保健食品在二十一世紀將持續獨領風騷，佔全球食品市場之重要地位，而水產保健食品則剛剛嶄露頭角極具開發潛力。聚球藻屬於藍綠藻，其富含的藻藍素為一光吸收色素-蛋白質複合體，已知具有抗氧化、抗發炎及抗癌的能力。本研究著重於從聚球藻中純化藻藍素進行抗腫瘤特性之評估，藉由下列方法：MTT assay、細胞型態變化、細胞凋亡的分析，探討藻藍素濃度及作用時間對腫瘤細胞的影響。實驗結果顯示，可以藉由添加溶菌酶結合凍結-解凍來進行萃取，並利用疏水性層析管柱與陰離子交換樹脂來純化藻藍素，回收率可達56%，並於1克的聚球藻粉末中可得到高純度的藻藍素3毫克。電泳分析結果證明，純化得到的藻藍素分子量為103 kDa，且是由分子量分別為20.4 kDa與18.5 kDa之次分子所組成。而細胞實驗顯示以500 μg/mL的藻藍素處理72小時後，可使Hep G2細胞的增殖減少74%，而其IC50値為299.1 µg/mL。而以倒立式位相差顯微鏡觀察發現細胞型態產生皺縮且形成群聚的圓形細胞。接著進一步利用流式細胞儀進行分析，細胞週期方面，結果指出經500 μg/mL的藻藍素處理72小時後，sub-G1期百分比達到20.16±0.57%。Annexin V-PI雙染分析則是以不同濃度藻藍素處理72小時後，細胞發生早期凋亡的比例介於5.39±0.3% ~ 12.62±0.69%。此外，在caspase-3活性分析方面發現處理之後活性增加6倍，其比例由2.99±0.58%增加至20.04±0.39%。
由以上的結果得知，藻藍素確實可以使Hep G2細胞走向凋亡。

Accordingly, nutraceutical will continuously leading the brand and position a critical issue of the global food market, while functional food derived from seafood just explode the highlight and exhibit a great potential at this special field. Synechococcus sp., a member of cyanobacteria family, containing phycocyanin (CPC) is one of the major light harvesting pigment-protein which exhibiting diverse antioxidant, anti-inflammatory and anticancerous properties. The main purpose of the current study is assessment of antitumor activity of CPC from Synechococcus sp. U2. The measurement of dose-dependent, and time course effect on antitumor activity of phycocyanin can be depicted by MTT assay, microscopic observation and cell apoptosis assay, respectively. The results showed that CPC was extracted with lysozyme and by freezing-thawing cycles, and purified by hydrophobic interaction chromatography with butyl sepharose column and anion chromatography using DEAE sepharose resin. The recovery efficiency of CPC from crude extract was above 56%. Three milligram pure CPC was obtained from 1 g of dried cell mass and the purity ratio (A620/A280) was 4.43.
The molecular mass of purified CPC was 103 kDa and has two subunits with apparent molecular mass of 20.4 and 18.5 kDa respectively by electrophoresis technology. In cell, the results indicated that 74% decrease in the proliferation of Hep G2 cells when treated with 500 µg/mL CPC up to 72 hrs and the IC50 was 299.1 µg/mL. By reverse phase microscope, cell morphology changed in shrinkaged and cluster of round cells were observed. The Hep G2 cell treated with CPC analyzed by Flow Cytometry suggested that in cell cycle, cells treated with 500 µg/mL CPC for 72h showed 20.16±0.57% in sub-G1. In annexin V-PI assay, the data suggested that 5.39±0.3% to 12.62±0.69% early apoptosis cells can be found when CPC were employed for 72 hours. Furthermore, the results demonstrated that the caspase 3 activity was enhanced by 6-fold from 2.99±0.58% to 20.04±0.39% when treated with the subject. Conclusively, the present study illustrated that CPC followed apoptosis pathway on Hep G2 cell lines.

