臺灣博碩士論文加值系統

牛樟芝為台灣特有的藥用菇蕈類。研究指出，樟芝菌絲體醣蛋白(Antrodan)具有抗發炎及抗氧化作用，此外，Antrodan已被證明在小鼠肺癌細胞(Lewis Lung carcinoma, LLC)中，可經由直接與免疫調節的作用抑制癌細胞轉移；然而，Antrodan在活體內是否具有抗癌轉移效果，目前仍不清楚。因此本研究利用腫瘤異體移植小鼠模式探討Antrodan與抗癌藥物順鉑(cisplatin)合併使用是否可減少cisplatin所造成的腎毒性。將LLC細胞以皮下注射方式接種入C57BL/6小鼠的背部，於癌細胞注射九天後，小鼠以管餵方式每日給予Antrodan (20及40 mg/kg)並以每周兩次的頻率以腹腔注射給予cisplatin (1 mg/kg)，持續28天。結果顯示，Antrodan可顯著地(1) 抑制肺臟與肝臟腫瘤轉移數量及原位腫瘤生長；(2) 降低血漿中尿激酶型血纖維蛋白溶解酶原活化因子(urokinase-type plasminogen activator, uPA)與基質金屬蛋白酶(matrix metalloproteinases, MMPs)-2與-9的酵素活性；(3) 降低肺臟及肝臟中信號轉導及轉錄激活蛋白3 (signal transducer and activator of transcription 3, STAT3)、降低有絲分裂原活化蛋白激酶(mitogen-activated protein kinase, MAPK)包括细胞外調節蛋白激酶 (extracellular regulated protein kinases, ERK)、c-Jun氨基末端激酶(c-Jun N-terminal kinase, JNK)及p38之磷酸化作用與MMP-2及MMP-9的蛋白質表現；(4) 減少血獎中介白素-6含量及增加血漿中IFN-γ含量。而cisplatin也具有類似抑制效果，除了無法抑制肺臟腫瘤轉移數目外。此外，於Antrodan改善cisplatin造成的腎毒性部分，Antrodan可有效減少小鼠血漿中尿素氮含量及降低腎臟中p38磷酸化作用。綜合以上結果，本研究證明Antrodan在活體內具有抑制腫瘤轉移的能力並改善cisplatin所造成的腎毒性。

Antrodia cinnamomea is a species known to be a treasured medicinal mushroom in Taiwan. Several studies have indicated that Antrodan, the glycoprotein from Antrodia cinnamomea mycelia, exhibits anti-inflammation and anti-oxidative actions. In addition, Antrodan has been shown to inhibit cancer metastasis in Lewis lung carcinoma (LLC) through direct action and immunomodulation. However, it is still unclear whether Antrodan has anti-metastatic effects in vivo. This study aimed to investigate the anti-metastatic effects of Antrodan and Antrodan in combination with cisplatin and to explore the protective effects of Antrodan against cisplatin-induced nephrotoxicity using tumor xenografted mice. LLC were injected (s.c.) into to C57BL/6 mice for 9 days, and mice were administered with Antrodan (20 and 40 mg/kg; p.o.) daily, cisplatin (1 mg/kg; i.p.) twice per week or their combined treatment for an additional 28 days. Results reveal that Antrodan treatment significantly (1) inhibited the number of tumor metastasis in lung and liver tissues and primary tumor growth; (2) decreased activities of urokinase-type plasminogen activator, matrix metalloproteinase (MMP)-2 and -9 in plasma; (3) reduced MMP-2/9 protein expression of and phosphorylation of signal transducer and activator of transcription 3 (STAT3) and mitogen-activated protein kinase (MAPK), including extracellular regulated protein kinases (ERK), c-Jun N-terminal kinase (JNK) and p38 in lung and liver tissues; (4) decreased plasma interleukin-6 level and increased interferon-γ level. Cisplatin exhibited similar inhibitory effects, except for the number of tumor metastasis in lung tissues. The combined treatment exhibited additive action on inhibition of primary tumor growth, plasma MMP-9 activity, and protein expression of MMP-2 and MMP-9 in lung and liver tissues. In addition, Antrodan effectively improved cisplatin-induced nephtotoxicity, as evidenced by decreased cisplatin-induced blood urea nitrogen levels in plasma and p38 phosphorylation in kidney. Overall, the present results demonstrate that Antrodan has abilities to inhibit cancer metastasis and to improve nephrotoxicity induced by cisplatin in vivo.

