雜交條紋鱸抗菌胜肽Piscidin-1在人類纖維瘤細胞誘導細胞凋亡與細胞壞死 暨 以Cre-loxP技術調控魚類生殖生理之基礎研究與應用

Piscidin-1, an antimicrobial peptide from hybrid striped bass Morone, induces apoptosis and necrosis activity in HT1080 cells & Study and development of Cre-loxP technology in regulation of fish reproduction

林恆如(Heng-Ju Lin)

指導教授 ： 段葉芳

國立臺北科技大學/工程學院/工程科技研究所/博士(2012年)

https://doi.org/10.6841/NTUT.2012.00289

摘要

鱸魚肥大細胞中分離出一條包含22個氨基酸的陽離子胜肽(cationic peptide)，名為Piscidin-1，具有對革蘭氏陽性和陰性細菌的抗菌活性。 目前，尚無任何腫瘤細胞株與piscidin-1抗腫瘤活性的報告。我們嘗試以人工合成piscidin-1，對幾種人類癌症細胞株以細胞活存試驗 (MTS assay)和軟瓊脂聚落形成實驗來測試 piscidin-1的抗腫瘤活性。結果顯示，低劑量的piscidin-1可誘導 HT1080細胞凋亡(apoptosis)和細胞壞死(necrosis);高劑量的piscidin-1則可在短時間內引發HT1080細胞壞死的細胞死亡途徑。我們將細胞以磷脂結合蛋白V (Annexin V)、碘化丙錠 (propidium iodide)、吖啶橙(acridine orange)和溴化乙錠 (ethidium bromide)染色，用穿透式電子顯微鏡觀察發現 piscidin-1對細胞膜破壞的證明。此外，piscidin-1也可以抑制HT1080細胞轉移。在我們的實驗中piscidin-1是第一個研究抗癌活性的抗菌胜肽，對未來用於治療癌症方面的研究深具潛力。

魚類基因轉殖研究至今，尚無法上市，其主要原因在於短欠基因轉殖田間隔離試驗場所，且尚無良好的基礎研究與技術來發展評估生物安全性。 基因轉殖魚的研究中，如何造成轉殖魚的後代不孕，是一個重要的生物安全性考量因子。 魚類在得到基因轉殖魚類後，在水產養殖場中與非基因轉殖魚混養，造成基因互換。 轉殖魚種不孕技術之研發，是保護生態環境重要的一個科技。 不孕之基因轉殖魚的產生，可以使得轉殖魚種逃脫人工飼育環境也無繁殖後代之能力，進而達到單一轉殖魚種無後代之觀念。 本研究以斑馬魚當做實驗魚種，針對魚類生殖生理做一系列研究與技術研，並以AMH（Anti-Müllerian hormone）啟動子在精巢組織與Zp（Zona pellucida）卵巢組織專一性表現之特性，轉殖出於精巢與卵巢組織專一性表現之基因轉殖斑馬魚，配合cre-loxp技術調控細胞凋亡，及gene targeting的技術抑制該基因功能，進而產生不孕之基因轉殖魚種。

關鍵字

抗菌胜肽piscidin-1 ； 細胞毒性 ； 基因轉殖魚 ； 斑馬魚 ； Cre-loxP ； 不孕

並列摘要

Piscidin-1, a 22-residue cationic peptide isolated from mast cells of hybrid striped bass, has potent antimicrobial activities against both gram-positive and -negative bacteria. To date, there is no report of its antitumor activity on any tumor cell lines. In this study, we examined the antitumor activity of a synthetic piscidin-1 peptide against several human cancer cell lines by an MTS assay and soft-agar colony formation assay. Results indicated that a low dosage of piscidin could induce both apoptosis and necrosis in HT1080 cells according to Annexin-V/propidium iodide and acridine orange/ethidium bromide staining, and triggered a necrotic cell death pathway in a short period of high-dosage treatment. The destruction of cell membranes by piscidin-1 was demonstrated by transmission electron microscopy. Furthermore, piscidin-1 could also inhibit the migration of HT1080 cells in a dose-dependent manner. To sum up, this study provides the first evidence of the anticancer activity of the antimicrobial peptide, piscidin-1, and illustrates a promising way to treat cancer.

Transgenic fish cannot seek permission to market is due to the reason that field test research area and basic research technology development for evaluation biosafety are not available. Infertility is a necessary adjunct to the exploitation of transgenic fish unless in completely secure land locked facilities. In addition, infertility is an important parameter in its own right, even aside from its use for containment of GM fish. In this research project, we applied Cre-loxP technology to zebrafish for infertility technology development. We produced fish carrying the plasmid that contained apoptosis genes, and these apoptosis genes are separated by two loxP sites under an ovary-specific promoter, AMH, or ZPC promoter control. After obtaining the transgenic lines with both transgenic fish crossed with each other, the floxed DNA would specifically be eliminated from the female, and apoptosis gene expression would be turned on in ovary cells. The infertility technology can be applied to transgene fish industry.

並列關鍵字

antimicrobial peptides (AMPs), ； piscidin-1 ； cytotoxicity ； transgenic fish ； zebrafish ； Cre-loxP ； infertility

參考文獻

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