Cu 2+胁迫对罗氏沼虾血细胞毒性及 caspase-3、α-2M基因表达的影响
Effects of Cu 2+ Stress on Hemocytes Toxicity and caspase-3, α-2M Gene Expression in Macrobrachium rosenbergii

中文摘要：本研究以Cu 2+（0、100 μg·L -1）对罗氏沼虾 Macrobrachium rosenbergii进行处理；分别在胁迫后的0 h、3 h、6 h、12 h、24 h和48 h收集血淋巴；检测血细胞中的细胞凋亡率、活性氧（ROS）含量、酯酶活性、一氧化氮（NO）含量以及细胞凋亡执行分子 caspase-3和免疫因子 α-2M基因的表达。结果显示；血细胞凋亡率和ROS含量的变化趋势一致；在胁迫后的6~48 h显著升高（ P<0.05）。酯酶活性在胁迫后的3~48 h均显著下降（ P<0.05）；最低值出现在48 h。在胁迫后的6~48 h；NO含量持续增加；并与对照组的差异有统计学意义（ P<0.05）。 caspase-3的相对基因表达量在胁迫后的6~48 h均显著升高（ P<0.05）；并在24 h达到最高值。在胁迫后的12 h； α-2M基因的表达量显著升高（ P<0.05）并达到峰值；在24 h有短暂降低；但与对照组相比仍显著增加（ P<0.05）；在48 h α-2M基因的表达量显著降低（ P<0.05）。相关性分析显示；血细胞凋亡率和NO以及ROS都呈显著的正相关（ P<0.001）。实验表明；Cu 2+胁迫显著抑制了机体的酯酶活性；诱导NO和ROS的产生以及免疫相关基因 α-2M的表达来对抗Cu 2+胁迫带来的损伤。随着胁迫时间的延长；过高浓度的NO和ROS对机体自身造成了伤害；并诱导凋亡相关基因 caspase-3的表达；最终导致细胞凋亡；说明Cu 2+对虾类具有免疫抑制性和毒性。NO和ROS的升高可能是细胞凋亡的主要诱因。
英文摘要：The present study analyzed the toxic effects of Cu 2+ on apoptotic cell ratio, ROS production, esterase activities, NO production, and the expression levels of caspase-3 and α-2M in hemocytes of Macrobrachium rosenbergii. The results showed that the changes of apoptotic cell ratio were consistent with ROS production which was increased significantly ( P<0.05) after exposure for 6~48 h. Esterase activities were significantly decreased ( P<0.05) at 3~48 h and the minimum was observed at 48 h. The NO production continued increasing and the level became significant difference ( P<0.05) after exposure for 6~48 h. The relative expression levels of caspase-3 were significantly induced ( P<0.05) after exposure for 6~48 h, and reached the peak at 24 h. The relative expression of α-2M was significantly increased ( P<0.05) and reached the peak at 12 h. Moreover, the expression level of α-2M reduced after exposure for 24 h and became significantly different( P<0.05) at 48 h. Correlation analysis showed that significant positive correlation ( P<0.001) between apoptotic cell ratio and ROS production was observed, as well as apoptotic cell ratio and NO production. All these results suggested that esterase activities were significantly inhibited after Cu 2+ exposure, while NO and ROS production as well as α-2M expression were induced against Cu 2+ exposure. Along with stress time increasing, overfull production of ROS and NO caused oxidative stress to prawn and then induced the expression level of caspase-3 which finally resulted in apoptosis, indicating that Cu 2+ stress may have immunosuppressive and toxic effects to M. rosenbergii, and the increased production of NO and ROS might be the main cause of apoptosis. 2017,36(3): 293-299 收稿日期：2017-02-08 DOI：10.11984/j.issn.1000-7083.20170035 分类号：X503.225;Q959.223