锰作用下PC12细胞的氧化应激机制研究

Oxidative stress mechanism of manganese-treated PC12 cell line

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 observe oxidative stress reaction of Mn-treated PC12 cell,cell morphology,biochemical changes and mitogen activated protein kinases（MAPKs） phosphorylated protein-38（p-p38）.Methods：PC12 cells in logarithm period incubated in culture media of 200,400,600,800μmol/L manganese（MnCl2） for 1day,2 days,3 days,4days respectively;The cell viability was examed by MTT [3-（4,5-Dimethylthiazol-2-yl）-2,5-diphenyl tetrasoliumBromide];Reduced glutathion and malonaldehyde（MDA） of PC12 cell were tested in 4days of Mn-treatment under 200-600μmol/L;morphological changes of PC12 cells was investigated by transmisssion electron microscope;Agarose gel electrophoresis tested the genomic DNA of Mn-treated PC12 cells.Western-blot was used to test p-p38 of manganese-treated PC12 cell in different time courses and concentrations.Results：The results of MTT revealed that manganese of different Concentrations（MnCl2 200,400,600,800μmol/L） could suppress the proliferation of PC12 cells in dose and time-dependent manner during 1d,2d,3d,4d respectively.The cell inhibited ratio on the fouthday in 600μmol/L MnCl2 culture medium approached 50% or or more.Reduced glutathion was decreased and MDA increased under the treatment of 200-600μmol/L MnCl2.In the same condition apoptosis was observed in transmisssion electron microscope as well as biochemical hallmark of DNA fragments.Western-blot tests show The phosphorylation of p38 of PC12 cells incubated in 600μmol/L MnCl2 culture medium was increasing gradually on the 1st,2nd,3rd and 4th day respectively.The activation of p38 of PC12 cells on the 3rd day is 6.6 times higher than that of control group（n=3,p0.05）.The phosphorylation of p38 enhanced by degrees with 200,400,600μmol/L MnCl2 treated PC12 cells in 4days.The activation of p38 of 400μmol/L MnCl2 treated group on the 4th day is 4.7times higher than that of control group（n=3,p0.05）.Conclusions：Mn-treatment has generated oxidative stress of PC12 cell,up-regulated p-p38,induced apoptosis and proliferation arrested.