甲醇对花鲈动脉球和脑细胞系细胞形态、活力及凋亡相关基因的影响

Effectsofmethanolonmorphology,viabilityandapoptosis-related genes in cells of arterial bulb and brain cell lines system of Lateolabrax maculatus

【目的】从细胞水平探究甲醇对花鲈(Lateolabrax maculatus)不同细胞系的影响,为揭示微塑料(甲醇为溶剂)对鱼类细胞的毒理作用打下基础。【方法】在体外分别以1%、3%、5%、7%、9%和11%的甲醇处理花鲈动脉球和脑细胞系细胞,倒置显微镜观察细胞形态变化,MTT检测细胞活力,通过实时荧光定量PCR检测半胱氨酸天冬氨酸蛋白酶3(Caspase3)、半胱氨酸天冬氨酸蛋白酶9(Caspase9)、Bcl2关联X蛋白(Bax)和B细胞淋巴瘤-2(Bcl-2)等细胞凋亡相关基因,并采用Caspase3活性检测试剂盒检测Caspase3活性。【结果】当甲醇体积分数超过7%后,花鲈动脉球细胞系细胞变圆缩小,失去贴壁能力,且出现死亡细胞;细胞活力呈显著降低趋势(P<0.05,下同),Caspase3、Caspase9、Bax和Bcl-2基因相对表达量均显著上调,Caspase3活性极显著升高(P<0.01,下同)。当甲醇体积分数超过1%后,花鲈脑细胞系细胞间出现空隙且细胞有回缩趋势,贴壁细胞数量明显减少,存在大量悬浮的死亡细胞;细胞活力显著降低,细胞凋亡相关基因的相对表达量均呈显著上调趋势,且随甲醇体积分数的增加其相对表达量呈上升趋势;甲醇对花鲈脑细胞系细胞Caspase3活性的影响呈浓度依赖性,即细胞Caspase3活性随甲醇体积分数的增加而升高,经5%~11%甲醇处理后极显著升高。【结论】甲醇可显著上调花鲈动脉球和脑细胞系细胞中的Caspase3、Caspase9、Bax和Bcl-2基因表达水平,并提高Caspase3活性。甲醇对不同种类细胞的毒性不同,尤其对脑细胞的毒性更强。因此,在选择甲醇为微塑料溶剂时应根据不同种类细胞配制不同的安全浓度。

【Objective】To investigate the effects of methanol on the morphology and viability on different cell lines of spotted sea bass(Lateolabrax maculatus)at the cellular level,and to lay the foundation for revealing the toxicological effects of microplastics(methanol as solvent)on fish cells.【Method】In vitro,different volume fractions of methanol(1%,3%,5%,7%,9%,11%)were used to treat the cells of arterial bulb and brain cell lines of L.maculatus,and the changes of cell morphology were observed by inverted microscopy,cell viability was detected by MTT,and apoptosisrelated genes like cysteine aspartate protease 3(Caspase3),cysteine aspartate protease 9(Caspase9),Bcl2-associated X protein(Bax)and B-cell lymphoma-2(Bcl-2)were detected by real-time fluoresceence quantitative PCR,and Caspase3 activity was detected by Caspase3 activity assay kit.【Result】When the volume fraction of methanol was more than 7%,the cells of arterial bulb cell line of L.maculatus became rounded and shrunk,lost the ability to adhere to the wall,and appeared dead cells;the cell viability showed a significant decrease(P<0.05,the same below),the relative expression of Caspase3,Caspase9,Bax and Bcl-2 genes were significantly up-regulated,and Caspase3 activity was highly significantly increased(P<0.01,the same below).When the volume fraction of methanol was more than 1%,there were gaps between the cells of the brain cell line and the cells tended to retract,the number of adherent cells was greatly reduced,and there were a large number of suspended dead cells;cell viability was significantly reduced,and the relative expression of apoptosisrelated genes were significantly up-regulated,and their relative expression tended to increase with the increase of methanol volume fraction;the effect of methanol on the caspase3 activity of the cells of the brain cell line was concentrationdependent.The caspase3 activity increased with the increase of methanol treatment,and it was highly significantly increased by 5%-11%methanol treatment.【Conclusion】Methanol significantly up-regulats the expression levels of Caspase3,Caspase9,Bax and Bcl-2 genes and increases Caspase3 activity in cells of arterial bulb and brain cell lines of L.maculatus,indicating that methanol has different toxicity levels to different cell types,and it is especially toxic to brain cells.Therefore,different safe concentrations of methanol should be formulated according to different kinds of cells when choosing methanol as a solvent for microplastics.