-apoptotic factors including Bax and Bad, and decreased the level of anti-apoptotic factors Bcl-2 and Bcl-xL. Another, K4-2 combined with chemotherapeutic agents, cisplatin、 doxorubicin、 etoposide and 5-FU showed significantly adjuvant anti-proliferation effect more than cisplatin、 doxorubicin、 etoposide and 5-FU alone on AGS and no more cytotoxic effect to H184B5F5/M10. Therefore, K4-2 may be a promising candidate for further development as a chemosensitizer in gastric cancer therapy.

中文摘要 I
Abstract III
目錄 V
表目錄 XI
圖目錄 XII
第一章、前言 1
第二章、 文獻回顧 3
一、 牛樟芝簡介 3
1. 生長特性 3
2. 分類法 3
3. 活性成分 5
4. 生理活性 9
4.1 保護肝臟 9
4.2 抗發炎 9
4.3 抗氧化 10
4.4 抗腫瘤 10
二、 胃癌介紹 12
1. 胃癌之死亡率排行 12
2. 胃癌種類 13
3. 胃癌分期 14
4. 危險因子 15
4.1 幽門螺旋桿菌 15
4.2 飲食習慣 16
4.3 生活習慣 16
4.4 遺傳 17
4.5 惡性貧血 17
4.6 胃切除手術 17
5. 治療方式 18
三、細胞凋亡 18
1. 細胞死亡型式與形態學特徵 18
2. 細胞凋亡之生化特性 20
3. 細胞凋亡之訊息傳遞路徑 20
3.1 Bcl-2 家族蛋白 (Bcl-2 family proetin) 23
3.2 Caspase 家族蛋白酶 (Caspase family proteases) 24
第三章、 研究目的 25
第四章、 實驗架構 27
第五章、 實驗材料與儀器 29
一、 實驗材料 29
1. 細胞株 29
2. 實驗樣品 29
3. 實驗藥品 29
4. 實驗耗材 32
二、 實驗儀器 33
第六章、 實驗方法 35
一、 牛樟芝子實體之樣品配製 35
1. 牛樟芝子實體萃取物 35
2. 管柱層析法 35
3. 半製備 HPLC 分離子實體萃取物 36
二、 人類胃腺癌細胞株 AGS 培養 36
三、 細胞存活率測試 ( MTT assay )36
四、 細胞型態觀察 37
五、 細胞凋亡測試 37
1. 細胞週期測試 37
2. 細胞 Annexin V-FITC / PI 雙染測試 38
3. DNA 片段化 39
4. 粒線體跨膜電位分析 40
5. Pan-caspase 活性分析 40
六、 細胞凋亡路徑分析 41
1. 反轉錄酵素聚合反應 41
1.1. RNA 萃取 41
1.2. 反轉錄酵素反應 41
1.3. 聚合酵素連鎖反應 42
2. 西方墨點法 42
2.1 蛋白質萃取 42
2.2 蛋白質定量 43
2.3 蛋白質電泳及轉漬 43
2.4 蛋白質免疫呈色 44
七、 統計分析 44
第七章、 實驗結果 45
一、 牛樟芝子實體之萃取與純化 45
1. 牛樟芝子實體萃取 45
2. 管柱層析 45
3. 半製備 HPLC 純化分離 K4-2 45
二、 細胞存活率測試 46
1. 乙醇溶劑對 AGS 細胞株存活率之影響 46
2. 牛樟芝子實體粗萃物 ( TCCE ) 對 AGS 細胞株存活率之影響 46
3. 牛樟芝子實體管柱分離萃取物 ( FK1~FK6 ) 對 AGS 細胞株存活率之影響 46
4. 半製備 HPLC 純化分離 K4-2 對 AGS 細胞株存活率之影響 47
5. 市售抗癌藥物 cisplatin、5-FU、etoposide、mitomycin c 與 doxorubicin 對 AGS 細胞株存活率之影響 47
6. 市售抗癌藥物 cisplatin、5-FU、etoposide、mitomycin C 及 doxorubicin 分別與 K4-2 結合AGS 細胞株存活率之影響 48
7. 乙醇溶劑對 H184B5F5/M10 細胞株存活率之影響 49
8. 半製備 HPLC 純化分離 K4-2 對 H184B5F5/M10細胞株存活率之影響 49
9. K4-2 與市售抗癌藥物 cisplatin、5-FU、etoposide、mitomycin c、doxorubicin對人類乳腺細胞 M10 細胞株存活率之影響 49
10. 市售抗癌藥物 cisplatin、5-FU、etoposide、mitomycin c 及 doxorubicin 分別與 K4-2 結合對人類乳腺細胞 M10 細胞株存活率之影響 50
三、 K4-2 對AGS 細胞型態之影響 51
四、 細胞凋亡測試 52
1. K4-2 對 AGS 細胞週期之影響 52
2. K4-2 誘導 AGS 細胞凋亡之分期比例 52
3. K4-2 對 AGS 細胞 DNA 片段化之影響 53
4. K4-2對 AGS 細胞粒線體膜電位之影響 53
5. K4-2對 AGS 細胞 Pan-caspase 蛋白活性之影響 54
五、 細胞凋亡路徑分析 54
1. K4-2 誘導 AGS 細胞株凋亡之 mRNA 表現 54
2. K4-2 誘導 AGS 細胞株凋亡之蛋白表現 55
第八章、 討論 111
第九章、 結論 121
第十章、 未來研究方向 123
第十一章、 參考文獻 125

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