- 期刊
魚類來源乳酸鏈球菌胞外產物之促細胞增生物質特性分析
Mitogenic Activity of Extracellular Products from Fish Lactococcus spp
摘要
胞外產物(extracellular products, ECP)是多種病原菌重要的致病因子且已知對細胞有細胞毒性(cytotoxic effect)，但有些細菌的ECP具有促進細胞增生的效果，本實驗室在鏈球菌的ECP中亦發現有此特性。故本論文的目標在初步探究魚類來源鏈球菌ECP中促使細胞增生之物質的特性，及其對體外培養細胞與魚類免疫細胞的增生效果。以透析膜覆蓋法收取分離自發病魚的鏈球菌菌株之ECP，使用吳郭魚卵巢細胞(TO-2)為主要測試細胞，以MTT (3-[4, 5-dimathylthiazol-2-yl]-2.5-diphenyltetrazolium bromide)試驗來量化ECP之促細胞增生效果，計算細胞增生百分比。結果顯示，鏈球菌ECP沒有明顯細胞毒性，在稀釋後都有促進TO-2增生的效果，將ECP以攝氏99℃加熱150分鐘、或以proteinase K處理或通過3 kDa分子篩之濾液皆不會影響其促細胞增生效果，且在細胞培養液中含有胎牛血清時，ECP才有促細胞增生效果。對吳郭魚周邊血管白血球及多種魚類及甲魚來源細胞株都有促增生效果，而對哺乳動物來源之細胞株Balb-3T3與BHK則無。魚類來源鏈球菌之ECP中含有促進魚類來源細胞株增生的物質，以膠體過濾進行此物質的初步純化，此物質有耐熱、不受proteinase K影響、分子量約為0.9-1.9 kDa的性質。
並列摘要
Extracellular products (ECP) are important virulence factors of most pathogenic bacteria and were proved to cause cytotoxic effect to cell, but some possess the mitogenic effect to induce cell proliferation. It was found that the ECP of Lactococcus spp. isolated from fish didn't cause cytopathic effect but enhance cell proliferation, contrary. Therefore, the aim of this study was to investigate initially the mitogenic activity and the characteristics of mitogenic factor(s) of Lactococcus garviae. ECP was harvested by culturing the bacteria on dialysis-membrane overlays, and mitogenic activity of ECP on tilapia ovary cell (TO-2) was evaluated by MTT colorimetric assay. The mitogenic activity was observed only when serum was added in the culture medium of tested cell and would not be affected when ECP was heated at 99℃, treated with proteinase K, and filtered through 3 kDa molecular weight cut-off. Furthermore, the growth of peripheral blood leukocyte of tilapia and all fish and softshell turtle cell lines studied in this research were stimulated by the ECP produced by Lactococcus spp. and Streptococcus sp. but the mammalian cell lines, such as Balb/3T3 and BHK, were not. The ECP was preliminarily purified by gel filtration and the mitogenic effect presented strongly in fraction 21, which posed the characteristics of thermo-resistance, unaffect by proteinase K and molecular weight about 0.9-1.9 kDa.