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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 investigate whether c-Jun N-terminal kinase (JNK) signal transduction pathway is involved in cadherin-induced apoptosis in mouse astrocytes using primary cultured mouse astrocytes (AS) . Methods Primary cultured mouse brain astrocytes were exposed to cadmium (0 ~ 20μmol / L) for 0 ~ 24 h. The morphological changes of cells were observed by inverted phase contrast microscope. Cadmium exposed to 0 ~ 20μmol / L cadmium for 9 and 12 h, Cell viability was detected by MTT assay. Apoptosis rate was detected by Annexin V-FITC / PI double staining flow cytometry after exposure to 0 ~ 20μmol / L cadmium for 12 hours. Cadmium 0 ~ 20μmol / L h, Western blotting was used to detect the expression of JNK phosphorylation protein. Results When the concentration of cadmium was 5 ~ 20μmol / L, cell morphology changed obviously with the increase of exposure dose and prolongation of exposure time. The cell connectivity was loosened, the processes disappeared, the cell body contracted into a sphere and the adhesion capacity decreased. Fall off, floating in the culture medium. Compared with the control group, the survival rate of astrocytes in mice after exposure to cadmium at various concentrations for 9 and 12 h decreased significantly (P <0.01), and with the increase of cadmium exposure And exposure time prolonged, the survival rate of mouse brain astrocytes showed a downward trend. Compared with the control group, the apoptotic rates of astrocytes in mice after exposure to cadmium at various concentrations for 12 h were significantly increased (P <0.01), and with the increase of cadmium exposure , The apoptotic rate of mouse brain astrocytes showed an upward trend. Compared with the control group, the expression of p-JNK in mouse astrocytes increased 5 ~ 20μmol / L cadmium exposure 3 hours after exposure to cadmium at 20μmol / L, the brain astrocytes The expression of p-JNK in the cells was increased. The expression of p-JNK in astrocytes of mice after 10 ~ 20μmol / L cadmium exposure for 9 and 12 h were significantly increased (P <0.05 ). Compared with 0 h, the expression of p-JNK in mouse astrocytes at 0 ~ 20 μmol / L 3 ~ 12 h after exposure was significantly increased (P <0.05); and With the increase of cadmium concentration and exposure time, the expression of p-JNK in mouse brain astrocytes increased. Conclusion Cadmium may induce the apoptosis of mouse brain astrocytes by up-regulating the expression of JNK phosphorylation protein.