米氏凯伦藻对鱼类FHM细胞的毒性作用及其致死机制

探讨米氏凯伦藻培养液(Karenia mikimotoi culture solution,KMCS)及细胞提取物(Cell extracts,KMCE)对胖头鲤(Fathead minnow,FHM)细胞的毒性作用及致死机制。用光学显微镜观察细胞形态变化来分析KMCS和KMCE对FHM细胞的毒性作用,并用CCK法(Cell counting kit)分析KMCS和KMCE对FHM细胞活力的影响;用DNA特异性染料Hoechst 33342检测FHM细胞的细胞核变化,分析KMCS及KMCE是否导致FHM细胞发生程序性死亡及凋亡小体的形成;检测caspase-3的活性,分析KMCS和KMCE是否导致相关凋亡程序的发生。结果发现,用稀释两倍的KMCS和KMCE分别处理FHM细胞4h后,观察到FHM细胞形态均出现明显改变;将未稀释和稀释两倍的KMCS以及稀释两倍的KMCE,分别与FHM细胞孵育,4h后用CCK法检测细胞活性,发现FHM细胞的细胞存活率分别下降了63.6%,22.2%和26.7%。Hoechst 33342染色结果表明,KMCS和KMCE都可以导致FHM细胞中出现凋亡小体,对凋亡相关蛋白因子caspase-3活性的检测,结果显示实验组细胞caspase-3活性水平显著升高,分别为对照组的2.32倍和1.49倍。KMCS和KMCE对鲤鱼FHM细胞生长具有明显的细胞毒性,会导致细胞发生细胞凋亡。

Toxicity and Lethal Mechanisms of Karenia mikimotoi on Fish FHM Cells

The toxic and lethal mechanisms of Karenia mikimotoi culture solution (KMCS) and Cell extracts (KMCE) on Fathead minnow (FHM) cell were explored.The morphological changes of cells were observed by light microscopy to analyze the toxic effects of KMCS and KMCE on FHM cells,and the effect of KMCS and KMCE on the viability of FHM cells was analyzed by CCK method.The nuclear specificity of FHM cells was detected by DNA specific dye Hoechst 33342 to analyze whether KMCS and KMCE caused programmed cell death and the formation of apoptotic bodies in FHM cells.The activity of caspase-3 was detected to analyze whether the occurrence of relevant apoptosis programs in FHM cells was caused by KMCS and KMCE.The results showed that significant morphological changes were observed in FHM cells after being infected with two times diluted of KMCS and KMCE for 4 h.Undiluted and diluted KMCS and twice diluted KMCE were incubated with FHM cells,and cell viability was measured by CCK method 4 h later.The cell viability of FHM cells was decreased by 63.6%,22.2% and 26.7%,respectively.The results of Hoechst 33342 staining showed that both KMCS and KMCE could lead to the occurrence of apoptosis bodies in FHM cells.The detection of apoptosis-related protein factor caspase-3 activity showed that the activity level of caspase-3 in the experimental group was significantly increased,which was 2.32 times and 1.49 times of that of the control group,respectively.KMCS and KMCE have obvious cytotoxic effect on the growth of cyprinid fish FHM cells,which will lead to the apoptosis of the cells.