维氏气单胞菌对草鱼背鳍细胞的毒性及凋亡研究

Study on Cytotoxicity and Apoptosis of Aeromonas veronii to Grass Carp Pectoral Fin Cell

草鱼(Ctenopharyngodon idellus)在我国是一种重要的淡水经济鱼类,但是由于近些年的集约化高密度养殖导致各种水产疫病暴发,不仅对养殖行业造成巨大的损失,同时对人类生命安全也存在着巨大威胁,维氏气单胞菌(Aeromonas veronii)就是其中一种人畜共患的条件致病菌。本研究旨在探索维氏气单胞菌导致宿主细胞死亡的致死机制,为今后进一步针对维氏气单胞菌开发抗菌渔用功能产品提供数据支持和理论依据。本文首先测定维氏气单胞菌胞外分泌物(Extracellular products,ECPs)的蛋白浓度,然后以不同浓度的ECPs为基准,根据细胞光镜观察结果及细胞活力检测结果确定维氏气单胞菌胞外ECPs的细胞毒性;再利用Hoechst 33342及TdT mediated dUTP Nick End Labeling(TUNEL)检测细胞核的变化情况,最终确定草鱼源维氏气单胞菌对宿主细胞的毒性致死机制。从毒性实验中可以看出高浓度的维氏气单胞菌ECPs对草鱼背鳍(Grass carp pectoral fin,GCPF)细胞具有明显的毒性,且从Hoechst 33342结果可以看到凋亡小体的出现,而TUNEL也出现了阳性结果,实验组出现了绿色荧光,而对照组没有出现荧光。说明维氏气单胞菌胞外分泌物对宿主细胞具有明显的毒性,而且会导致宿主细胞发生细胞凋亡。

Grass carp(Ctenopharyngodon idellus)is an important commercially cultured freshwater fish in China.In recent years,with the advent of intensive aquatic farming,various diseases emerged in commercial populations,which caused enormous economic loss,even threatened human safety.Aeromonas veronii is one of the zoonotic pathogens.This study aimed to explore the lethal mechanism of A.veronii causing to host cell death and provide data supports and theoretical basis for further development of anti bacterial fishery functional products against A.veronii.This paper first determined the protein concentration of extracellular products(ECPs)of A.veronii.Then,based on the ECPs of different concentrations,the cell toxicity of extracellular ECPs of A.veronii was determined according to the results of light microscopy and cell viability.Hoechst 33342 and TdT mediated dUTP Nick End Labeling(TUNEL)were used to detect the changes of nucleus,and finally the toxic lethal mechanism of A.veronii to host cells was determined.It can be seen from the toxicity experiment that high concentrations of A.veronii ECPs have obvious toxicity to grass carp pectoral fin(GCPF)cells,and the appearance of apoptotic bodies can be seen from Hoechst 33342 results.Meanwhile,TUNEL showed positive results with green fluorescence in test group and no fluorescence in the control group.It is indicated that A.veronii ECPs is obviously toxic to host cells and can cause apoptosis of host cells.