中文摘要 I
英文摘要 III
謝 誌 V
目 錄 VI
1. 前言 1
2. 文獻回顧 3
2.1 藻類 3
2.1.1 海洋藻類 5
2.1.2 藍藻 6
2.1.3 藍藻的應用 7
2.1.4 聚球藻 10
2.2 藻藍素 10
2.2.1 藻藍素之應用 13
2.2.2 藻藍素之生理活性 13
2.2.2.1 抗氧化活性 13
2.2.2.2 抗發炎活性 14
2.2.2.3 保護肝臟 15
2.2.2.4 抗腫瘤活性 16
2.3 肝癌 17
2.3.1 肝癌高風險群 17
2.3.2 肝癌症狀 18
2.3.3 肝癌診斷 19
2.3.4 肝癌治療 19
2.3.5 肝癌預防 22
2.4 常用之抗癌藥物 23
2.4.1 Doxorubicin 23
2.4.2 5-flurouracil 24
2.5 細胞週期 26
2.5.1 細胞週期在癌症治療上的應用 28
2.6 細胞死亡 28
2.6.1 細胞壞死 28
2.6.2 細胞凋亡 29
2.6.2.1 細胞凋亡之分期 30
2.6.2.2 細胞凋亡之途徑 32
2.6.2.2.1 死亡接受體途徑 33
2.6.2.2.2 粒腺體途徑 33
3. 材料與方法 37
3.1 實驗設計 37
3.2 實驗材料來源 39
3.2.1 聚球藻來源 39
3.2.2 細胞株來源 39
3.3 實驗試藥及設備 39
3.3.1 藻藍素純化試藥 39
3.3.2 電泳分析試藥 39
3.3.3 細胞培養及分析試藥與套組 40
3.3.4 常用儀器設備 40
3.4 實驗方法 41
3.4.1 聚球藻培養 41
3.4.2 聚球藻前處理 42
3.4.3 藻藍素分離與純化 42
3.4.3.1 聚球藻粗萃液製備 42
3.4.3.2 疏水性層析 42
3.4.3.3 陰離子交換層析 43
3.4.3.4 蛋白質含量測定 43
3.4.3.5 藻藍素含量測定 43
3.4.3.6 純化總表製作 44
3.4.4 分子量測定 44
3.4.4.1 電泳樣品處理 44
3.4.4.2 電泳分析試劑配製 44
3.4.4.3 電泳膠體製備 45
3.4.4.4 電泳操作步驟 47
3.4.5 細胞培養 47
3.4.5.1 培養基配製 47
3.4.5.2 緩衝溶液配製 48
3.4.5.3 細胞解凍 48
3.4.5.4 細胞繼代培養 48
3.4.5.5 細胞計數 49
3.4.5.6 細胞冷凍保存 49
3.4.6 細胞試驗 50
3.4.6.1 細胞存活率測試 50
3.4.6.2 細胞型態觀察 50
3.4.6.3 流式細胞儀分析 50
3.4.6.3.1 細胞週期分析 51
3.4.6.3.2 Annexin V-PI雙染分析 51
3.4.6.3.3 Caspase 3蛋白酶活性分析 52
3.4.7 統計分析 52
4. 結果與討論 53
4.1 聚球藻培養與前處理 53
4.2 藻藍素之純化 57
4.2.1 粗萃取液製備 57
4.2.2 疏水性交換層析 58
4.2.3 陰離子交換層析 60
4.2.4 純化總表 62
4.3 分子量測定 67
4.3.1 Native-PAGE 67
4.3.2 SDS-PAGE 67
4.4 細胞試驗 70
4.4.1 細胞存活率測試-MTT assay 70
4.4.2 細胞型態觀察 75
4.4.3 流式細胞儀分析 82
4.4.3.1 細胞週期分析 82
4.4.3.2 Annexin V-PI雙染分析 85
4.4.3.3 Caspase 3蛋白酶活性分析 93
5. 結論 98
參考文獻 99
作者簡介 113

圖表目錄
圖 1、藻膽色體其概要構造圖 12
圖 2、藻藍素之bilin發色基團與膽紅素之化學結構 14
圖 3、Doxorubicin化學結構式 25
圖 4、5-flurouracil化學結構式 25
圖 5、細胞週期 27
圖 6、細胞凋亡與細胞壞死 30
圖 7、細胞凋亡之三種途徑 32
圖 8、細胞凋亡之死亡接受器途徑 35
圖 9、細胞凋亡之粒線體途徑 36
圖 10、藻藍素抗腫瘤活性之實驗設計 38
圖 11、聚球藻之菌種觀察：聚球藻培養液 54
圖 12、聚球藻粉末外觀 55
圖 13、 Synechococcus sp. U2中藻藍素之butyl-sepharose管柱層析圖 59
圖 14、 Synechococcus sp. U2中藻藍素之DEAE-sepharose管柱層析圖 61
圖 15、A：聚球藻培養液；B：經純化後所得之藻藍素純化液 65
圖 16、經純化後之藻藍素吸收波長曲線圖 66
圖 17、Synechococcus sp. U2中藻藍素之Native-PAGE電泳圖 68
圖 18、Synechococcus sp. U2中藻藍素之SDS-PAGE電泳圖 69
圖 19、聚球藻中藻藍素對人類肝癌細胞株Hep G2之毒殺效果 72
圖 20、不同濃度之藻藍素處理Hep G2細胞24小時後的細胞型態變化 (200x) 77
圖 21、不同濃度之藻藍素處理Hep G2細胞48小時後的細胞型態變化 (200x) 78
圖 22、不同濃度之藻藍素處理Hep G2細胞72小時後的細胞型態變化 (200x) 79
圖 23、經5FU處理不同時間後之AGS細胞的型態變化 (200x) 80
圖 24、不同濃度之藻藍素處理AGS細胞後的細胞型態變化 (200x) 81
圖 25、不同濃度之藻藍素處理Hep G2細胞24小時後annexin V-PI分析 87
圖 26、不同濃度之藻藍素處理Hep G2細胞48小時後annexin V-PI分析 88
圖 27、不同濃度之藻藍素處理Hep G2細胞72小時後annexin V-PI分析 89
圖 28、不同濃度之藻藍素處理Hep G2細胞24小時後caspase 3活性變化 94
圖 29、不同濃度之藻藍素處理Hep G2細胞48小時後caspase 3活性變化 95
圖 30、不同濃度之藻藍素處理Hep G2細胞72小時後caspase 3活性變化 96

表 1、藻類主要特徵與類型 4
表 2、細胞凋亡與細胞壞死之差異 31
表 3、MN培養基 42
表 4、聚球藻粉末之產率 56
表 5、Synechococcus sp.U2藻藍素之純化總表 64
表 6、聚球藻中藻藍素對人類腫瘤細胞株Hep G2之毒殺效果 73
表 7、聚球藻中藻藍素對人類腫瘤細胞株AGS之毒殺效果 74
表 8、 以不同濃度藻藍素處理24至72小時後其Sub-G1期百分比 84
表 9、 以不同濃度藻藍素處理24小時後其細胞死亡之情形 90
表 10、以不同濃度藻藍素處理48小時後其細胞死亡之情形 91
表 11、以不同濃度藻藍素處理72小時後其細胞死亡之情形 92
表 12、以不同濃度藻藍素處理不同時間後其促凋亡蛋白產生之百分比 97

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