N-乙酰半胱氨酸和微囊藻毒素-LR联合暴露对中国仓鼠卵巢细胞凋亡的影响

Effects of N-acetylcysteine and microcystin-LR in combination on apoptosis of Chinese hamster ovary cells

目的研究N-乙酰半胱氨酸(NAC)和微囊藻毒素-LR(MC-LR)联合暴露对中国仓鼠卵巢(CHO)细胞凋亡的影响。方法取对数生长期细胞,分别加入0(对照)、1、5、10、20、30、40、50、60、80 mmol/L的NAC溶液以及终浓度为0、1、5 mmol/L的NAC溶液+终浓度为0、2.5、5、10μg/ml的MC-LR溶液培养24 h。采用四甲基偶氮唑蓝(MTT)试验检测细胞活性,采用2,7-二氯荧光乙酰乙酸盐(DCFH-DA)法检测细胞内活性氧(ROS)含量的变化,采用四氯四乙基苯并咪唑基羰花青碘化物(JC-1)荧光探针法检测线粒体膜电位的变化,采用膜联蛋白V-异硫氰酸荧光素/碘化丙啶(Annexin VFITC/PI)双染法检测细胞凋亡率。结果 1、5 mmol/L NAC单独作用对细胞活性无明显抑制作用,且细胞存活率均在80%以上;NAC与MC-LR同时作用时,细胞活性增加,ROS含量减少,线粒体膜电位升高,凋亡率减少。结论 MC-LR可诱导CHO细胞产生大量ROS从而引起细胞凋亡,抗氧化剂NAC可拮抗MC-LR诱导ROS生成导致的细胞凋亡。

Objective To study the effects of N-acetylcysteine （NAC） and microcystin-LR in combination on apoptosis of Chinese hamster ovary cells. Methods The logarithmic phase CHO cells were used and treated by different concentrations of NAC （0,1,5,10,20,30,40,50,60 and 80 mmol/L） alone,simuhaneously treated by NAC （0,1,5 mmol/L） and MC-LR （0,2.5,5,10 tLg/ml） for 24 h. The changes of cell viability were determined by MTr method. The changes of ROS in CHO cells were measured by DCFH-DA. The changes of mitochondrial membrane potential （MMP） were judged by a fluorescent probe JC-1 staining. The apoptosis rate of CHO ceils was measured by Annexin V-PI staining. Results After CHO cells were exposed to NAC alone for 24 h, cell viability remained at more than 80% at the concentrations of 1,5 mmol/L NAC. After CHO cells were treated by different concentration of NAC and MC-LR together,cell viability increased,ROS content decreased, mitochondrial membrane potential elevated, and the apoptosis of cells was inhibited at a certain extent. Conclusion MC-LR can induce the apoptosis of CHO cells by creating plenty of reactive oxygen species ,which can be antagonized by antioxidant NAC. Key words： Microcystin-LR; N-acetylcysteine ; Reactive oxygen species ; Apoptosis ; Chinese hamster ovary ceils