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研究生:法冠寧
研究生(外文):Kuan-Ning Fa
論文名稱:樟芝菌絲體醣蛋白可藉由直接作用及免疫調節作用抑制小鼠肺癌細胞轉移
論文名稱(外文):Glycoprotein from mycelia of Antrodia cinnamomea inhibits metastasis of Lewis lung carcinoma 1 cells through direct action and immunomodulation
指導教授:胡淼琳胡淼琳引用關係
指導教授(外文):Miao-Lin Hu
口試委員:林金源喬長誠
口試日期:2013-07-18
學位類別:碩士
校院名稱:國立中興大學
系所名稱:食品暨應用生物科技學系所
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2013
畢業學年度:101
語文別:中文
論文頁數:110
中文關鍵詞:樟芝菌絲體醣蛋白免疫調節小鼠肺癌細胞轉移
外文關鍵詞:Glycoprotein from mycelia of Antrodia cinnamomea (AC-GP)ImmunomodulationLewis Lung carcinoma 1 (LLC1)Metastasis
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癌細胞轉移為癌症病患致死的主要原因,且為一連串複雜的過程。因此,有效抑制癌細胞轉移為主要抗癌的策略之一。牛樟芝又名樟芝或牛樟菇,為台灣特有的藥用蕈類。研究指出,樟芝菌絲體醣蛋白(AC-GP)具有抗發炎及抗氧化作用,但是否具有抗癌活性仍不清楚。因此,本論文擬探討AC-GP對於癌轉移的影響。本論文研究目的分為兩部分,第一部分將AC-GP直接與具有高度轉移能力之小鼠肺癌細胞株(Lewis lung carcinoma 1, LLC1)培養,探討AC-GP是否具有直接抑制LLC1轉移的能力。結果顯示AC-GP在培養24小時可顯著抑制LLC1侵襲、黏附及移行的作用,其最佳抑制濃度及抑制率分別為60 μg/mL (17%, P < 0.05)、50 μg/mL (19%, P < 0.05)及50 μg/mL (15%, P < 0.05)。作用機制方面,AC-GP可顯著地:(1)抑制整合素β1所調節的黏著斑激酶(focal adhesion kinase, FAK)磷酸化作用及其調控的有絲分裂活化蛋白質激酶家族(mitogen-activated protein kinases family, MAPKs family)的磷酸化作用並進一步抑制Rho small GTPases如Rho及Rac的蛋白質表現;(2)促進基質金屬蛋白酶內生性抑制劑(tissue inhibitor of matrix metalloproteinases, TIMP)-1和-2蛋白質表現,並抑制基質金屬蛋白酶(matrix metalloproteinase, MMP)-2和-9酵素活性及蛋白質表現;(3)提升胞漿素原活化子抑制物(plasminogen activator inhibitor, PAI)-1蛋白質表現,抑制尿素激酶型胞漿素原活化子(urokinase plasminogen activator, uPA)酵素活性;(4)促進抗轉移蛋白(nm23-H1)蛋白質表現。
本論文第二部分則利用AC-GP與人類周邊血液分離出的人類單核細胞培養三天後所得到的條件培養液(conditioned medium, CM)與LLC1培養,探討AC-GP是否具有增加免疫調節能力而抑制癌細胞轉移。結果顯示AC-GP可促進單核細胞增加分泌Th1型細胞激素如介白素(interleukin, IL)-12及IL-1β,並抑制Th2型細胞激素如IL-6與趨化素(chemokine) IL-8的分泌。此外,AC-GP-CM在培養24小時可顯著抑制LLC侵襲、黏附及移行作用,其最佳抑制濃度皆為60 μg/mL,抑制率分別為51% (P < 0.05)、27% (P < 0.05)及43% (P < 0.05)。作用機制方面,AC-GP-CM可顯著地: (1)促進TIMP-1和-2蛋白質表現,並抑制MMP-2和-9酵素活性及蛋白質表現;(2)提升PAI-1蛋白質表現,並抑制uPA酵素活性;(3)增加nm23-H1蛋白質表現。
綜合上述實驗結果,我們證明AC-GP具有直接作用以及促進免疫調節而達到抑制癌細胞轉移的能力。而在相同濃度(50及60 μg/mL)下的AC-GP藉由免疫調節作用抑制LLC1轉移的能力優於直接作用。


Metastasis, a complex and a multi-step process, is the major cause of death in cancer patients. Therefore, inhibition of cancer metastasis is considered one of the therapeutic strategies. Antrodia cinnamomea (AC), also called “zhan-chi” or “niu-chang-ku”, is a unique medicinal fungus in Taiwan. Glycoprotein from mycelia of AC (AC-GP) was previously shown to possess anti-inflammation and antioxidant activity, but it is still unclear whether AC-GP has anti-cancer actions. Herein, we