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通过给大鼠肝细胞培养液中加入乐果(染毒终浓度分别为0、3、10、30、100和300μmol·L-)1,染毒12、24h后,AnnexinV/PI双染法检测肝细胞凋亡率;分别用Fluo-2/AM、双氢-乙酰乙酸二氯荧光黄(DCFH-DA)和罗丹名123检测细胞内Ca2+浓度、活性氧(ROS)和线粒体膜电位(Δψm)变化,并在扫描电镜和荧光显微镜下观察凋亡细胞情况,研究乐果对大鼠肝细胞凋亡的影响。结果表明,肝细胞染毒12和24h后,出现了明显的细胞凋亡的形态学变化,细胞凋亡率明显升高,除3μmol·L-1组外,与对照组相比差异显著(P<0.05或P<0.01),且呈时间-剂量效应。3μmo·lL-1组细胞内Ca2+浓度极显著高于对照组(P<0.01),之后随染毒剂量的增加,细胞内Ca2+浓度逐渐下降;细胞内ROS水平在3~100μmo·lL-1范围内随染毒剂量的增大和染毒时间的延长而升高,而在300μmo·lL-1组略有下降,除3μmo·lL-1组外,与对照组相比均差异极显著(P<0.01);Δψm除24h300μmol·L-1组外均出现持续下降。表明低剂量乐果染毒可诱导肝细胞发生凋亡,细胞内Ca2+、ROS和Δψm可能参与了这一过程。
By adding dimethoate (final concentration of 0, 3, 10, 30, 100 and 300 μmol·L-) to rat hepatocyte culture medium 1, the cells were stained with Annexin V / PI for 12 and 24 hours, respectively The apoptosis rate of hepatocytes was detected by flow cytometry. The intracellular Ca2 + concentration, reactive oxygen species (ROS) and mitochondrial membrane potential (Δψm) were detected by Fluo-2 / AM, DCFH- The changes of apoptotic cells were observed under scanning electron microscopy (SEM) and fluorescence microscopy. The effects of dimethoate on the apoptosis of hepatocytes in rats were studied. The results showed that the morphological changes of apoptotic cells were observed at 12 and 24 h after hepatotoxicity, and the apoptosis rate of hepatocytes increased obviously. Compared with the control group, the difference was significant (P <0.05 or P <0.01) and showed a time-dose effect. The intracellular Ca2 + concentration in 3μmo · L-1 group was significantly higher than that in the control group (P <0.01), and then decreased with the increase of the dose of intracellular Ca2 +. The level of intracellular ROS was in the range of 3-100μmo · L-1 But decreased in 300μmo · L-1 group, except for 3μmo · L-1 group, the difference was significant compared with the control group (P < 0.01); Δψm continued to decline except 24h300μmol·L-1 group. The results showed that low doses of dimethoate induced apoptosis in hepatocytes, and intracellular Ca2 +, ROS and Δψm might be involved in this process.