铅对原代培养海马细胞形态及酶活力的影响

Effects of Lead on Morphology and Enzyme Activity of Primary Cultured Hippocampus Neurons

目的观察铅对原代培养海马神经元形态及酶活力的影响。方法原代培养乳大鼠海马神经元，经不同浓度醋酸铅（50、100、200μg／ml）作用1～8d，用活细胞倒置相差显微镜、HE染色和尼氏体染色观察细胞形态改变；光密度法检测细胞外液中的乳酸脱氢酶（lactic dehydrogenase，LDH）和胆碱酯酶（cholesterase，CHE）活力；激光扫描共聚焦显微镜定量分析神经元胞浆中游离Ca^2＋的平均荧光强度。结果形态学观察发现，低醋酸铅（50、100μg／ml）组海马神经元表面粗糙，胞体变小，突起细小，细胞间网络疏松，胞体与胞浆之间界限逐渐模糊不清，核仁不清晰；200μg／ml组，神经元数量减少，细胞之间聚集呈团簇状，可见细胞膜损伤破裂，即神经元解体坏死。HE染色观察低浓度醋酸铅作用24h后，可见散在单个细胞核回缩，胞浆、突起着色不均，甚至有的出现染色质凝集、核边移，但核膜完整；高浓度醋酸铅则使细胞质空泡变性，突起减少，核边移呈半月形，甚至细胞出现解体坏死，细胞之间聚集成团。尼氏体染色显示醋酸铅能使海马神经元的尼氏体减少，着色浅淡，且有剂量依赖关系。另外，醋酸铅作用于海马神经元24h后，培养上清中LDH释放增加（P〈0．01），CHE活力明显下降（P〈0．01）。激光共聚焦显微镜检测结果显示，随着醋酸铅浓度的增大，细胞内液Ca^2＋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组（P〈0．01），并有剂量反应关系。结论原代培养的海马神经元随着醋酸铅浓度的加大，呈现不同程度的损伤。

Objective To observe effects of lead on morphology and enzyme activity of primary cultured hippocampus neurons. Methods Primary cultured hippocampus neurons of rats were treated with different concentrations of Pb （C2 H3O2 ）2 （ 10, 50, 100 and 200 μg/ml） respectively for 1 - 8 days. Morphological changes of cells were observed under inverted phase contrast microscope after HE dyeing and tigroid body dyeing. The activities of lactic dehydrogenase （LDH）and cholesterase （CHE）in extracellular fluid were detected by photodensitometry. The average fluorescence intensity of free Ca^2＋ in cytoplasm was determined under laser scanning confocal microscope （LSCM）. Results The morphological findings in low level Pb （CaHaO2 ）2 （50 and 100 μg/ml） were rough surface, shrunk cell body, tiny ecphyma, intercellular porous network, vague fuzzy limes between cell body and endochylema and indistinctive nucleolus. When the dose increased to 200 μg/ml, nerve cells decreased in quantity and integrated into elump, and the plasma membrane was damaged and disrupted resulting to necrosis of nerve cells. The results of HE dyeing showed that after treated with low level Pb（C2H3O2）2 for 24h the recovery of scattered single cell nucleus and uneven coloring of endochylema and ecphyma were observed. Moreover, some cells appeared chromatic agglutination and nucleus moving aside, however, earyotheca of which still kept integrity. In high dose of Pb（C2HaO2）2, vacuolar degeneration of cytoplasm occurred, ecphyma decreased, nucleus moved aside presenting crescent shape and even resolved into cell necrosis and integrated into lumps. Pb（C2H3O2）2 could cause the decrease and color lightening of tigroid body in hippocampus neurons showing dose-dependent relationship. In addition, after hippocampus neurons treated with Pb（C2H3O2 ）2 for 24h, the increase of LDH release and obvious decrease of CHE activity in culture supernatant showed that Pb （C2H3O2）2 might lead to the damage and functional disturbance of hippocampus cytomembrane. The LSCM results showed that the concentration of intracytoplasmic Ca^2＋ also increased with the increase of the dose of Pb（C2HaO2 ）2. The average fluorescence intensity in groups of three concentrations was all significantly higher than that of normal control group（P〈0.01） showing dose-response relationship. Conclusions Primary cultured hippoeampus neurons show different degrees of damage with the increase of Pb（C2HaO2 ）2 dosage.