摘要
目的利用原代培养的小鼠脑星形胶质细胞（astrocytes,AS）,探讨c-Jun氨基末端激酶（JNK）信号转导通路是否参与镉致小鼠脑星形胶质细胞凋亡的过程。方法原代培养的小鼠脑星形胶质细胞给予0-20μmol/L镉染毒0-24 h,倒置相差显微镜观察细胞形态变化;给予0-20μmol/L镉染毒9、12 h,采用MTT法检测细胞存活率;给予0-20μmol/L镉染毒12 h,采用Annexin V-FITC/PI双染流式细胞术检测细胞凋亡率;给予0-20μmol/L镉染毒0-12 h,采用蛋白印迹（Western blotting）法检测JNK磷酸化蛋白的表达水平。结果镉浓度为5-20μmol/L时,随染毒剂量的升高及作用时间的延长,细胞形态发生明显改变：细胞间网络连接松散,突起消失,胞体收缩成球形,贴壁能力下降,最终脱落,漂浮于培养液中。与对照组比较,各浓度镉染毒9、12 h后小鼠脑星形胶质细胞的存活率均下降,差异有统计学意义（P〈0.01）;且随着镉染毒浓度的升高和染毒时间的延长,小鼠脑星形胶质细胞的存活率均呈下降趋势。与对照组比较,各浓度镉染毒12 h后小鼠脑星形胶质细胞的凋亡率均升高,差异有统计学意义（P〈0.01）;且随着镉染毒浓度的升高,小鼠脑星形胶质细胞的凋亡率均呈上升趋势。与对照组比较,5-20μmol/L镉染毒3 h后小鼠脑星形胶质细胞内p-JNK表达量均升高,20μmol/L镉染毒6 h后小鼠脑星形胶质细胞内p-JNK表达量升高,10-20μmol/L镉染毒9、12 h后小鼠脑星形胶质细胞内p-JNK表达量均升高,差异有统计学意义（P〈0.05）;与0 h比较,0-20μmol/L镉染毒3-12 h后小鼠脑星形胶质细胞内p-JNK表达量均升高,差异有统计学意义（P〈0.05）;且随着镉浓度的升高和染毒时间的延长,小鼠脑星形胶质细胞内p-JNK的表达量均呈上升趋势。结论镉可能通过上调JNK磷酸化蛋白的表达水平来诱导小鼠脑星形胶质细胞的凋亡。
Objective To understand whether the c-Jun N-terminal kinase（JNK） signal transduction pathway participates in the process of cadmium-induced mice astrocytes apoptosis. Methods The primary cultured mice astrocytes were treated with cadmium at the doses of 0-20 μmol/L, for 0-24 h,and the morphological changes of cells were observed by inverted phase contrast microscope. Astrocytes were exposed to 0-20 μmol/L cadmium for 9,12 h,then cell survival rate was detected by MTT method;Astrocytes were exposed to 0-20 μmol/L cadmium for 12 h and the apoptosis rate was detected by Annexin V-FITC/PI flow cytometry. Astrocytes were treated by 0-20 μmol/L cadmium for 0-12 h,the expression level of JNK phosphorylated protein was detected by Western blotting. Results With the increase of exposure dose and prolongation of exposure time,the cells morphology changed obviously：cell network connection was loose,neuronal processes vanished,cell body shrank into sphericity,and the adherent ability decreased,eventually fall off,floating in the culture medium. Compared with the control group,the survival rate of astrocytes significantly decreased after 9,12 h of 5-20 μmol/L cadmium exposure（P〈0.01）;As the exposure dose increased and exposure time prolonged,the survival rate of astrocytes showed a declining trend. Compared with control group,after 5-20 μmol/L cadmium treatment for 12 h,the apoptosis rate of astrocytes was significantly higher（P〈0.01）,with a rising trend with the cadmium dose increased. Compared with control group,the expression of p-JNK increased after 5-20 μmol/L cadmium treatment for 3 h,p-JNK expression increased after 20 μmol/L cadmium exposure for 6 h,the expression of p-JNK cadmium increased after 10-20 μmol/L cadmium exposure for 9,12 h（P〈0.05）;Compared with exposure for 0 h,the expression of p-JNK significantly increased after 0-20 μmol/L cadmium exposure for 3-12 h（P〈0.05）. With the increase of cadmium（5-20 μmol/L） concentration and prolongation of exposure time（3-12 h）,the expression of p-JNK showed a rising trend. Conclusion Cadmium may induce the apoptosis of mice astrocytes through up-regulating the expression of JNK phosphorylation protein.
出处
《环境与健康杂志》
CAS
北大核心
2016年第6期487-492,F0003,共7页
Journal of Environment and Health
基金
贵州省科技厅社会发展攻关项目(黔科合SY字[2014]3024号)
贵阳市科技计划项目(筑科合同[20141001]21号)
贵州省科学技术基金(黔科合J字[2015]2012号)
贵州省科技合作计划项目(黔科合LH字[2015]7348号)
