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研究生: 陳信益
Chen, Hsin-Yi
論文名稱: 探討具有保護正常細胞免於順鉑毒殺作用的中草藥
Screening of the herb extracts with cisplatin-protection effects on normal cells
指導教授: 賴韻如
Lai, Yun-Ju
學位類別: 碩士
Master
系所名稱: 生命科學系
Department of Life Science
論文出版年: 2019
畢業學年度: 107
語文別: 中文
論文頁數: 148
中文關鍵詞: 肝癌卵巢癌順鉑化學治療藥物神經毒性中草藥
英文關鍵詞: adjuvant therapy, supportive therapy, herbal medicine chemotherapy
DOI URL: http://doi.org/10.6345/NTNU201900049
論文種類: 學術論文
相關次數: 點閱:201下載:0
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  • 順鉑為最常用於治療癌症的化學治療藥物,但具有腎臟毒性和神經毒性。因此,找到能夠與順鉑協同抑制癌細胞生長,又能降低順鉑對正常細胞毒性(特別是神經細胞)的藥物是很重要的。中草藥在中國已被廣泛使用數千年,其藥效相較於西藥來得溫和,現今也有研究指稱它具有保護正常細胞免於化療藥物的毒殺效果。因此,我們以肝癌細胞株Hep3B和卵巢癌細胞株SKOV3建立癌症模型,並使用神經瘤細胞株SH-SY5Y建立神經細胞平台,以篩選可保護神經細胞免於順鉑毒殺作用的中草藥萃取物。初步篩選後,茯苓及天麻兩種中草藥萃取物能夠抑制Hep3B和SKOV3的生長,並且對於經維生素A酸刺激分化過的神經母細胞瘤SH-SY5Y細胞沒有毒性。根據以上實驗結果,茯苓及天麻可望成為與順鉑一起作用的複方,並期許其未來可應用於臨床治療。

    Cisplatin is one of the most often used chemotherapeutics in clinical. However, it often induces renal and neural toxicity. Therefore, it is important to establish an adjuvant therapy that reduces the cytotoxicity of cisplatin to normal cells, especially neuron, but does not affect its inhibitory effects on cancer cells. Herbal medicine has been widely used for thousand years in Asia and presents relatively moderate side-effects compared to western medicine. Recent studies show that it has potential to protect cells against cytotoxicity caused by drugs. Here we used hepatocellular carcinoma (HCC) Hep3B cell line and ovarian neoplasms SKOV3 cell line as cancer model, and neurocytoma SH-SY5Y as a neuron cell platform to screen the Chinese herbal extracts which act synergistically with cisplatin to inhibit survival of cancer cells, but weaken the cytotoxicity of cisplatin to neuron. Our results found that two Chinese herbal extracts, Wolfiporia extensa and Gastrodia elata, that can inhibit the growth of Hep3B and SKOV3 cells but do not have cytotoxicity to retinoic acid-differentiated SH-SY5Y neuroblastoma cells. From these results, Wolfiporia extensa and Gastrodia elata extracts may serve as an adjuvant treatment for cisplatin clinically in the future.