investigated the effect of AC-GP on cancer metastasis in this thesis. The aim of the first part of this thesis was to determine the direct anti-metastatic efficacy of AC-GP by incubation with the highly invasive Lewis lung carcinoma 1 (LLC1) cells. Results reveal that AC-GP significantly inhibited metastasis, as evidenced by inhibition of invasion, adhesion and migration in LLC1 cells at 24 h of incubation, and the most effective concentration was 60, 50 and 50 μg/mL, respectively, with an inhibiton of 17% (P < 0.05), 19% (P < 0.05) and 15% (P < 0.05), respectively. Mechanistically, AC-GP significantly: (1) decreased protein expression of integrin β1-mediated phosphorylation of FAK followed by reduced phosphorylation of mitogen-activated protein kinases (MAPKs) leading to inhibition of Rho small GTPases, such as Rho and Rac protein expression; (2) increased protein expression of tissue inhibitor of MMP (TIMP)-1 and -2, but decreased protein expression and activities of matrix metalloproteinase (MMP)-2 and -9; (3) increased protein expression of plasminogen activator inhibitor-1 (PAI-1), but decreased activities of urokinase plasminogen activator (uPA); and (4) increased protein expression of nm23-H1 in LLC1 cells.
In the second part of this thesis, we isolated mononuclear cells (MNCs) from human peripheral blood, and MNCs were then treated with AC-GP for 3 d to obtain AC-GP-conditioned medium (AC-GP-CM). Results reveal that AC-GP treatment significantly increased Th1 cytokines (IL-12 and IL-1β), but decreased Th2 cytokine (IL-6) and chemokine (IL-8) levels in MNC-CM. We also found that AC-GP-CM significantly inhibited invasion, adhesion and migration of LLC1 cells at 24 h of incubation, and the most effective concentration was 60 μg/mL, with an inhibition of 51% (for invasion, P < 0.05 ), 27% (for adhesion, P < 0.05) and 43% (for migration P < 0.05). In addition, we found that AC-GP-CM significantly: (1) increased protein expression of TIMP-1 and -2, but decreased protein expression and activities of MMP-2 and -9; (2) increased protein expression of PAI-1, but decreased activities of uPA; (3) increased protein expression of nm23-H1 in LLC1 cells.
In conclusion, we have demonstrated the inhibitory effect of AC-GP on cancer metastasis through both direct action and immunomodulation in LLC1 cells. It should be noted that the anti-metastatic efficacy of AC-GP via immunomodulation is stronger than that of direct action on LLC1 cells at the same concentrations (50 and 60 μg/mL).


