CAJ | 학술논문

目的探讨苯并(a)芘[B(a)P]致神经元凋亡中线粒体膜电位和胞质中细胞色素C(CytC)蛋白变化.方法选用新生1～3 d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,选取生长良好的同批次神经细胞,以B(a)P同时加S9分别对神经元染毒,使B(a)P终浓度分别为0、10、20、40 μmol/L.继续培养40 h,应用Annexin V和碘丙啶(PI)双染法进行细胞凋亡的检测,加入Rh123,应用流式细胞仪测定细胞线粒体膜电位,免疫印迹(Western blot)法测定神经元胞质中Cyt C蛋白的表达.结果随着B(a)P浓度的增加,神经细胞的早期、晚期凋亡率逐渐增高,中、高剂量组与对照组比较,差异均有统计学意义(P<0.05或P<0.01),趋势检验表明,早期凋亡率增高具有剂量依赖性.随B(a)P染毒浓度的增加,神经细胞线粒体膜电位下降,低、中、高剂量组神经细胞膜电位分别为5.02±1.32、4.36±1.26、3.15±1.47,与对照组(8.89±2.18)比较,差异均有统计学意义(P<0.05),线粒体膜电位与早期凋亡率之间呈负相关(r=-0.763,P<0.05);随着B(a)P浓度的增加,胞质中Cyt C蛋白的表达逐渐升高,Cyt C蛋白表达与早期凋亡率间呈正相关(r=0.831,P<0.01).结论 B(a)P可致神经细胞凋亡,线粒体膜电位降低以及Cyt C的释放可能是诱发凋亡的主要因素.
목적 탐토분병(a)비[B(a)P]치신경원조망중선립체막전위화포질중세포색소C(CytC)단백변화.방법 선용신생1～3 d적SD대서분리대뇌피질진행신경원배양,재세포배양제5천,선취생장량호적동비차신경세포,이B(a)P동시가S9분별대신경원염독,사B(a)P종농도분별위0、10、20、40 μmol/L.계속배양40 h,응용Annexin V화전병정(PI)쌍염법진행세포조망적검측,가입Rh123,응용류식세포의측정세포선립체막전위,면역인적(Western blot)법측정신경원포질중Cyt C단백적표체.결과 수착B(a)P농도적증가,신경세포적조기、만기조망솔축점증고,중、고제량조여대조조비교,차이균유통계학의의(P<0.05혹P<0.01),추세검험표명,조기조망솔증고구유제량의뢰성.수B(a)P염독농도적증가,신경세포선립체막전위하강,저、중、고제량조신경세포막전위분별위5.02±1.32、4.36±1.26、3.15±1.47,여대조조(8.89±2.18)비교,차이균유통계학의의(P<0.05),선립체막전위여조기조망솔지간정부상관(r=-0.763,P<0.05);수착B(a)P농도적증가,포질중Cyt C단백적표체축점승고,Cyt C단백표체여조기조망솔간정정상관(r=0.831,P<0.01).결론 B(a)P가치신경세포조망,선립체막전위강저이급Cyt C적석방가능시유발조망적주요인소.
Objective To investigate the changes of mitochondria membrane potential and cytoplasmal cytochrome C as the mechanism of neuron apoptosis induced by B (a)P. Methods Primary neurons were dissociated from cerebral cortex of 1-3 days old SD rats and cultured with DMEM incubator at 37 ℃. After 5 days' cultivation, the neurons were added S9 and B(a)P, and the concentrations of treated B(a)P were 0, 10, 20 and 40 μmol/L respectively. After administering of B (a)P, the neurons were cultivated for 40 hours. Apoptosis rate was measured by flow cytometry using Annexin V-FITC and propidium iodide(PI) staining,and the changes in mituchondrial potential (△ψm) were tested with Rhodamine fluorescence(R2123) technique. Preparation of cytosolic extracts by centrifugation. Western blotting analysis was used to evaluate the level of cytochrome C of cytoplasm. Results The apoptotic rate of neuron increased in beth the middle dose group and the high dose group compared with controls, and had a dose-response tendency with the concentration of B(a)P. Moreover mitochondrial potential decreased in a dose dependent manner. There was a negative correlation between △ψm and the apoptotic rate of neurons (r=-0.763, P<0.05); Western blotting analysis showed cytoplasmic cytochrome C level increased significantly, which was positively related with neuron apoptosis (r=0.831, P<0.01). Conclusion Loss of mitochondria membrane potential and increase of cytoplasmal cytochrome C may be the main cause of neuron apoptosis induced by B(a)P.