臺灣博碩士論文加值系統

俗稱動物人蔘的海馬，藥用價值相當高，由於全球人類所造成環境極度威脅，已視為易危物種。針對巴西吻海馬(Hippocampus reidi)之人工繁養殖試驗發現，從2022年7月開始，以六對公母海馬群體可以穩定地繁殖2000隻小海馬，且仍持續增量中，存活率高達80%，預試驗結果顯示，三種海馬(三斑、庫達及巴西吻海馬)萃取物的細胞毒殺實驗，發現Hexane萃取物對於血癌細胞(Molt 4 及 K562 細胞)具有很好的毒殺活性，因此，本論文針對這巴西吻海馬進行人工繁養殖之標準化、定性定量分析活性成分及深入探討其抗癌作用，利於海洋天然物與抗癌藥物開發之應用。
方法: 使用氣相色譜-質譜分析儀 (gas-chromatography-mass spectrometry)分析微藻、浮游生物及糠蝦之脂肪酸組成分之差異，以MTT、流式細胞儀、西方轉漬及共軛焦顯微鏡探討海馬萃取物對於血癌細胞毒殺作用之機制，進一步利用次世代定序出分析海馬萃取物抗癌相關蛋白質之變化。
結果:從脂肪酸組成分分析結果發現利用三種微藻培養混合液提供海馬所需之餌料生物生長發育以提高海馬繁養殖之穩定度。MTT結果發現養殖型海馬HE萃取物細胞毒殺效果較野生海馬萃取物更好，以3.125及6.25 μg/mL可以誘導血癌K562及Molt4細胞凋亡及粒線體膜電位下降約94.91&78.64% 及97.02&80.19%；共軛焦顯微鏡結果發現，HE萃取物可促使血癌細胞形成不規則性皺縮凋亡小體細胞，西方轉漬分析結果證實，經由HE萃取物可增加PARP及caspase3/8/9蛋白質之裂解。從HPLC 化學圖譜及NMR分析比較下，確實巴西吻海馬HE萃取物具有活性成分。
結論: 本研究已成功建立人工繁養殖海馬系統，未來將大規模繁殖以利深入探討抗癌機制及其活性化合物之結構解析。

The seahorse, commonly known as the animal ginseng, has high medicinal value and has been considered a vulnerable species due to the extreme threat to the environment caused by humans around the world. The artificial breeding experiment of Hippocampus reidi was in Brazil. Our previous study suggested that from July 2022, six pairs of male and female seahorse groups can stably breed 2000 young seahorses, and the survival rate is as high as 80%, and the preliminary results showed that hexane extract of sea horse had good anti-proliferative effect against leukemia (Molt4 and K562) cells. Therefore, we establish the aquacultural platform to conduct the application of marine natural substances and anti-cancer drug development.
Methods: The differences in fatty acid composition of microalgae, zooplankton and mysis shrimp were analyzed by gas chromatography-mass spectrometry, and the anticancer mechanism of hippocampal extract on the cytotoxicity of leukemia cells was investigated by MTT, flow cytometry, western blotting and confocal microscopy, and the changes of proteins related to the anticancer effect of hippocampal extract were further analyzed by next generation sequencing.
Results: From the analysis of fatty acid composition, it was found that three microalgae culture mixture was used to provide the food required by seahorse biological growth and development, and improve the stability of seahorse breeding. MTT results showed that the cytotoxicity effect of HE extract from aquacultured sea horse was better than that of wild ones. The apoptosis induction and disruption of mitochondrial membrane potential was showed about 94.91&78.64% and 97.02&80.19% in K562 and Molt4 cells with treatment at 3.125 and 6.25 μg/mL, respectively. Confocal microscopic examination was showed that HE extract could promote the formation of irregular apoptotic bodies in leukemia cells, and Western blotting analysis was confirmed that the cleavage of PARP and caspase3/8/9 protein was increased by treatment of HE extract. Result of HPLC chemical profiles and NMR analysis suggested that HE extract of Hippocampus reidi has active ingredients.
Conclusion: In this study, a seahorse breeding system has been successfully established, and large-scale aquaculture will be carried out in the future to facilitate in-depth exploration of the anti-cancer mechanism and the structure analysis of its active compounds.