目錄
中文摘要 i
Abstract iii
表目錄 x
圖目錄 xi
縮寫表 xiii

第一章 文獻回顧
1. 肺癌 2
2. 癌轉移 2
3. 癌轉移生化指標 3
3.1. 基質金屬蛋白酶(Matrix metalloproteinase, MMPs) 3
3.2. 基質金屬蛋白酶內生性抑制劑(Tissue inhibitor of matrix metalloproteinases,
TIMPs) 4
3.3. 尿素激酶型胞漿素原活化子系統(Urokinase plasminogen activator system) 5
3.3.1. 尿素激酶型胞漿素原活化子(Urokinase plasminogen activator, uPA) 5
3.3.2. 胞漿素原活化子抑制物(Plasminogen activator inhibitor, PAI)-1 5
3.4. 抗轉移蛋白(Non-metastatic protein 23 homologue 1, nm23-H1) 6
3.5. Rho small GTPases 6
3.6. 黏著斑激酶(Focal Adhesion Kinase, FAK) 6
3.7. 有絲分裂原活化蛋白激酶(Mitogen-activated protein kinases, MAPKs) 7
3.8. 整合素β1 (Integrin β1) 8
4. 細胞激素 8
5. 細胞激素與免疫反應 9
6. 多醣體吸收及作用途徑 10
7. 樟芝(Antrodia cinnamomea) 11
7.1. 成分分析 12
7.2. 樟芝生理功能 12
7.2.1. 樟芝子實體生理功能 12
7.2.1.1. 抗癌細胞增生 12
7.2.1.2. 抗發炎 12
7.2.1.3. 促癌細胞凋亡 13
7.2.1.4. 抗氧化 13
7.2.1.5. 抑制癌細胞侵襲及轉移 13
7.2.2. 樟芝菌絲體生理功能 14
7.2.2.1. 抗癌細胞增生 14
7.2.2.3. 抗發炎 14
7.2.2.4. 促癌細胞凋亡 15
7.2.2.5. 抗氧化 15
7.2.2.6. 抗轉移 15
8. 研究動機與目的 16
9. 試驗設計 17
9.1. AC-GP製備 17
9.2. 第二章試驗架構 18
9.3. 第三章試驗架構 19
10. 參考文獻 20

第二章 樟芝菌絲體醣蛋白可藉由直接作用抑制小鼠肺癌細胞轉移
Glycoprotein from mycelia of Antrodia cinnamomea inhibits metastasis of Lewis lung carcinoma 1 cells through direct action.
摘要 31
1. 前言 32
2. 材料與方法 33
2.1. 樟芝菌絲體醣蛋白之萃取 33
2.2. 細胞培養 33
2.3. 酸性磷酸酶分析法 34
2.4. 細胞侵襲能力分析 34
2.5. 傷口癒合分析 35
2.6. 細胞黏附能力分析 35
2.7. 同功酵素圖譜分析(zymography) 36
2.8. 西方墨點法 36
2.8.1. 藥品配製 36
2.8.2. 抗體來源 36
2.8.3. 蛋白質萃取 37
2.8.4. 樣品製備及電泳 37
2.8.5. 轉漬及Blocking 38
2.9. 統計分析 38
3. 結果 38
3.1. 樟芝菌絲體醣蛋白(AC-GP)對小鼠肺癌細胞(LLC1)細胞存活率的影響 38
3.2. AC-GP抑制LLC1細胞侵襲作用 39
3.3. AC-GP抑制LLC1細胞移行作用 39
3.4. AC-GP抑制LLC1細胞黏附作用 39
3.5. AC-GP抑制基質金屬蛋白酶(matrix metalloproteinase, MMP)-9和-2及尿素激
酶型胞漿素原活化子(urokinase plasminogen activator, uPA)酵素活性……...40
3.6. AC-GP抑制MMP-9及MMP-2蛋白質表現……..………………………….….40
3.7. AC-GP促進基質金屬蛋白酶內生性抑制劑(tissue inhibitor of matrix
  metalloproteinases, TIMP)-1和-2蛋白質表現……………...…….…………….41
3.8. AC-GP促進胞漿素原活化子抑制物(plasminogen activator inhibitor, PAI)-1及
抗轉移蛋白(nm23)-H1蛋白質表現 41
3.9. AC-GP抑制Rho及Rac蛋白質表現 41
3.10. AC-GP抑制有絲分裂活化蛋白質激酶(MAPKs)的磷酸化作用 42
3.11. AC-GP抑制FAK的磷酸化作用 43
3.12. AC-GP抑制整合素β1蛋白質表現 43
4. 討論 44
4.1. AC-GP抑制LLC1細胞轉移之分子機制探討 44
4.1.1. AC-GP造成MMPs/TIMPs比例失衡而抑制LLC1細胞轉移 44
4.1.2. AC-GP向下調節uPA system而抑制LLC1細胞轉移 .45
4.1.3. AC-GP活化nm23-H1而抑制LLC1細胞轉移 .45
4.1.4. AC-GP弱化整合素β1所調控的訊號傳遞路徑而抑制LLC1細胞轉移 .46
4.2. 多醣直接與細胞作用之探討 .46
4.3. AC-GP抑制轉移作用呈現U型效應 47
4.4. 結論 47
5. 參考文獻 49

第三章 樟芝菌絲體醣蛋白可藉由免疫調節作用抑制小鼠肺癌細胞轉移
Glycoprotein from mycelia of Antrodia cinnamomea inhibits metastasis of Lewis lung carcinoma 1 cells through immunomodulation.
