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目的探讨锰对嗜铬细胞瘤细胞(PC12cells)凋亡的诱导作用及与抑癌基因P53、原癌基因MDM2蛋白表达的关系。方法以PC12细胞作为多巴胺能神经元的模型细胞,取对数生长期的PC12细胞,用含200,400,800μmol/LMnCl2的培养液分别染毒培养24,36,48h。四甲基偶氮噻唑蓝(MTT)法检测细胞的生长情况,流式细胞仪(FMC)和原位缺口末端标记法(TUNEL)检测细胞凋亡状况;用免疫细胞化学法检测细胞P53、MDM2蛋白表达强度。结果氯化锰可呈时间和剂量依赖性抑制PC12细胞的增殖和诱导细胞凋亡,抑制率为11.8%～73.6%,凋亡率为8.72%～53.60%。免疫化学法检测结果显示,随锰浓度的增高,P53蛋白的表达增加(P<0.01),而MDM2蛋白表达下降,差异有统计学意义(P<0.01),且呈剂量依赖关系。结论锰可诱导PC12细胞凋亡,上调P53基因的表达和下调MDM2基因的表达可能在其诱导PC12细胞凋亡中起着重要的作用。
Objective To investigate the effect of manganese on the apoptosis of pheochromocytoma cells (PC12cells) and its relationship with the expression of tumor suppressor gene P53 and proto-oncogene MDM2. Methods PC12 cells were used as model cells of dopaminergic neurons. PC12 cells in logarithmic growth phase were harvested and cultured for 24, 36 and 48 h with 200, 400 and 800 μmol / L MnCl2. Cell growth was detected by MTT assay. Cell apoptosis was detected by flow cytometry (FMC) and terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL). The expressions of P53 and MDM2 were detected by immunocytochemistry Protein expression intensity. Results MnCl2 could inhibit the proliferation and induce the apoptosis of PC12 cells in a dose-dependent and time-dependent manner. The inhibition rate was 11.8% -73.6% and the apoptosis rate was 8.72% -53.60%. The result of immunochemistry showed that with the increase of Mn concentration, the expression of P53 protein increased (P <0.01), while the expression of MDM2 protein decreased (P <0.01), and there was a dose-dependent relationship. Conclusion Manganese can induce apoptosis of PC12 cells. Upregulation of P53 gene expression and down-regulation of MDM2 gene expression may play an important role in inducing PC12 cell apoptosis.