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目的探讨鱼藤酮诱发内质网(endoplasmicreticulum,ER)应激的钙离子机制及其超微结构的变化。方法应用流式细胞仪(flowcytometry,FCM)检测鱼藤酮对体外培养大鼠肾上腺嗜铬细胞瘤(PC12)细胞株活性氧的影响,应用激光扫描共聚焦显微镜观察细胞内钙离子变化及透射电镜观察超微结构。结果0·1、1·0、2·0和3·0μmol/L浓度鱼藤酮诱导细胞内活性氧的生成,荧光指数(FI)值分别为1·55±0·17、2·16±0·10、1·77±0·20和1·41±0·12,相同浓度鱼藤酮所致细胞内钙离子荧光强度变换值分别为0·6029±0·0685、1·0902±0·1127、0·7479±0·0820和0·5614±0·0870,分别与对照组比较差异有统计学意义(P<0·01)。当鱼藤酮浓度大于1·0μmol/L时,其作用呈剂量依赖性递减。预先用超氧化物歧化酶(SOD)1200U/ml干预24h,可有效减弱1·0μmol/L鱼藤酮引发的细胞内钙离子升高(P<0·01)。电镜观察到滑面内质网大量增生,粗面内质网轻、中度扩张及脱颗粒。结论鱼藤酮可通过活性氧途径耗竭ER腔钙离子,诱发ER应激,并引起其超微结构的相应变化。
Objective To investigate the mechanism of calcium ion induced by rotenone induced endoplasmic reticulum (ER) stress and its ultrastructure changes. Methods The effect of rotenone on reactive oxygen species in rat adrenal pheochromocytoma (PC12) cell line was detected by flowcytometry (FCM). The changes of intracellular Ca2 + were observed by laser scanning confocal microscopy and electron microscopy microstructure. Results Rotenone induced intracellular reactive oxygen species (ROS) production at 0.1,1.0,2.0 and 3.0 μmol / L, with FI values of 1.55 ± 0.17 and 16.16 ± 0. respectively 10,1 · 77 ± 0 · 20 and 1 · 41 ± 0 · 12, the same concentration of rotenone intracellular calcium fluorescence intensity conversion values were 0.6029 ± 0.0685,1.0902 ± 0.127,0,0 · 7479 ± 0 · 0820 and 0 · 5614 ± 0 · 0870, respectively, compared with the control group, the difference was statistically significant (P <0.01). When rotenone concentrations greater than 1.0 μmol / L, its role in a dose-dependent decline. Pretreatment with superoxide dismutase (SOD) at 1200 U / ml for 24 h effectively attenuated the increase of intracellular Ca2 + induced by rotenone 1.0 μmol / L (P <0.01). Electron microscopy showed a significant proliferation of endoplasmic reticulum, rough endoplasmic reticulum light, moderate expansion and degranulation. Conclusion Rotenone can excrete ER calcium through reactive oxygen species pathway, induce ER stress and induce the corresponding changes of its ultrastructure.