摘要…………………………….…………………………………………………….70
1. 前言…………………………………………………………………………….....71
2. 材料與方法………………………………………………………………….…....72
2.1. 樟芝菌絲體醣蛋白之萃取……………………………………………………72
2.2. 製備條件培養液(conditioned medium, CM)…………………………………73
2.3. 組合微球免疫分析技術及流式細胞儀測定(Cytometric bead array, CBA)...73
2.4. 細胞培養……………………………………………………….……………...74
2.5. 酸性磷酸酶分析法…………………………………………………………....74
2.6. 細胞侵襲能力分析……………………………………………………….…...74
2.7. 傷口癒合分析……………………………………………………….………...75 2.8. 細胞黏附能力分析……………………………………………………….…...75
2.9. 同功酵素圖譜分析(zymography) ……………………………………….…...76
2.10. 西方墨點法…………………………………………………….…………….77
2.10.1. 藥品配製………………………………………………….………………77
2.10.2. 抗體來源…………………………………………………….……………77
2.10.3. 蛋白質萃取…………………………………………………….…………77
2.10.4. 樣品製備及電泳………………………………………………………….77
2.10.5. 轉漬及Blocking………………………………………….……………….78
2.11 統計分析...……………………………………………………….…………....78
3. 結果……..………………………………………………………………………....80
3.1. AC-GP對MNCs分泌細胞激素的影響…………………………...………........80
3.2. AC-GP-CM對LLC1細胞存活率的影響…………..…………………………...80
3.3. AC-GP-CM抑制LLC1細胞侵襲作用…………..……………………….…......81
3.4. AC-GP-CM抑制LLC1細胞移行作用……………..………………………..….81
3.5. AC-GP-CM抑制LLC細胞黏附作用……………..………………………..…...81
3.6. AC-GP-CM抑制基質金屬蛋白酶(matrix metalloproteinase, MMP)-9和-2
及尿素激酶型胞漿素原活化子(urokinase plasminogen activator, uPA)酵素
活性……………………..…………………………………..................................82
3.7. AC-GP-CM抑制MMP-9及MMP-2蛋白質表現..…………………………….....82
3.8. AC-GP-CM促進基質金屬蛋白酶內生性抑制劑(tissue inhibitor of matrix metalloproteinases, TIMP)-1和-2蛋白質表現……………………………...…....82
3.9. AC-GP-CM促進胞漿素原活化子抑制物(plasminogen activator inhibitor, PAI)-1及抗轉移蛋白(nm23)-H1蛋白質表現…...………………………………………….83
4. 討論…………………………………………………………………………….…..83
4.1. 多醣體促進免疫調節………………………..……………………………..…..83
4.2. 細胞激素與癌細胞轉移相關的分子機制探討 ………………………………..84
4.2.1. Th1型細胞激素(IL-12及IL-1β)……………..……………………….…........84
4.2.2. Th2型細胞激素(IL-6)及趨化素(IL-8) ...........................................................85
4.3. 多醣可經由人體消化吸收且直接作用與免疫調節作用同時發生…..…........86
4.4. 結論………………………………………………………………………….….86
5. 參考文獻……………………………………………………………………….…..87
總結……………………………………………………………………………….….102


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