臺灣博碩士論文加值系統

順鉑(cisplatin)為目前最被廣泛使用的化療藥物之一，可用於治療不同種類的癌症。儘管順鉑具有顯著的抗腫瘤活性，但其在臨床上受到限制主要是因為產生副作用如腎毒性及肝毒性，然而，鮮少有文獻探討順鉑誘導肝毒性。許多研究指出牛樟芝具有多重生理活性如：抗癌、抗發炎、抗氧化、抗高血壓、免疫調節及保護肝臟等。然而，牛樟芝對於順鉑誘導肝毒性之保護效果仍不清楚。我們的研究發現牛樟芝減緩順鉑對於HepG2的毒殺作用可能是藉由抗發炎以及抗氧化所導致。我們進一步證明牛樟芝可以減緩化療小鼠體重流失及減少血清中GOT和GPT的含量可能是藉由減少發炎細胞激素(IL-6、TNF-α及iNOS)及抑制肝細胞凋亡。此外，腫瘤過度表現抗細胞凋亡蛋白(Bcl-2)與化療抗性及預後不良有關，餵食牛樟芝後可以向下調節NF-κB增加促細胞凋亡蛋白(Bax)的表現量並減少抗細胞凋亡蛋白(Bcl-2)的表現量。研究結果顯示牛樟芝可以藉由調節Bcl-2及Bax基因表現來抑制腫瘤生長。因此，鑑於我們的研究結果，牛樟芝合併抗癌藥物可能有助於提高癌症化療的結果。

Cisplatin is one of the major anticancer drugs used against different human cancer. In spite of its significant anticancer activity, the clinical used of cisplatin is often limited by its side effects such as nephrontoxicity and hepatotoxicity. However cisplatin-induced hepatotoxicity has been rarely paid attention to. Many reports show that antrodia camphorata (AC) possesses a number of bioactive properties such as anti-cancer, anti-inflammatory, anti-oxidant, anti-hypertensive, immunomodulatory and hepatoprotection. However, the effects of antrodia camphorata (AC) protects liver from cisplatin have not been studied. We find that AC reduces cisplatin-induced cytotoxicity in HepG2 through inhibition of inflammatory and oxidative stress. We further demonstrate that AC attenuates body weight loss and reduces serum GOT and GPT in cisplatin-treated mice through decreasing inflammatory cytokines (IL-6, TNF-α and iNOS) and apoptotic reaction. In addition, overexpression of the anti-apoptotic protein Bcl-2 has been associated with chemo-resistance and poor prognosis. The down-regulation of NF-kB was followed by the increasing of pro-apoptotic Bax and decreasing of anti-apoptotic Bcl-2, after AC treatment. Analysis of the data suggests that AC growth inhibition on tumor cells through targeting Bcl-2 and Bax genes. Consequently, given our present findings, we propose that the combination of anticancer drugs with AC contributes to the enhancement of clinical outcomes in cancer chemotherapy.

