铅致原代培养海马神经元凋亡及Bcl-2、Bax、p53和NOS1基因表达的影响

The effects of lead exposure on primary cultured hippocampus neurons apoptosis and the expressions of Bcl-2,Bax,p53 and NOS1

目的观察铅对原代培养海马神经元的致凋亡作用并初步探讨其机制。方法原代培养乳大鼠海马神经元,不同浓度的醋酸铅(50、100和200μg/ml)染毒后,Giemsa染色观察铅诱导海马细胞凋亡的形态学改变,流式细胞仪检测铅诱导海马神经元的凋亡率,免疫细胞化学染色观察各浓度组细胞Bcl-2、Bax和p53表达,激光扫描共聚焦显微镜定量分析各浓度组神经元胞浆中游离Ca2+的平均荧光强度。结果Giesma染色观察发现一定浓度醋酸铅作用于海马神经元后出现凋亡的形态学改变,且与剂量相关;流式细胞术检测结果铅浓度分别为50、100、200μg/ml作用细胞24h后,可致海马神经元凋亡率增加,与对照组相比均有统计学意义(P<0.01);免疫细胞化学染色结果表明Bcl-2蛋白表达随染铅剂量增大而减少,染色强度逐渐降低,Bax、p53表达随染铅剂量增大而上调,与对照组相比,细胞积分光密度(IOD)值有统计学意义(P<0.01);激光共聚焦显微镜检测结果显示随着醋酸铅剂量的增大,细胞内液Ca2+浓度也随着增大,醋酸铅3个浓度组的平均荧光强度均显著高于正常对照(P<0.01),并有剂量反应关系。结论铅可诱导海马神经元不同程度的凋亡,提示细胞内Ca2+浓度升高可能与铅致海马神经元损伤和诱导其凋亡有重要关系。推测铅在一定浓度范围内可能影响海马神经元在学习记忆等功能的正常发挥。

Objective To observe effects of plumbum on the apoptosis of primary cultured hippocampus neurons. Methods After primary cultured hippocampus neurons of rats treated with different concentration of Pb （C2H3O2） （10,50,100 and 200μg/ml）,to observe the morphological changes of the apoptosis of hippocampus cells by Giemsa dyeing,to detect the rate of apoptosis by flow cytometry,to observe the expression of Bcl-2、Bax and p53 by immunocytochemical stain and to analyze quantitatively the average fluorescence intensity of free Ca2 ＋ in kytoplasm of each concentration group by laser scanning confocal microscope （ LSCM）. Results The results of Giemsa dyeing showed typically morphological changes of apoptosis after hippocampus neurons treated with certain concentration of Pb（C2 H3 O2 ）,and had dose-dependent; The results of flow cytometry showed,after cells treated respectively in 50、100、200μg /ml for 24h,that the rate of apoptosis increased and compared with the control group the difference of were all statistical significance（P 0. 01）; The results of immunocytochemical stain showed that, with dose of Pb increasing,the expression of Bcl-2 protein decreased、the intensity of dyeing increasingly degraded and the expression of Bax and p53 was up-regulation,and compared with the control group,IOD value of cells had statistical significance（P 0. 01）; The results of LSCM showed that the concentration of Ca2 ＋ in kytoplasm was also increased with the dose of Pb （ C2 H3 O2 ） increasing,and the average fluorescence intensity in groups of three concentration of Pb （ C2 H3 O2 ） was all significantly higher than in the control group （ P 0. 01 ） and had dose- response relation. Conclusion Plumbum might lead to different degrees of the apoptosis of hippocampusneurons. It is supposed that Pb in certain concentration range might affect the normal function of hippocampus neurons in learning and memory,and so on.