臺灣博碩士論文加值系統

　　樟芝(Antrodia cinnamomea, Antrodia camphorata, Taiwanofungus camphoratus或Ganoderma comphoratum)，俗稱牛樟菇，是牛樟樹(Cinnamomum kanehirai)上目前唯一被發現的木材腐朽菌，具專一性依賴生長之關係，唯牛樟樹已被列為保育樹種，因此利用深層液態培養方法，為目前獲得此稀有資源之最佳方式，然而其培育方式與野生明顯不同，兩者間之組成亦顯差異。由於乳癌已成為已成為國人婦女癌症死亡之第一位，如何由天然物中開發成為輔助治療的方法，是目前發展重點之一，本研究之目的即擬由液態培養之樟芝菌絲體中製備植化成分，包括多醣體及二級代謝物，結合化療或放療方法，探討對MCF-7乳腺癌細胞增殖活性之影響。實驗分為三部分，第一與第二部分為二級代謝物之製備，分別以（1）溶劑萃取及配合SephadexTM LH-20管柱區分方式製備，以及（2）溶劑萃取物經Amberlite XAD-7管柱吸附後，再以溶劑溶離後，製備之萃洗物，以分離製備其中具較高三萜類含量之粗萃物，進行對乳腺癌細胞MCF-7抗癌活性之研究。此外，由於樟芝菌絲體中含大量之多醣體，其中我們發現在鹼萃取及酸沉澱製備之樟芝多醣體（AC-2）具明顯之抗發炎活性，因此，本研究之另一目的（3），即以此多醣體進行相關之抗癌活性試驗，並結合放射線處理或乳癌化療藥Doxorubicin，探討其是否具增加抑制癌細胞之活性，以及了解其抑制之作用機制。
　　實驗（1）結果中，發現七種區分物中Sub-Fraction 4具較高之粗三萜與總酚化合物濃度，藉由液相層析串聯質譜分析，鑑定出主要之成分為：camphorataimide A與C, antrocinnamomin C, dehydrosulphurenic acid及sulphurenic acid；此區分物在濃度34.6–53.4 µg/mL下，及輻射劑量（15 Gy）之合併處理，可顯著性降低MCF-7細胞之存活率，抑制率分別達29.65 %及37.95 %。以流式細胞儀分析證實此癌細胞之死亡是藉由晚期凋亡及細胞壞死所造成，並以細胞凋亡為主要途徑。以西方墨點法分析，證實在合併處理輻射顯著性增加細胞毒性及細胞凋亡之效果，並且升高促凋亡蛋白之表現，如p53（202.22 %）、p21（272.75 %）和Bax（197.84 %）。於實驗（2）部分，結果指出樟芝菌絲體以甲醇萃取，及利用 Amberlite XAD-7管柱層析之萃洗物，其成分經鑑定主要為camphorataimide C, antroquinonol及sesamin；此萃取物單獨處理時即對抑制MCF-7癌幹細胞有明顯效果，低劑量處理（24.4 µg/mL）抑制率為25 %，高劑量（61.8 µg/mL）處理抑制率可達50 %，於低劑量IC25（24.4 µg/mL）合併臨床化療藥物Doxorubicin處理MCF-7細胞，亦能也顯著性抑制細胞之存活率抑制率可達59.23 %，並使癌細胞停滯於G2/M期；流式細胞儀分析顯示，低劑量處理會造成早期凋亡，而隨著劑量增加，其晚期凋亡隨之增加；以西方墨點法分析，證實可以藉由降低抗凋亡的Bcl-2蛋白（61.25 %）表達，並且升高促凋亡蛋白之表現，如p53（219.5 %）、p21（119.65 %）和Bax（155.47 %）。此外，於實驗（3）部分，發現樟芝菌絲多醣體AC-2 在濃度0.6–1.2 µg/mL和輻射（0–6 Gy）合併處理，證實隨著劑量提升，能有顯著性減少細胞增生及存活率，同時利用流式細胞儀分析，證實細胞死亡原因主要是藉由誘導癌細胞走向細胞凋亡。以西方墨點法分析，證實處理多醣體AC-2顯著性降低抗凋亡蛋白Bcl-2（14.74 %）表達，合併處理輻射發現藉由合併處理明顯造成細胞毒性及細胞凋亡增加，並促進凋亡蛋白之表現，如p53（271.04 %）、p21（314.32 %）及Bax（213.51 %）蛋白表現量上升，於 TUNEL染色分析亦證實造成癌細胞核內DNA之片斷化；藉由螢光物質（fluorescein isothiocyanate, FITC）標誌多醣體AC-2堆積於細胞膜上，進一步以西方墨點法分析，發現多醣體AC-2於1.2 µg/mL濃度即抑制組合蛋白（Integrin β1）達64.05 %之表現，並促進凋亡蛋白Caspase 9之表現達181.91 %，此一發現推測多醣體AC-2對MCF-7細胞之抑制活性，部分來自對膜蛋白之影響。
　　綜合以上三項實驗顯示，不論採用菌絲體二級代謝物（小分子）或構成菌絲體結構之多醣體（大分子），配合藥物Doxorubicin及輻射皆能促使乳癌細胞MCF-7走向凋亡途徑，其中又以樟芝多醣體AC-2被認為最具有可能被開發為放射線療法之輔助治療替代物。此研究之結果，認為在開發有效治療乳癌疾病方面可提供另一新的發展策略。

