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目的:以鼠嗜铬神经瘤细胞(PC12)为模型,筛选锰对神经细胞增殖抑制作用的时间及剂量,观察锰作用下PC12细胞的氧化应激反应与细胞形态学、生化指标改变和丝裂原活化蛋白激酶pp38(p38MAPKs)的活化表达。方法:用200,400,600,800μmol/LMnCl2的培养液,分别作用对数生长期PC12细胞1,2,3,4d后,用MTT筛选锰的细胞毒性剂量;测定200-600μmol/L MnCl2作用4d后,PC12细胞还原型谷胱甘肽和丙二醛含量;透射电镜观察细胞形态学变化;琼脂糖凝胶电泳检测MnCl2对PC12细胞基因组DNA的影响。western-blot法检测p-p38。结果:MTT实验显示200~800μmol/LMnCl2作用1,2,3,4d对PC12有显著的抑制作用,呈剂量和时间依赖趋势,600μmol/LMnCl2作用4d对PC12的抑制率可达50%以上。200-600μmol/LMnCl2作用于细胞4d后,随着浓度的升高,还原型GSH逐渐降低,MDA的含量逐渐升高;600μmol/LMnCl2作用4d电镜可见细胞凋亡,同样条件下细胞DNA碎片化。Western-blot实验显示600μmol/LMnCl2作用1,2,3,4dp-p38逐渐升高,3d时较对照组增加6.6倍(n=3,p<0.05),200,400,600μmol/L MnCl2作用4d时,磷酸化蛋白38(p-p38)也逐渐升高,400μmol/L MnCl2作用4d时较对照组升高了4.7倍(n=3,p<0.05)。结论:PC12细胞在锰作用下发生氧化应激反应,上调p-p38,诱导细胞凋亡,细胞增殖抑制。
OBJECTIVE: To screen the time and dose of Mn2 + on the proliferation inhibition of PC12 cells in a rat model of PC12. To observe the effects of Mn on the oxidative stress response and cell morphology, biochemical changes and mitosis of PC12 cells Activation of the pro-active protein kinase pp38 (p38MAPKs). Methods: PC12 cells were treated with 200,400,600 and 800μmol / L MnCl2 for 1, 2, 3 and 4 days respectively. MTT was used to screen the cytotoxic dose of manganese. After treated with 200-600μmol / L MnCl2 for 4 days, Glutathione and malondialdehyde, the morphological changes of cells were observed by transmission electron microscope, and the effect of MnCl2 on genomic DNA of PC12 cells was detected by agarose gel electrophoresis. Western-blot detection of p-p38. Results: MTT assay showed that PC12 was significantly inhibited by 200 ~ 800μmol / L MnCl2 for 1, 2, 3 and 4 days, and dose-dependent and time-dependent. After treated with 200-600μmol / L MnCl2 for 4 days, the reduced GSH gradually decreased and the content of MDA gradually increased with the increase of the concentration. Cell apoptosis was observed under the condition of 600μmol / LMnCl2 for 4 days and DNA fragmentation under the same conditions. The results of Western-blot showed that the effect of 600μmol / L MnCl2 on 1,2,3,4dp-p38 increased gradually, that of the control group increased 6.6 times (n = 3, p <0.05) The protein 38 (p-p38) also increased gradually. Compared with the control group, the level of protein 38 (p-p38) was increased by 4.7 times (n = 3, p <0.05) after treated with 400μmol / L MnCl2 for 4 days. Conclusion: PC12 cells undergo oxidative stress reaction under the action of manganese, up-regulate p-p38, induce cell apoptosis and inhibit cell proliferation.