    縮寫表 I ABSTRACT IV 摘要 V 1. 緒論 1 1.1. 癌症特徵 1 1.2. 肝癌 (LIVER CANCER) 2 1.3. 卵巢與卵巢癌 10 1.4. 神經細胞實驗模式 13 1.5. 癌症與化學治療法 14 1.6. 順鉑 14 1.7. 中草藥 ( CHINESE HERBAL MEDICINE ) 18 1.7.1. 茯苓 18 1.7.2. 天麻 21 1.8. MTT ASSAY作用原理 24 1.9. 細胞凋亡 25 1.10. ERK訊息傳遞途徑 30 1.11. PI3K/AKT 訊息傳遞途徑 31 1.12. 實驗動機 34 1.13. 實驗目的與假說 34 2. 研究材料與方法 36 2.1. CISPLATIN藥物原液 36 2.2. 中草藥萃取物 36 2.1.1. 水萃取中草藥 36 2.1.2. 酒精萃取中草藥 36 2.2. 細胞培養與繼代 36 2.2.1. 人類肝癌細胞株Hep3B 36 2.2.2. 卵巢癌細胞株SKOV3 37 2.2.3. 神經瘤細胞株SH-SY5Y 37 2.3. 神經細胞分化 37 2.4. 細胞計數 38 2.5. 免疫螢光細胞染色 38 2.6. 藥物處理 39 2.6.1. 人類肝癌細胞株Hep3B 39 2.6.2. 卵巢癌細胞株SKOV3 39 2.6.3. 未分化神經瘤細胞株SH-SY5Y 39 2.6.4. 分化後神經瘤細胞株SH-SY5Y 40 2.7. MTT ASSAY 40 2.8. 半抑制濃度定義與計算方式 40 2.9. 西方墨點法 41 2.9.1. 蛋白質萃取 41 2.9.2. 蛋白質濃度測定與樣品製備 41 2.9.3. 聚丙烯醯胺膠體之製備 42 2.9.4. 蛋白質電泳 43 2.9.5. 轉漬及Blocking 43 2.10. 統計分析 44 2.11. 實驗設計 44 3. 結果 49 3.1. 探討順鉑對於癌細胞株的影響 49 3.1.1. 順鉑溶於DMSO對於肝癌細胞株Hep3B的殺傷效果 49 3.1.2. 順鉑溶於PBS對於肝癌細胞株Hep3B的殺傷效果 49 3.1.3. 順鉑溶於PBS對於卵巢癌細胞株SKOV3的殺傷效果 50 3.2. 探討順鉑對於神經細胞株的影響 50 3.2.1. 順鉑溶於PBS對於未分化神經瘤細胞株SH-SY5Y的殺傷效果 50 3.2.2. 順鉑溶於PBS對於已分化神經瘤細胞株SH-SY5Y的殺傷效果 51 3.3. 探討中草藥水萃液對於癌細胞株的影響 51 3.3.1. 中草藥水萃液對於肝癌細胞株Hep3B的殺傷效果 51 3.3.2. 茯苓/天麻水萃液對於卵巢癌細胞株SKOV3的殺傷效果 53 3.4. 探討中草藥水萃液對於神經細胞株的影響 54 3.4.1. 茯苓水萃液對於未分化神經瘤細胞株SH-SY5Y的影響 54 3.4.2. 茯苓水萃液對於已分化神經瘤細胞株SH-SY5Y的影響 54 3.4.3. 天麻水萃液對於未分化神經瘤細胞株SH-SY5Y的影響 55 3.4.4. 天麻水萃液對於已分化神經瘤細胞株SH-SY5Y的影響 55 3.5. 探討順鉑及中草藥水萃液共同給藥對於肝癌細胞株的影響 56 3.5.1. 順鉑及茯苓共同給藥對於Hep3B的影響 56 3.5.1.1. 順鉑及茯苓共同給藥對於Hep3B生長的影響 56 3.5.1.2. 順鉑及茯苓共同給藥對於Hep3B蛋白質表現的影響 57 3.5.2. 順鉑及天麻共同給藥對於Hep3B的影響 58 3.5.2.1. 順鉑及天麻共同給藥對於Hep3B生長的影響 58 3.5.2.2. 順鉑及天麻共同給藥對於Hep3B蛋白質表現的影響 60 3.6. 探討順鉑及中草藥水萃液共同給藥對於卵巢癌細胞株的影響 60 3.6.1. 順鉑及茯苓共同給藥對於SKOV3的影響 60 3.6.1.1. 順鉑及茯苓共同給藥對於SKOV3生長的影響 60 3.6.1.2. 順鉑及茯苓共同給藥對於SKOV3蛋白質表現的影響 62 3.6.2. 順鉑及天麻共同給藥對於SKOV3的影響 63 3.6.2.1. 順鉑及天麻共同給藥對於SKOV3生長的影響 63 3.6.2.2. 