Antrodia cinnamomea (AC, also named Antrodia camphorata, Taiwanofungus camphoratus and Ganoderma comphoratum), “niu-chang-chih” or “niu-chang-ku” in Chinese, is an exclusive fungus parasitic on the rot wood of the endemic species Cinnamomum micranthum. The liquid submerged culture has been recognized as the most potential method for producing and replacing the rare sources of wild growing fruiting bodies. However, the secondary metabolites of the mycelia from submerged liquid cultures are quite different from those of the wild fruiting bodies due to the different cultivation methods. According to the Health Annual Report, breast cancer has become the first leading cause of death from cancer among women in Taiwan. The purpose of this study was to evaluate the effects of mycelia extracts and/or polysaccharides from the liquid culture of A. cinnamomea in combined with the chemotherapy or radiotherapy on the proliferative activity of MCF-7 breast cancer cell line. In the study, total of three parts of experiments are conducted to examine the bioactivities of A. cinnamomea mycelia, in which the prepared extracts from secondary metabolites of mycelia are conducted in the first and second part of studies. In Part I, the solvent extraction and SephadexTM LH-20 column fractionation were used to prepare extracts from secondary metabolites of mycelia. While the liquid-liquid partition and Amberlite XAD-7 column adsorption methods for fractionating the extracts with high quantities of triterpenoids were conducted in the Part II experiment. The prepared extracts from these two experiments were applied to evaluate the anticancer activities on breast cancer cell line MCF-7. Additionally, the huge amounts of polysaccharides are enriched in the mycelia. In which, the isolated polysaccharides AC-2 performed by the method of “alkali extraction and acid precipitation” has been found with significant anti-inflammatory effects. In hence, the Part III experiment was to examine the anticancer activities of polysaccharides AC-2 in combining with the chemotherapy drug Doxorubicin on breast cancer for underlying the inhibition mechanisms.
