柱孢藻毒素对草鱼淋巴细胞毒效应及氧化损伤机理研究

针对淡水水体拟柱孢藻水华产生的柱孢藻毒素,以我国典型食用鱼种草鱼(Ctenophar yngodon idellus)为实验材料,研究了其对鱼体免疫细胞毒效应及机理.结果表明,随着体外暴露剂量的增加,草鱼淋巴细胞活性逐渐降低,暴露24h后100μg·L-1柱孢藻毒素诱导组细胞活性仅为对照组的20%;对诱导组淋巴细胞的DNA检测发现呈现阶梯状电泳的典型细胞凋亡特征;应用流式细胞仪进一步检测了细胞凋亡率,表明1～100μg·L-1柱孢藻毒素均能够诱导细胞凋亡,并且其凋亡毒效应呈现明显的时间-效应和剂量-效应关系;诱导12h后,氧化应激产物活性氧(ROS)和丙二醛(MDA)含量均明显上升,100μg·L-1柱孢藻毒素诱导组ROS含量为对照组的2.5倍,MDA含量为对照组的2倍,诱导24h后,1、10、50和100μg·L-1实验组氧化应激产物含量仍然明显上升,这说明氧化应激是柱孢藻毒素导致草鱼淋巴细胞DNA损伤的重要原因;RT-PCR技术对重要凋亡基因的检测表明,1～100μg·L-1实验组凋亡促进基因Bax表达显著增强(p<0.05),50和100μg·L-1高剂量组凋亡抑制基因Bcl-2表达显著降低(p<0.01).本研究从细胞水平揭示柱孢藻毒素对草鱼免疫细胞具有明显的毒性,该毒效应通过氧化应激介导的DNA损伤表现,凋亡是柱孢藻毒素对草鱼免疫细胞毒性的主要机制.

Cytotoxicities and oxidative damage mechanisms of Ctenopharyngodon idellus lymphocytes to cylindrospermopsin

The cytotoxicities and mechanisms of cylindrospermopsin (CYN) on the isolated Ctenopharyngodon idellus lymphocytes in vitro were studied. Lymphocyte cell viabilities significantly decreased with the increasing of exposure doses, and the viability of lymphocytes induced by 100 μg · L-1 CYN in 24 hours was only 20% of control group. DNA analysis by agarose gel electrophoresis for CYN induced lymphocytes revealed DNA ladder which is the most important characteristic of apoptosis. Then, the results of flow cytometry analysis further showed that the apoptosis percentage of lymphocytes induced by CYN in a time- and dose-dependent manner. Reactive oxygen species (ROS) and Malondialdehyde (MDA) analysis found their levels increased after 12 hours exposure to CYN. The concentrations of ROS and MDA for 100 μg · L-1 exposure group were 2.5 and 2 folds higher than that of the control group, respectively. The concentrations of ROS and MDA further increased for 1, 10, 50 and 100 μg · L-1 exposure group after 24 hours. These results confirmed that oxidative stress is the main mechanism of Ctenopharyngodon idellus lymphocytes DNA damage induced by cylindrospermopsin. RT-PCR analysis found that the expressions of Bax gene in concentration of 1~100 μg · L-1 exposure group were significantly increased (p<0.05), and the expressions of Bcl- 2 gene for 50 and 100 μg · L-1 exposure group were significantly decreased (p<0.01). There results demonstrated that the cytotoxicities of CYN on Ctenopharyngodon idellus lymphocytes were mainly performed by apoptosis mechanism with DNA damage resulted by oxidative stress.