順鉑及天麻共同給藥對於SKOV3蛋白質表現的影響 64 3.7. 探討順鉑及中草藥水萃液共同給藥對於已分化神經瘤細胞株的影響 65 3.7.1. 順鉑及茯苓共同給藥對於已分化SH-SY5Y的影響 65 3.7.1.1. 順鉑及茯苓共同給藥對於已分化SH-SY5Y生長的影響 65 3.7.1.2. 順鉑及茯苓共同給藥對於已分化SH-SY5Y蛋白質表現的影響。 67 3.7.2. 順鉑及天麻共同給藥對於已分化SH-SY5Y的影響 69 3.7.2.1. 順鉑及天麻共同給藥對於已分化SH-SY5Y生長的影響 69 3.7.2.2. 順鉑及天麻共同給藥對於已分化SH-SY5Y蛋白質表現的影響。 71 4. 討論 73 5. 參考文獻 85 6. 圖表 104 6.1. 表格 104 表 3. 順鉑溶於DMSO對於肝癌細胞株Hep3B的殺傷效果。 104 表 4. 順鉑溶於PBS對於肝癌細胞株Hep3B的殺傷效果。 104 表 5. 中草藥水萃液對於肝癌細胞株Hep3B的殺傷效果。 105 表 6. 各中草藥對於Hep3B細胞株之半抑制濃度。 106 表 7. 順鉑、茯苓、天麻對於卵巢癌細胞株SKOV3的影響。 106 表 8. 順鉑、茯苓、天麻對於未分化神經瘤細胞株SH-SY5Y的影響。 107 表 9. 順鉑、茯苓、天麻對於已分化神經瘤細胞株SH-SY5Y的影響。 107 表 10. 順鉑及茯苓共同處理對於Hep3B細胞株的影響。 108 表 11. 順鉑及天麻共同處理對於Hep3B細胞株的影響。 108 表 12. 順鉑及茯苓共同處理對於SKOV3細胞株的影響。 109 表 13. 順鉑及天麻共同處理對於SKOV3細胞株的影響。 109 表 14. 順鉑及茯苓共同處理對於已分化神經瘤細胞株SH-SY5Y的影響。 110 表 15. 順鉑及天麻共同處理對於已分化神經瘤細胞株SH-SY5Y的影響。 110 6.2. 圖片 111 圖 1. 順鉑溶於DMSO對於肝癌細胞株Hep3B的殺傷效果。 112 圖 2. 順鉑溶於PBS對於肝癌細胞株Hep3B的殺傷效果。 113 圖 3. 順鉑溶於PBS對於卵巢癌細胞株SKOV3的殺傷效果。 114 圖 4. 順鉑溶於PBS對於未分化神經瘤細胞株SH-SY5Y的殺傷效果。 115 圖 5. 順鉑溶於PBS對於已分化神經瘤細胞株SH-SY5Y的殺傷效果。 116 圖 6. 各中草藥水萃液對於肝癌細胞株Hep3B的殺傷效果。 118 圖 7. 各中草藥水萃液對於卵巢癌細胞株SKOV3的殺傷效果。 119 圖 8. 茯苓水萃液對於未分化神經瘤細胞株SH-SY5Y的影響。 120 圖 9. 茯苓水萃液對於已分化神經瘤細胞株SH-SY5Y的影響。 121 圖 10. 天麻水萃液對於未分化神經瘤細胞株SH-SY5Y的影響。 122 圖 11. 天麻水萃液對於已分化神經瘤細胞株SH-SY5Y的影響。 123 圖 12. 順鉑及茯苓共同處理對於Hep3B細胞株生長的影響。 125 圖 13. 順鉑及茯苓共同給藥對於Hep3B蛋白質表現的影響。 127 圖 14. 順鉑及天麻共同處理對於Hep3B細胞株生長的影響。 129 圖 15. 順鉑及天麻共同給藥對於Hep3B蛋白質表現的影響。 131 圖 16. 順鉑及茯苓共同給藥對於SKOV3生長的影響。 133 圖 17. 順鉑及茯苓共同給藥對於SKOV3蛋白質表現的影響。 135 圖 18. 順鉑及天麻共同給藥對於SKOV3生長的影響。 137 圖 19. 順鉑及天麻共同給藥對於SKOV3蛋白質表現的影響。 139 圖 20. 順鉑及茯苓共同給藥對於已分化神經瘤細胞株SH-SY5Y生長的影響。 141 圖 21. 順鉑及茯苓共同給藥對於已分化神經瘤細胞株SH-SY5Y蛋白質表現的影響。 143 圖 22. 順鉑及天麻共同給藥對於已分化神經瘤細胞株SH-SY5Y生長的影響。 145 圖 23. 順鉑及天麻共同給藥對於已分化神經瘤細胞株SH-SY5Y蛋白質表現的影響。 147 圖 24. Retinoic acid誘導SH-SY5Y細胞株分化之免疫螢光染色。 148

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