Results in the Part I indicate that camphorataimides A and C, antrocinnamomin C, dehydrosulphurenic acid and sulphurenic acid identified by LC/MS/MS analysis were the major components in the sub-fraction 4, which was the highest concentrations of triterpenoids and polyphenolics found in the seven fractions of mycelia extracts. Under the concentrations of 34.6 and 53.4 g/mL of sub-fraction 4, significant inhibition activities up to 29.65 and 37.95 %, respectively were shown in the cell viability when combining treatment with radiotherapy (15 Gy) on MCF-7 cells. The cell death appeared in a late growth phase was found from the cell apoptosis and necrosis by the flow cytometric analysis with the cell apoptosis as the major cause. Western blot analysis reveal that the effects of extract in combining treatment with radiotherapy significantly promote the expressions of pro-apoptotic proteins, such as p53 (202.22 %), p21 (272.75 %) and Bax (197.84 %). In the Part II work, the Amberlite XAD-7 eluate (identified sesamin, camphorataimide C and antroquinonol as the major components) from mycelia extracts presented significant inhibition activity on cancer stem cells with marked effects in low dosage of IC25 (24.4 g/mL) and high dosage of IC50 (61.8 g/mL). In a low dosage IC25 (24.4 µg/mL) of Amberlite XAD-7 eluate and combining treatment with doxorubicin on MCF-7 cells resulted in an inhibition rate of 40.91% and caused a G2/M phase arrest in cell cycle. The apoptosis occurred in early growth phase when a low dosage used, while a higher dosage resulted in an increasing of cell apoptosis in the late growth phase. The combined treatment reduced the anti-apoptotic Bcl-2 protein (61.25 %) expression, and increased expressions of pro-apoptotic proteins such as p53 (219.5 % ), p21 ( 110.65 % ) and Bax ( 155.47 % ). In the Part III study, the combined treatment with AC-2 (1.2 µg/mL) and ionizing radiation (0  6 Gy), resulted in the decreasing of cell proliferation and clonogenic survival in a dose-responsive manner. The cell apoptosis was simultaneously proved by the flow cytometry. By western blot analysis, the expression of apoptotic regulatory molecules were induced through the down-regulation expression of anti-apoptotic