锰致PC12细胞凋亡作用及与p-Erk关系  

Relationship between apoptosis and pErk in manganese-treated PC12 cell line

作　　者：徐文[1] 徐强 董大海 缪珊[3] 李金翠 高琨[1] 高丽莉[1] 侯顺利[1] 左晶[1] 刘红[1] 闫文[1] 杨银书[1] 卢娟[1] 

机构地区：[1]兰州军区疾病预防控制中心中心实验室,甘肃兰州730020 [2]兰州市中医医院 [3]中国人民解放军第四军医大学药物研究所 [4]兰州军区第一医院

出　　处：《中国公共卫生》2012年第1期51-53,共3页Chinese Journal of Public Health

基　　金：甘肃省自然基金(3ZS05-A25-093)

摘　　要：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 observe apoptosis related cell morphology,biochemical changes and phosphrylations of phos- phoralated extracellularc signal-regulated kinase （p-Erk）in pheochromocytoma cells （PC12）exposed to manganese at different concentration and exposure time. Methods PC12 cells in logarithm growth period were incubated in culture media with 200,400,600, and 800μmol/L manganese （MnC12 ）for 1,2,3 and 4 days, respectively. The cell viability was examined with 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyl tetrasolium bromide （ MTT ） and morphological changes of PC12 cells were investigated with transmission electron microscope. Agarose gel electrophoresis was adopted to detect the genomic DNA of Mn-treated PC12 cells. Western blot was used to test p-Erk in manganese-treated PC12 cell at different time and concentra- tion of the exposure. Results Manganese at different concentrations could suppress the proliferation of PC12 cells in dose- and time-dependent manner at 1,2,3,4 days ,respectively. The cell inhibited ratio on the fouth day in 600 μmol/L MnC12 group approached 50% or more and the apoptosis was observed with transmission electron microscope as well as biochemi- cal hallmark of DNA fragments. The results of western blot showed that the phosphorylation of Erk of PC12 cells exposed to 600 izmol/L MnC12 increased gradually on the lst,2nd,3rd,and 4th day,respectively. The activation of Erk on the 3rd day was 6. 6 times higher than that of control group （ n = 3, P 〈 0. 05 ）. The phosphorylation of Erk was enhanced by the expo- sures of 200,400 ,and 600 μmol/L MnC12 in PC12 cells within 4 days. The activation of Erk of 400 μmol/L MnC12 treated group at the 4th day was 4. 7 times higher than that of control group（ n = 3,P 〈 0. 05 ）. Conclusion The neuron toxicity of manganese could induce apoptosis in PC12 cells by down-regulaton of p-Erk.

关 键 词：锰 鼠嗜铬神经瘤细胞 氧化应激 凋亡 磷酸化的胞外信号调节激酶 

分 类 号：Q593.1[生物学—生物化学] R135[医药卫生—劳动卫生]