锰诱导PC12细胞凋亡与p-38MAPKs的关系

Relationship between apoptosis and molecular mechanism of p-38 MAPKS activation in manganese-treated PC12 cells

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 ,观察细胞形态学、细胞周期和生化指标改变与丝裂原活化蛋白激酶 (p38MAPKs)活化表达间的关系。方法 用 2 0 0 ,4 0 0 ,6 0 0 ,80 0μmol/LMnCl2 的培养液 ,分别作用对数生长期PC12细胞 1,2 ,3,4d后 ,用噻唑蓝比色 (MTT)筛选锰的细胞毒性剂量 ;流式细胞仪检测细胞周期分布 ;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。蛋白印迹 (western -blot)法检测 p -p38。结果 MTT实验结果显示 ,2 0 0～ 80 0 μmol/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 ,6 0 0 μmol/LMnCl2 作用 4d对PC12的抑制率可达 5 0 %以上。流式细胞仪检测实验表明 :6 0 0 μmol/LMnCl2 作用 4d将PC12细胞周期阻滞在S期 ,诱导细胞凋亡 ,与电镜结果一致 ,同样条件下细胞DNA碎片化。Western -blot实验显示 6 0 0 μmol/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 0 5 ) ,2 0 0 ,4 0 0 ,6 0 0 μmol/LMnCl2 作用 4d时 ,磷酸化蛋白 38(p - p38)也逐渐升高 ,4 0 0 μmol/LMnCl2 作用 4d时较对照组升高 4 7倍 (n =3,P <0 0 5 )。结论 锰通过MEK3/

Objective To employ PC12 cell line as a model in vitro to test the concentration and time of maganese on proliferation arrest,and to o bserve the changes of cell morphology,cell cycle and biochemical marker and furt her to study the relationships between above aspects and activiations of p38MAPK s pathway.PC cells in logarithm period incubated in culture media with 200,400,600,800?μmol /L manganese (MnCl 2) for 1,2,3,4 days respectively.Methods Cell viability was examined by MTT.Cell cycle was m onitored by FCM (flow cytometry).Morphological changes of PC12 cells was investi gated by transmisssion electron microscope.Agarose gel electrophoresis was used to test the genomic DNA. Western-blot was used to test p-p38.Results MTT revealed that 200,400,600,800?μmol/L MnCl 2 could suppress the proliferation of PC12 cells in dose and time-dependent tren d.The cell inhibited ratio on the fourth day in 600?μmol/L MnCl 2 approached 50% or more.FCM showed that the cell cycle of the PC12 cells could be inhibite d in S period at the concentration of 600?μmol/L MnCl 2 on the 4th day and ap optosis was observed through FCM and transmisssion electron microscope Biochem ical hallmark of DNA fragments was observed.Western-blot tests showed the p-p38 in 6 00?μmol/L MnCl 2 culture medium was increasing gradually on the 1st,2nd,3rd a nd 4th day.the p-p38 on the 3rd day was 6.6 times higher than that of control group (n=3,P<0.05).The p-p38 was enhanced by degrees in 200,400,600?μ mol/ L MnCl 2-treated PC12 cells in 4 days.The p-p38 of 400?μmol/L MnCl 2 treated group on the 4th day was 4.7 times higher than that of control group (n=3, P<0.05).Conclusion Manganese upregulated p-p38 through MEK3/6 dow nstream to induce proliferation arrest and apoptotic cell death.