| Summary: | 碩士 === 國立臺灣大學 === 獸醫學研究所 === 91 === Extracellular products (ECP) are important virulence factors of many fish pathogens including Streptococcus spp., most pathogenic bacteria and their products were proved to cause cytotoxic effect to cell but some possess the mitogenic effect to induce cell proliferation, such as Pasteurella multocida and Helicobacter pylori etc. It was found that the ECP of streptococcus isolated from fish didn’t cause cytopathic effect but the mitogenic effect causing cell proliferation was observed contrary. Therefore, the aim of this study was to investigate initially the mitogenic activity of streptococcus ECP on various fish cell line and fish leukocyte and the characteristics of mitogenic factor(s). ECP was harvested by culturing the streptococcus on dialysis-membrane overlays. Mitogenic activity of ECP on fish cell line (TO-2) was evaluated by MTT colorimetric assay. ECP of Streptococcus spp. promoted TO-2 cell proliferation significantly reaches 150% compared to PBS control (100%). ECP could enhance DNA synthesis, stimulate cell proliferation and influence cell growth curve. The mitogenic activity was observed only when serum was contained in the medium and would not be affected when ECP was heated at 99oC, treated with proteinase K, and filtered through 3kDa molecular weight cut-off. Furthermore, the growth of peripheral blood leukocyte of tilapia and all fish cell lines studied stimulated by ECP but the mammalian cell lines, such as Balb/3T3 and BHK, were not. The effect on plating efficiency and ability to grow on soft agar were not elevated after long-term treatment of ECP (24 passages) on TO-2 cells. These results indicate that the ECP from fish streptococcus possess mitogenic factor(s), which enhance the proliferation of fish cell lines. 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.
|
|---|