Bcl-2 protein (14.74 %), and the up-regulation of pro-apoptotic protein, including p53 (271.04 %), p21 (314.32 %) and Bax (213.51 %). The cell apoptosis was further confirmed by TUNEL staining assay in DNA fragmentation. The observation of the fluorescence from the labeling of polysaccharides AC-2 through the fluorescein isothiocyanate method revealed that the accumulation of polysaccharides AC-2 on MCF-7 cell membranes was time responsive. Furthermore, the inhibition expression of integrin 1 64.05% and apoptotic protein caspase 9 significant increase 181.91% induced by polysaccharides AC-2 was presented in the western blot analysis. Thus, it was concluded that AC-2 could sensitize the effect of radiation in breast carcinoma partly from the regulating effects on cell membrane protein which involved in cell cycle and apoptosis.
It is concluded that the apoptosis of MCF-7 cells were enhanced by the combination of doxorubicin or radiotherapy regardless of the sample from secondary metabolites (small molecules) or polysaccharides (macromolecules) according to the above three studies. In which, the choice of polysaccharide AC-2 in combination of radiotherapy might be a new approach for developing effective complementary therapy on breast cancer.

目錄 I
圖目錄 VI
表目錄 IX
附圖目錄 X
中文摘要 1
英文摘要 4
第一章 緒論 7
第二章 文獻回顧 8
　2.1樟芝介紹 8
　　2.1.1 樟芝植物分類學 8
　　2.1.2 樟芝命名 9
　　2.1.3 樟芝藥理應用 10
　　2.1.4 樟芝分布與特徵 13
　2.2 樟芝成分分析 14
　　2.2.1 樟芝主要植化成分 14
　　2.2.2 樟芝活性成分 15
　　2.2.3 層析管柱SephadexTM LH-20與Amberlite XAD-7樹脂特性 25
　2.3 乳腺癌及其治療 26
　　2.3.1 形成 26
　　2.3.2 乳腺癌治療 26
　　2.3.3 乳腺癌好發性 27
　2.4 放射線治療 29
　2.5 合併治療 29
　2.6 輻射增敏 30
　2.7 TUNEL染色分析 31
　2.8 粒線體膜電位之測定 34
　2.9 細胞死亡 34
　2.10 癌幹細胞 38
第三章 實驗架構 40
第四章 材料與方法 41
　4.1 實驗材料 41
　　4.1.1 實驗藥品 41
　　4.1.2 細胞培養試藥 43
　　4.1.3 抗體 44
　　4.1.4 儀器設備 45
　4.2 實驗方法 47
　　4.2.1 樟芝菌絲體製備與萃取 47
　　　4.2.1.1 樟芝菌絲體之製備 47
　　　4.2.1.2 樟芝菌絲體之萃取 47
　　4.2.2 樟芝子實體萃取 48
　　　4.2.2.1 樣品來源與萃取方法 48
　　　4.2.2.2 樟芝菌絲多醣體AC-2之製備 49
　　4.2.3 高效液相層析（HPLC）與液相層析質譜分析（LC-MS） 50
　　4.2.4 總酚類化合物含量測定 51
　　4.2.5 粗三萜含量之比色測定法 51
　　4.2.6 細胞培養 52
　　　4.2.6.1 細胞株 52
　　　4.2.6.2 磷酸緩衝液（PBS）製備 52
　　　4.2.6.3 細胞培養液配置 52
　　　4.2.6.4 細胞株的培養 52
　　　4.2.6.5 細胞之解凍與保存 53
　　　4.2.6.6 MCF-7 乳癌細胞株生長曲線之測定 54
　　　4.2.6.7 細胞存活率實驗（MTT assay） 54
　　　4.2.6.8 放射線照射 55
