水產鏈球菌胞外產物分離之促細胞增生物質定性研究

The Characterization of The Mitogenic Factor Isolated from Extracellular Products of Aquatic Streptococcaceae

林洺佐(Ming-Tzuo Lin)

指導教授 ： 陳媺玫

國立臺灣大學/獸醫專業學院/獸醫學研究所/碩士(2009年)

https://doi.org/10.6342/NTU.2009.03375

摘要

由細菌產生，分泌至細胞外的物質通稱為胞外產物（extracellular product, ECP），多種魚類病原菌及其他動物來源的鏈球菌會分泌出的胞外產物都已證實為重要的致病因子。而本實驗室先前進行鏈球菌之ECP進行細胞毒性研究時，發現其具有促使細胞增生之效果。此外前人之研究也發現多種不同的病原菌也會分泌促使細胞增生的物質，如Pasteurella multocida、Bartonella hanselae和Helicobacter pylori等。本論文以Lactococcus garvieae之 ECP為研究對象，進行具有細胞增生特性之ECP的特性分析，並推測與疾病的相關性。本研究使用透析膜覆蓋法收取臨床分離株的粗製ECP，以快速蛋白質液相層析（Fast protein liquid chromatography, FPLC）純化分離有增生功能的ECP，再進行毛細管電泳（capillary electrophresis, CE）及串聯式液相層析質譜儀（liquid chromatography-mass spectrometry, LC/MS）分析具有增生特性的物質，並使用魚類來源細胞株TO-2為主要測試細胞，以MTT試驗量化ECP及純化增生物質的細胞增生效果，並計算細胞增生百分比，以及測試ECP對巨噬細胞的趨化活性。結果顯示ECP具有細胞增生的特性並具有巨噬細胞的趨化活性，而經由FPLC純化後的ECP具有細胞增生特性的部份為一小分子量的物質，該物質偵測不出蛋白質的含量，因此認為此種促細胞增生物質並非蛋白質，而是胜

關鍵字

鏈球菌 ； 胞外產物

並列摘要

Bacteria secreted substances into environment are named extracellular products ( ECPs ). They are important virulence factors of many fish pathogens including Streptococcus spp. Most pathogenic bacteria and their products are tend to cause cytotoxic effect of cell, but some may have the mitogenic effect to induce cell proliferation, such as Pasteurella multocida, Bartonella hanselae and Helicobacter pylori etc. It was found that the ECP of streptococcus isolated from fish have mitogenic effect causing cell proliferation. Therefore, the this study focuses on the biology activity of the ECP of Lactococcus garvieae, including the mitogenic activity on fish cell line and chemotaxis effect on fish macrophage, to realize the relationship between ECP and pathogenic mechanism. First, ECP was produced by culturing L. garvieae on dialysis-membrane overlays, and purified using fast protein liquid chromatography (FPLC) technique purify the crude ECP, and then collecting the fractionated ECP. The fractions shown significant cell proliferation effect were further analyzed by using capillary electrophoresis (CE) and liquid chromatography-mass spectrometry (LC/MS). Mitogenic activity of ECP on fish cell line ( TO-2 ) was evaluated by MTT colorimetric assay. To demonstrate macrophage chemotaxis effect, we count the number of migrated macrophage with chemotaxis assay. The results showed that the ECP of L. garvieae significantly promoted TO-2 cell proliferation and caused macrophage migration. The biological activity with mitogenic and chemotaxis effect was found to be a group of low molecular weight peptides. The electrical neutral peptides analyzed with CE and LC/MS have been shown to be similar to the fragment of plantaricin SA6 secreted by Lactobacillus plantarum. Two peptides (ECP-P1, ECP-P2) were chemically synthesized demostrated TO-2 cell mitogenic activity and macrophage chemotaxis effect. Cell proliferation effect shows no dose dependency with the peptide concentration. However, low concentration (1 ppm) showed better mitogenic effect than high concentration. On the other hand, the chemotaxis effect was dose dependent on concentration. High concentration can cause more macrophage migration than low one.

並列關鍵字

Lactococcus ； ECP

參考文獻

林鈺倩。魚類來源鏈球菌胞外產物之促細胞增生效果。碩士論文，台灣大學獸醫

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