中文摘要 I
Abstract II
致謝 IV
圖目錄 VII
表目錄 VIII
一、前言 1
二、文獻回顧 3
1. 海馬研究之發展 3
2. 脂肪酸對於海洋生物營養之重要性 5
3. 海洋微藻之應用 5
4. 以「2020國家海洋政策白皮書」、「2021海洋科技政策綱領」為發展藍圖，繁養殖海馬為素材，開發癌症醫藥用途之「海洋生物材料」為目標： 7
4.1.國外的海洋生3物材料(marine biomaterials)研究情況： 7
4.2. 國內的海洋生物材料(marine biomaterials)研究情況： 8
5. 癌細胞死亡特徵 9
6. 論文研究目標: 繁養殖海馬萃取之建立及抗癌活性分析 12
三、材料及方法 14
1. 進行人工繁養殖海馬之平台建立 14
2. 脂肪萃取與脂肪酸分析 15
3. 細胞存活率試驗（MTT assay） 15
4. DAPI染色 (4, 6-diamidino-2-phenylindole stain) 16
5. 檢測細胞內粒線體膜電位之變化 16
6. Annexin V/PI 雙重染色檢測細胞凋亡之程度 16
7. 西方墨點法 (Western Blotting Assay) 16
8. 海馬活性化合物之HPLC分析及其細胞毒殺活性測試 17
9. 數據統計 17
四、結果 18
1. 分析海洋微藻脂肪酸組成分之差異 18
2. 比較不同品種海馬之萃取物對於血癌細胞生長之影響 21
3.養殖型巴西吻海馬己烷萃取物對於血癌Molt4及K562細胞生長之影響 24
4.養殖型巴西吻海馬己烷萃取物細胞毒殺作用與細胞凋亡誘發之相關性 28
5.養殖型巴西吻海馬己烷萃取物對於細胞凋亡所誘發相關蛋白之影響 33
五、討論 36
六、結論 39
參考文獻 40

圖目錄
圖一、三種微藻的脂肪酸組成模式。誤差線(Error bars)代表平均值的標準誤差………………………………………………………......….20
圖二、微藻中前三個主成分PC1、PC2和PC3的評分圖.......................................................................................................21
圖三、不同提取物（己烷，EA和MeOH萃取物）24小時處理，對於Molt4和K562細胞生長之作………………………………….………………………...…...…23
圖四、分別處理血癌Molt4細胞24及48小時，己烷萃取物對於癌細胞(Molt4)生長之影響…………………………………………………………......….…26
圖五、分別處理血癌Molt4細胞24及48小時，己烷萃取物對於癌細胞(K562)生長之影響………………………………………………………………........27
圖六、利用共軛焦顯微鏡分別檢測己烷萃取物對於血癌K562及Molt4細胞核型態上之影響………………………………………...….....29
圖七、HE萃取物處理24小時對於血癌K562及Molt4細胞誘導細胞凋亡之影響……...................................................................................31
圖八、HE萃取物處理24小時對於血癌K562及Molt4細胞粒線體完整性之影響…………………………..…..…………………………...33
圖九、HE萃取物處理24小時對於血癌K562及Molt4細胞蛋白半胱天冬酶活化之影響……………………………..……...…………......35

表目錄
表一、三種微藻中脂肪酸組成的差異…………………………………….19
表二、三種海馬不同萃取物處理Molt4及K562細胞24小時IC50值之比較………………………………………………………………....22
表三、將養殖型海馬利用三種不同極性溶劑Hexane、EA及Methanol)萃取物處理Molt4及K562細胞24小時IC50值之比較………….25

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