　　　4.2.6.9 細胞增殖試驗 55
　　　4.2.6.10 細胞群落試驗（Clonogenic assay） 55
　　　4.2.6.11 細胞內ROS之測定 56
　　　4.2.6.12 細胞凋亡分析 57
　　　4.2.6.13 凋亡分析–細胞週期分析 57
　　　4.2.6.14 粒線體膜電位之測定（MMP） 58
　　　4.2.6.15 TUNEL染色分析 59
　　　4.2.6.16 西方墨點法（Western blot） 59
　　　　4.2.6.16.1 蛋白質萃取 59
　　　　4.2.6.16.2 蛋白質定量 60
　　　　4.2.6.16.3 蛋白質凝膠製備與電泳分析 60
　　　　4.2.6.16.4 蛋白質轉漬 61
　　　　4.2.6.16.5 抗體反應與壓片 61
　　4.2.7 螢光標示法分析多醣體AC-2細胞內含量 62
　　4.2.8 幹細胞之含量測定 62
　　4.2.9 統計分析 63
第五章　實驗結果 64
　5.1 樟芝菌絲體與子實體之萃取與分析 65
　　5.1.1 樟芝菌絲體萃取物配合SephadexTM LH-20管柱製備之區分物
　　 　　中三萜類及酚類化合物含量及HPLC與LC/MS分析 66
　　5.1.2樟芝菌絲體萃取物於Amberlite XAD-7管柱吸附之萃洗物組成
　　 　　 70
　　5.1.3 樟芝子實體萃取物HPLC之分析 72
　　5.2.1 樟芝菌絲多醣體AC-2合併化療藥物Doxorubicin對MCF-7細
　　　　 胞之細胞存活率試驗 73
　　5.2.2 樟芝菌絲體溶劑萃取物合併化療藥物Doxorubicin對MCF-7
　　 　　細胞之細胞存活率試驗 75
　　5.2.3 樟芝子實體萃取物對MCF-7細胞之細胞存活率試驗 79
　　5.2.4 樟芝菌絲多醣體AC-2抑制MCF-7細胞增殖活性 82
　　5.2.5 樟芝菌絲多醣體AC-2對MCF-7細胞形態之影響 84
　　5.2.6 多醣體AC-2對MCF-7細胞內ROS含量之影響 86
　　5.2.7 螢光標示法分析多醣體AC-2細胞內含量 88
　　5.2.8 西方墨點法分析樟芝多醣體AC-2對MCF-7乳癌細胞integrin
　　　　 β1及caspase 9蛋白質表現量之影響 89
　　5.3.1 樟芝菌絲體Amberlite XAD-7萃洗物及合併化療藥物
　　　　 Doxorubicin對MCF-7細胞存活率試驗 91
　　5.3.2 樟芝菌絲體Amberlite XAD-7萃洗物抑制MCF-7癌幹細胞之
　　　　 生成 94
　　5.3.3 樟芝菌絲體Amberlite XAD-7萃洗物及化療藥物Doxorubicin
　　　　 處理對MCF-7乳癌細胞週期之影響 97
　　5.3.4 樟芝菌絲體Amberlite XAD-7萃洗物及化療藥物Doxorubicin
　　　 　處理對MCF-7乳癌細胞凋亡現象之影響 100
　　5.3.5 利用TUNEL染色分析樟芝菌絲體Amberlite XAD-7萃洗
物及化療藥物Doxorubicin處理對MCF-7乳癌細胞DNA斷裂
之現象 104
　　5.3.6 利用西方墨點法探討樟芝菌絲體Amberlite XAD-7萃洗物及
　　 化療藥物Doxorubicin處理對MCF-7乳癌細胞凋亡之相關蛋
　　 白質表現 106
　　5.4.1 樟芝多醣體AC-2對MCF-7乳癌細胞的輻射增敏作用之細胞
　　 群落分析 108
　　5.4.2 樟芝多醣體AC-2及放射線處理對MCF-7乳癌細胞凋亡現象
　　 　　之探討 110
　　5.4.3 利用粒線體膜電位（MMP）探討樟芝多醣體AC-2及放射線
　　　　 處理對MCF-7乳癌細胞凋亡之位移 113
　　5.4.4 利用TUNEL染色分析樟芝多醣體AC-2及放射線處理對
　　　　 MCF-7乳癌細胞DNA斷裂之現象 115
　　5.4.5 利用西方墨點法探討樟芝多醣體AC-2及放射線處理對
　　　 　MCF-7乳癌細胞凋亡之相關蛋白質表現 117
　　5.5.1 樟芝菌絲體萃取物之Sephadex LH-20 管柱區分物
　　　　 Sub-Fraction 4及放射線處理對MCF-7乳癌細胞凋亡現象之
　　　　 探討 119
　　5.5.2 利用西方墨點法探討樟芝菌絲體萃取物之Sephadex LH-20
　　　　 管柱區分物Sub-Fraction 4及放射線處理對MCF-7乳癌細胞凋
　　　　 亡之相關蛋白質表現 122
　5.6 樟芝菌絲體及子實體萃取物其抑制50%細胞存活率之濃度 124
第六章 討論 125
第七章 結論 129
第八章 參考文獻 131


圖目錄
圖一、 樟芝（Antrodia cinnamomea）形態圖 8
圖二、 Maleic anhydride及其衍生物：camphorataimides A, B與C 20
圖三、 實驗架構 40
圖四、 樟芝菌絲體酒精萃取物(A)及其液—液相區分物與SephadexTM
LH-20管柱層析區分物Sub-Fraction 1－Sub-Fraction 7 (B) 之高
效液相層析圖 67
圖五、 液相層析質譜分析Sub-Fraction 4之液相層析（A圖）與總離子
層析圖（B與C圖） 69