摘要............................................................................................................................... I
Abstract........................................................................................................................II
總目錄.........................................................................................................................III
結果圖表目錄............................................................................................................VII
縮寫表...........................................................................................................................X

第一章文獻回顧........................................................................................................... 1
1. 文獻回顧.................................................................................................................. 2
1.1 肺癌 2
1.2 癌轉移 2
1.3 癌轉移生化指標. 2
1.3.1 基質金屬蛋白酶 2
1.3.2 尿素激酶型胞漿素原活化子 (Urokinase plasminogen activator;uPA)…………………………………………………………………...……..3
1.3.3 轉錄訊息傳遞活化子(Signal transducer and activator of transcription, STAT)……………………………………………………………………………….....3
1.3.3.1 轉錄訊息傳遞活化子3 (STAT3) 4
1.3.3.1.1 STAT3與癌細胞增生和凋亡 4
1.3.3.1.2 STAT3與血管新生 4
1.3.3.1.3 STAT3與免疫逃脫(Immune Evasion) 5
1.3.3.1.4 STAT3與癌轉移 5
1.4 有絲分裂活化之蛋白質激酶(Mitogen-activated protein kinases, MAPKs) 5
1.5 樟芝(Antrodia cinnamomea) 6
1.5.1 樟芝生物功能 6
1.5.2 樟芝有效成分部位 6
1.5.2.1 子實體 6
1.5.2.1.1 抑制癌細胞增生 6
1.5.2.1.2 調節免疫反應 6
1.5.2.1.3 抗轉移 7
1.5.2.1.4 抗氧化 7
1.5.2.2 菌絲體 7
1.5.2.2.1 抗癌細胞增生 7
1.5.2.2.2 抗血管新生 7
1.5.2.3 發酵液 8
1.5.2.3.1 抗發炎 8
1.5.2.4 醣蛋白................................................................... 8
1.5.2.4.1 抗轉移 8
1.5.2.4.2 抗發炎 8
1.6 順鉑(Cisplatin) 8
1.6.1 順鉑抗癌機制 9
1.6.2 順鉑副作用 9
1.6.2.1 腎毒性機制 9
2. 目的與假說 11
3. 實驗架構 12
4. 參考文獻 13

第二章、樟芝菌絲體醣蛋白Antrodan合併順鉑在小鼠異體移植肺癌細胞株(LLC1)之模式中抑制腫瘤轉移並改善順鉑的腎毒性 17
1. 緒論 18
2. 材料與方法 19
2.1 樟芝菌絲體醣蛋白(Antrodan)之萃取 19
2.2 實驗試劑與Antrodan配置…………………………………………………….19
2.3 細胞培養 20
2.4 動物分組…………………………………………………...……………....……20
2.5 Antrodan及順鉑配製與給予小鼠方次及頻率 21
2.6腫瘤面積測量和肺臟與肝臟腫瘤轉移數目計數 21
2.7 酵素圖譜同功分析 23
2.8 西方墨點法 23
2.8.1蛋白質萃取 23
2.8.2蛋白質濃度測定與樣品製備 23
2.8.3聚丙烯醯胺膠體之製備 24
2.8.4電泳 25
2.8.5轉漬及Blocking 25
2.9細胞激素測定 26
2.10免疫組織化學染色 26
2.11 血漿中尿素氮含量測定 27
2.12 統計分析 27
3. 結果 ...…………………………………………………………………………….28
3.1 單獨Antrodan或合併cisplatin對於小鼠體重、肝臟及腎臟相對重量的影響………………………………………………………………………….…....…….28
3.2 單獨Antrodan或合併cisplatin對於小鼠原位腫瘤面積及重量的影響 28
3.3 單獨Antrodan或合併cisplatin對於小鼠肺臟與肝臟腫瘤轉移數目的影響.. 28
3.4 單獨Antrodan或合併cisplatin對於小鼠血漿中MMP-2、MMP-9和uPA酵素活性之影響 29
3.5 單獨Antrodan或合併cisplatin對於小鼠肺臟及肝臟組織中MMP-2與MMP-9蛋白質表現之影響 30
3.6 單獨Antrodan或合併cisplatin對於小鼠肺臟及肝臟組織中STAT3磷酸化作用之影響 .... 30
3.7 單獨Antrodan或合併cisplatin對於小鼠肝臟及肺臟組織中MAPK磷酸化作用之影響 ……………………………………………………………………….......31
3.7.1 ERK1/2 31
3.7.2 JNK1/2 32
3.7.3 p38 32
3.8 單獨Antrodan或合併cisplatin對於小鼠血漿細胞激素的影響…..………….…...............…………………………………………………………33
3.8.1 IL-6 ...........................33
3.8.2 IFN-γ 33
3.9 單獨Antrodan或合併cisplatin對於小鼠肝臟及肺臟組織的影響 33
3.10 單獨Antrodan或合併cisplatin對於小鼠血漿中尿素氮的影響 34
3.11 單獨Antrodan或合併cisplatin對於小鼠腎臟組織之影響 34
3.12 單獨Antrodan或合併cisplatin對於小鼠腎臟組織中p38磷酸化作用之影響………………………………………………………..........………………………34
4. 討論.... 36
4.1 結論 39
5. 參考文獻 40
 
