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目的探讨苯并(a)芘[B(a)P]对神经细胞毒性、细胞凋亡与细胞色素P4501A1(CYP1A1)诱导表达的关系。方法选用新生1～3d的SD大鼠,分离大脑皮质,进行神经元培养,在细胞培养第5天左右,选取生长良好的同批次神经细胞,以苯并(a)芘分别对神经细胞染毒,使苯并(a)芘终浓度分别为0μmol/L、10μmol/L、20μmol/L、40μmol/L。继续培养40h,应用cck8试剂盒检测神经细胞活力、Annexin V和PI双染法进行细胞凋亡的检测,应用RT-PCR法检测神经细胞CYP1A1mRNA的表达,免疫组织化学SABC法检测神经元CYP1A1蛋白的表达。结果随着苯并[a]芘浓度的增加,神经细胞活力降低,早期凋亡率逐渐增高,细胞活力中高剂量组与对照组比较差异均有统计学意义,早期凋亡率仅高剂量组与对照组比较差异有统计学意义,趋势检验表明,细胞活力降低、凋亡率增高具有剂量依赖性。而且随B(a)P剂量的增高,CYP1A1mRNA及蛋白表达增多,有剂量-反应关系,CYP1A1基因和蛋白表达与神经细胞凋亡率的相关分析表明,神经细胞凋亡率与CYP1A1mRNA表达呈正相关(r=0.831,P<0.01);与CYP1A1蛋白表达呈正相关(r=0.780,P<0.01)。结论苯并[a]芘可致神经细胞凋亡,神经细胞CYP1A1诱导表达是神经细胞损伤的关键因素。
Objective To investigate the relationship between the cytotoxicity of neurons and the induction of CYP1A1 expression by benzo (a) pyrene [B (a) P]. Methods Sprague Dawley (SD) rats of 1 ~ 3 days old were used to separate the cerebral cortex and conduct neuronal culture. On day 5 of cell culture, the same batch of nerve cells with good growth were selected, The final concentrations of benzo (a) pyrene were 0μmol / L, 10μmol / L, 20μmol / L and 40μmol / L, respectively. After cultured for 40h, the viability of neurons was detected by cck8 kit. The apoptosis of neurons was detected by Annexin V and PI staining. The expression of CYP1A1 mRNA was detected by RT-PCR. The expression of CYP1A1 protein in neurons was detected by immunohistochemical SABC expression. Results As the concentration of benzo [a] pyrene increased, the viability of nerve cells decreased and the early apoptotic rate gradually increased. The cell viability of medium and high dose groups was significantly different from that of the control group The difference between the control group was statistically significant, trend test showed that cell viability decreased, the apoptosis rate increased in a dose-dependent manner. Moreover, with the increase of the dose of B (a) P, the expression of CYP1A1 mRNA and protein increased, there was a dose-response relationship. The correlation analysis between the expression of CYP1A1 gene and protein and the apoptosis rate of nerve cells showed that the apoptosis rate of neurons was positively correlated with the expression of CYP1A1 mRNA r = 0.831, P <0.01), but positively correlated with CYP1A1 protein expression (r = 0.780, P <0.01). Conclusions Benzo [a] pyrene can induce neuronal apoptosis. The induction of CYP1A1 by neurons is the key factor of neuronal injury.