圖六、 液相層析及液相層析質譜分析Amberlite XAD-7管柱萃洗物 71
圖七、 樟芝一至四年生之子實體萃取物之高效液相層析圖 72
圖八、 樟芝菌絲多醣體AC-2（A）合併化療藥物Doxorubicin（B）對
MCF-7細胞24、48及72小時存活率之影響 74
圖九、 樟芝菌絲體酒精萃取物之區分物Sub-Fraction 1－Sub-Fraction 7
對MCF-7細胞24、48及72小時存活率之影響 75
圖十、 樟芝子實體一至四年生對MCF-7細胞24、48及72小時存活率
之影響 78
圖十一、 樟芝子實體一至四年生對MCF-7細胞24、48及72小時存活
　　　　 率之影響 81
圖十二、 樟芝菌絲多醣體AC-2對MCF-7細胞24、48、72及96小時
　　　　 細胞增殖之活性 83
圖十三、 樟芝菌絲多醣體AC-2對MCF-7細胞0、24、48及72小時細
　　　　 胞形態之影響 85
圖十四、 多醣體AC-2對MCF-7細胞經72小時共同培養後細胞內反應
　　　　 活性氧物質含量之影響 87
圖十五、 以螢光顯微鏡分析FITC標誌之樟芝菌絲多醣體AC-2於
　　　　 MCF-7細胞膜之堆積 88
圖十六、 以西方墨點法分析，樟芝多醣體AC-2處理MCF-7細胞經72
　　　　 小時之蛋白質表現量 90
圖十七、 （A）樟芝菌絲體Amberlite XAD-7樹脂萃洗物對MCF-7細胞
　　　　 24、48及72小時存活率之影響（B）合併化療藥物Doxorubicin
　　　　 對MCF-7細胞24、48及72小時存活率之影響 93
圖十八、 以ALDEFLUOR染色分析樟芝Amberlite XAD-7萃洗物處理
　　　　 MCF-7細胞24、48及72小時後抑制癌幹細胞之生成 94
圖十九、 以ALDEFLUOR染色分析樟芝Amberlite XAD-7萃洗物處理
　　　　 MCF-7細胞24、48及72小時後癌幹細胞增生之抑制作用 96
圖二十、 PI單染分析樟芝Amberlite XAD-7萃洗物及Doxorubicin
　　　　 (Dox)對MCF-7細胞處理24、48及72小時之細胞週期之
　　　　 影響 98
圖二十一、 以PI單染分析，樟芝Amberlite XAD-7萃洗物處理MCF-7
　　　　　 細胞經24小時之細胞週期之影響 99
圖二十二、 以Annexin V-FITC/PI雙染色法分析樟芝Amberlite XAD-7
　　　　　 萃洗物處理MCF-7細胞24、48及72小時之影響 102
圖二十三 、 以Annexin V-FITC/PI雙染色法分析，樟芝Amberlite XAD-7
　　　　　 萃洗物處理MCF-7細胞經24小時之影響 103
圖二十四、 以TUNEL染色法分析，樟芝菌絲體Amberlite XAD-7萃洗
　　　　　 物及化療藥Doxorubicin處理24小時之影響 105
圖二十五、 以西方墨點法分析，樟芝菌絲體Amberlite XAD-7萃洗物及
　　　　　 化療藥物Doxorubicin處理MCF-7細胞經24小時之影響
　　　　　 107
圖二十六、 以細胞群落分析經樟芝菌絲多醣體AC-2處理MCF-7細胞
　　　　　 經14天培養之影響 109
圖二十七、 以Annexin V-FITC/PI雙染色法分析，樟芝菌絲多醣體AC-2
　　　　 　處理MCF-7細胞經48及72小時後造成凋亡之活性 111
圖二十八、 以Annexin V-FITC/PI雙染色法分析，樟芝菌絲多醣體AC-2
　　　　 處理MCF-7細胞經72小時後造成凋亡之活性 112
圖二十九、 樟芝菌絲多醣體AC-2合併放射線15 Gy處理72小時對
　　　　 MCF-7細胞膜電位之影響 114
圖三十、 以TUNEL染色法分析，樟芝多醣體AC-2、15 Gy處理
　　　　 MCF-7胞經72小時之影響 116
圖三十一、 以西方墨點法分析，樟芝多醣體AC-2、15 Gy及20 Gy處
　　　　 　理MCF-7細胞經72小時之蛋白質表現量 118
圖三十二、 以Annexin V-FITC/PI雙染色法分析，樟芝菌絲體溶劑萃取
　　　　　 物Sub-Fraction 4處理MCF-7細胞經48及72小時之影響
　　　　　 120
圖三十三、 以Annexin V-FITC/PI雙染色法分析，樟芝菌絲體溶劑萃取
　　　　　 物Sub-Fraction 4處理MCF-7細胞經48和72小時之影響
　　　　　 121
圖三十四、 以西方墨點法分析樟芝菌絲體萃取物之Sephadex LH-20管
　　　　　 柱區分物Sub-Fraction 4及15 Gy輻射處理MCF-7細胞後經
　　　　　 48小時之影響 123


表目錄
表一、 野生樟芝三萜類化合物中主要成分及其活性 15
表二、 樟芝菌絲體中主要化學成分及其活性 17
表三、 常用於人類乳癌研究之細胞株種類及其生長特徵 28
表四、 抗體條件 44
表五、 樟芝子實體及菌絲體萃取物其三萜類及酚類化合物之含量分析
　　　 65
表六、 樟芝子實體及菌絲體萃取物其抑制50%細胞之濃度 124

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