結果圖表目錄
Table 1. Effects of Antrodan alone, cisplatin alone or combined treatment on body weight, relative weight, tumor weight, and relative organ weight in LLC-bearing C57BL/6 mice. 44
Table 3. Effects of the Antrodan alone, cisplatin alone or combined treatment combination on plasma IL-6 and IFN-γ of LLC-bearing C57BL/6 mice. 45

Figure 1. Figure 1. Effects of Antrodan alone, cisplatin alone or combined treatment on body weight in LLC-bearing C57BL/6 mice. 46
Figure 2. Effects of Antrodan alone, cisplatin alone or combined treatment on tumor area in LLC-bearing C57BL/6 mice. 47
Figure 3. Effects of Antrodan alone, cisplatin alone or combined treatment on lung metastasis in LLC-bearing C57BL/6 mice. LLC were injected subcutaneously into the right flank of C57BL/6 mice. 48
Figure 4. Effects of Antrodan alone and cisplatin alone or combined treatment on liver metastasis in LLC1-bearing C57BL/6 mice. LLC were injected subcutaneously into the right flank of C57BL/6 mice. 49
Figure 5. Effects of Antrodan alone, cisplatin alone or combined treatment on MMP-2, MMP-9 and uPA activity in plasma of LLC-brearing mice. 50
Figure 6. Effects of Antrodan alone, cisplatin alone or combined treatment on MMP-2 protein expression in lung tissues of C57BL/6 mice. 51
Figure 7. Effects of Antrodan alone, cisplatin alone or combined treatment on MMP-2 protein expression in liver tissues of C57BL/6 mice. 52
Figure 8. Effects of Antrodan alone, cisplatin alone or combined treatment on MMP-9 protein expression in lung tissues of C57BL/6 mice.. 53
 
Figure 9. Effects of Antrodan alone, cisplatin alone or combined treatment on MMP-9 protein expression in liver tissues of C57BL/6 mice.. 54
Figure 10. Effects of Antrodan alone, cisplatin alone or combined treatment on phosphorylation and protein expression of STAT3 in lung tissues of C57BL/6 mice.. 55
Figure 11. Effects of Antrodan alone, cisplatin alone or combined treatment on phosphorylation and protein expression of STAT3 in liver tissues of C57BL/6 mice. .56
Figure 12. Effects of Antrodan alone, cisplatin alone or combined treatment on phosphorylation and protein expression of ERK1/2 in lung tissues of C57BL/6 mice. 57
Figure 13. Effects of Antrodan alone, cisplatin alone or combined treatment on phosphorylation and protein expression of ERK1/2 in liver tissues of C57BL/6 mice. 58
Figure 14. Effects of Antrodan alone, cisplatin alone or combined treatment on phosphorylation and protein expression of JNK1/2 in lung tissues of C57BL/6 mice .59
Figure 15. Effects of Antrodan alone, cisplatin alone or combined treatment on phosphorylation and protein expression of JNK1/2 in liver tissues of C57BL/6 mice .60
Figure 16. Effects of Antrodan alone, cisplatin alone or combined treatment on phosphorylation and protein expression of p38 in lung tissues of C57BL/6 mice....... 61
Figure 17. Effects of Antrodan alone, cisplatin alone or combined treatment on phosphorylation and protein expression of p38 in liver tissues of C57BL/6 mice...... 62
Figure 18. Histopathological findings of lung of C57BL/6 mice after subcutaneous inoculated with Lewis lung carcinoma.. 63
Figure 19. Histopathological findings of lung of C57BL/6 mice after subcutaneous inoculated with Lewis lung carcinoma.. 64
Figure 20. Histopathological findings of liver of C57BL/6 mice after subcutaneous inoculated with Lewis lung carcinoma.. 65
Figure 21. Histopathological findings of liver of C57BL/6 mice after subcutaneous inoculated with Lewis lung carcinoma.. 66
Figure 22. Effects of Antrodan alone, cisplatin alone or combined treatment on BUN levels of C57BL/6 mice... 67
Figure 23.Histopathological findings of lung of C57BL/6 mice after subcutaneous inoculated with Lewis lung carcinoma... 68
Figure 24.Histopathological findings of lung of C57BL/6 mice after subcutaneous inoculated with Lewis lung carcinoma... 70
Figure 25. Effects of Antrodan alone, cisplatin alone or combined treatment on phosphorylation and protein expression of p38 in kidney tissues of C57BL/6 mice... 71
Figure 26. Proposed anti-metastatic mechanisms of Antrodan (20,40 mg/kg) in combination with cisplatin (1 mg/kg) in LLC1-bearing mice. ... 72

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