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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细胞的细胞形态学、生化指标改变和磷酸化的胞外信号调节激酶(p-Erk)的表达。方法用200、400、600、800μmol/L MnCl2的培养液,分别作用对数生长期PC12细胞1、2、3、4 d后,四甲基偶氮塞唑蓝(MTT)筛选锰的细胞毒性剂量;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。免疫印迹法(western blot)检测p-Erk。结果 MTT显示200～800μmol/L MnCl2作用1、2、3、4 d对PC12有显著的抑制作用,呈剂量和时间依赖趋势,600μmol/L MnCl2作用4 d对PC12的抑制率50%;600μmol/L MnCl2作用4 d电镜可见细胞凋亡,同样条件下细胞DNA碎片化;Western blot显示600μmol/L MnCl2作用1、2、3、4 d可见p-Erk2逐渐降低,其中作用2 d时较对照降低75%(n=3,P<0.05),200、400、600μmol/L MnCl2分别作用4 d时,p-Erk亦逐渐降低,当400μmol/L MnCl2作用4 d时较对照明降低78%(n=3,P<0.01);使用Erk通路MEK1/2特异性阻制剂PD98059实验结果表明:锰可能通过MEK1/2磷酸化下游的Erk,下调p-Erk。结论锰对PC12细胞的毒性作用可能是通过关闭胞外信号调节激酶ErK通路诱导细胞凋亡。
OBJECTIVE: To screen the time and dose of manganese on the inhibition of proliferation of nerve cells in mouse model of PC12. To observe the changes of cell morphology, biochemical indexes and phosphorylation of extracellular signal-regulated kinase (p-Erk) expression. Methods The cytotoxicity of manganese was screened by tetramethylazosulfuron (MTT) after treated with 200, 400, 600 and 800 μmol / L MnCl2 for 1, 2, The morphological changes of cells were observed by transmission electron microscope. The effect of MnCl2 on the genomic DNA of PC12 cells was detected by agarose gel electrophoresis. Western blot was used to detect p-Erk. Results MTT showed that PC12 was significantly inhibited by 200 ~ 800μmol / L MnCl2 for 1, 2, 3, 4, and 4 days. The dose-dependent and time-dependent effects of 600μmol / Cell apoptosis was observed by L-MnCl2 for 4 days. DNA fragmentation was also observed under the same conditions. Western blot showed that the expression of p-Erk2 was gradually decreased after treated with 600μmol / L MnCl2 for 1, 2, 3 and 4 days, (P <0.05). After treated with 200,400,600μmol / L MnCl2 for 4 days, p-Erk also decreased gradually. When 400μmol / L MnCl2 for 4 days, the content of p-Erk decreased by 78% = 3, P <0.01). Using the MEK1 / 2 specific inhibitor PD98059 in Erk pathway, it was found that manganese could down-regulate p-Erk by phosphorylating MEK downstream of MEK1 / 2. Conclusion The toxic effects of manganese on PC12 cells may be induced by turning off the extracellular signal-regulated kinase ErK pathway.