目錄..........I
表目錄..........V
圖目錄..........VI
中文摘要..........VIII
Abstract..........IX
壹、前言..........1
貳、文獻回顧..........3
一、癌症的特徵..........4
二、肺癌..........5
1. 肺癌病理學..........5
2. 肺癌流行病學..........5
三、 化學療法..........6
1. 癌症與化學療法..........6
2. 順鉑(cisplatin)..........6
3. 順鉑(cisplatin)與肝毒性..........6
4. 氧化性壓力(oxidative stress)..........7
5. 發炎反應 (inflammatory)..........7
6. 細胞凋亡(apoptosis)..........8
四、牛樟芝(Antrodia camphorata)..........9
1. 牛樟芝簡介..........9
2. 牛樟芝的歷史..........9
3. 牛樟芝之生理活性..........9
3.1 牛樟芝之抗氧化功效..........9
3.2 牛樟芝具抗發炎之功效..........9
3.3 牛樟芝具免疫調節之功效..........9
3.4 牛樟芝之抗病毒功效..........10
3.5 牛樟芝之抗癌功效..........10
3.6 牛樟芝之保肝功效..........10
參、實驗材料與方法..........11
一、實驗材料..........12
1. 試驗細胞株..........12
2. 細胞培養相關試劑..........12
3. 實驗動物..........12
4. 牛樟芝來源..........12
5. 抗氧化能力試驗..........12
6. 細胞存活率試劑..........13
7. 一氧化氮(NO)濃度分析試劑..........13
8. SDS-PAGE..........13
9. 細胞裂解液..........14
10. 西方墨點法相關試劑..........14
11. 組織切片..........14
12. 抗體..........14
13. RNA萃取..........14
14. 反轉錄反應試劑..........15
15. 即時定量聚合酶連鎖反應試劑..........15
二、 儀器設備..........16
三、 實驗方法..........17
1. 細胞株之保存活化與培養..........17
1.1 細胞株之保存..........17
1.2 細胞株之活化..........17
1.3 細胞培養(subculture)..........17
2. 牛樟芝抗氧化能力分析..........17
2.1 清除α,α-diphenyl-β-picryhydrazyl (DPPH)自由基能力測定..........17
2.2 清除超氧陰離子能力測定..........18
2.3 螯合亞鐵離子能力測定..........18
3. 牛樟芝(Antrodia camphorata)及順鉑(cisplatin)對細胞之存活率測試..........19
4. 一氧化氮(NO)濃度分析..........19
5. 動物實驗設計..........19
5.1 實驗小鼠模式之建立..........19
5.1.1 化療組..........19
5.1.2 腫瘤組..........19
5.2 小鼠血液生化指數..........20
6. 病理組織切片..........20
6.1 石蠟切片..........20
6.2 H&;E stain..........20
7.以西方墨點法分析小鼠肝臟發炎及細胞凋亡相關蛋白質之表現量..........20
7.1 萃取組織內蛋白質..........20
7.2 蛋白質定量..........21
7.3 SDS-PAGE與轉漬..........21
7.4 西方墨點法..........21
8. 以Real-time PCR分析小鼠肝臟發炎及細胞凋亡相關之基因表現量..........21
8.1 萃取組織之total RNA..........21
8.2 Total RNA之定性與定量..........22
8.3 反轉錄反應..........22
8.4 以Real-time PCR偵測目標基因之表現量..........22
9. 統計分析..........21
肆、結果..........23
一、牛樟芝減緩順鉑誘導肝細胞之毒殺作用..........25
1. 牛樟芝及順鉑對於正常細胞及癌細胞之存活率..........25
2. 牛樟芝對於正常細胞及癌細胞因順鉑誘導毒殺作用之影響..........25
3. 牛樟芝抑制LPS誘導發炎反應..........25
4. 牛樟芝抗氧化能力分析..........25
二、牛樟芝藉由減少促發炎激素及抑制細胞凋亡減緩化療小鼠因順鉑誘導肝毒性..........26
1. 牛樟芝對於順鉑注射小鼠血液生化指數之影響..........26
2. 牛樟芝對於順鉑注射小鼠體重及臟器重量之影響..........26
3. 牛樟芝對於順鉑誘導小鼠肝臟損傷之影響..........26
4. 牛樟芝對於順鉑注射小鼠肝臟發炎相關基因及蛋白質表現之影響..........26
5. 牛樟芝對於順鉑注射小鼠肝臟細胞凋亡相關基因及蛋白質表現之影響..........27
三、牛樟芝減緩腫瘤小鼠因順鉑誘導肝臟損傷並能增加腫瘤對於化療的敏感性..........28
1. 牛樟芝對於順鉑注射腫瘤小鼠血液生化指數之影響..........28
2. 牛樟芝對於順鉑注射腫瘤小鼠腫瘤生長、體重及臟器重量之影響..........28
3. 牛樟芝對於順鉑誘導腫瘤小鼠肝臟損傷之影響..........28
4. 牛樟芝對於順鉑注射腫瘤小鼠肝臟發炎相關基因及蛋白質表現之影響..........29
5. 牛樟芝對於順鉑注射腫瘤小鼠肝臟細胞凋亡相關基因及蛋白質表現之影響..........29
6. 牛樟芝合併順鉑對於抑制腫瘤生長之機制..........29
伍、討論..........31
陸、結論..........35
柒、參考文獻..........37
捌、圖表..........44

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