氯吡硫磷染毒撤除对原代培养大鼠海马神经元细胞毒性的延迟效应

摘要

目的：探讨低浓度氯吡硫磷（毒死蜱，CPF）染毒撤除对原代培养海马神经元细胞毒性的延迟效应。方法原代培养海马神经元经CPF 10和30μmol·L-1连续染毒72 h，或连续染毒48 h后更换无CPF的培养液继续培养24 h，运用CCK-8试剂盒检测海马神经元的存活；用神经元核（NeuN）、5-溴脱氧尿苷（BrdU）和βⅢ微管蛋白免疫荧光染色法检测海马不同发育阶段神经元数量。结果原代培养海马神经元经CPF 10和30μmol · L-1连续染毒72 h，与正常对照组相似，未检测到明显神经元死亡；而CPF 10和30μmol·L-1连续染毒48 h撤除后24 h，与正常对照组相比，出现细胞破裂、突触断裂现象，且海马神经元数目明显减少（P<0.05），神经元的存活率明显下降（P<0.05），BrdU和βⅢ微管蛋白表达阳性细胞的数量明显减少（P<0.05）。结论 CPF 10和30μmol·L-1染毒撤除后对大鼠海马神经元细胞毒性具有延迟效应。%OBJECTIVE To investigate the delayed cytotoxicity effect of chlorpyrifos (CPF) with⁃drawal on primary hippocampal neurons. METHODS Hippocampal neurons were prepared from SD rat fetuses on the 17th day of gestation. Seven days after culture,neurons were exposed to CPF 10 and 30 μmol · L-1,respectively,for 72 h or for 48 h followed by CPF withdrawal for 24 h. CCK-8 kit and neuronal nuclei(NeuN), 5-bromodeoxyuridine(BrdU) and β Ⅲ tubulin immunofluorescence expression methods were used to evaluate the cell viability. RESULTS Compared with normal control, no significant cytotoxicity was found after CPF 72 h continuous exposure. However,CPF 48 h expo⁃sure followed by CPF withdrawal for 24 h induced evident cytotoxicity. The amount of BrdU positive and β Ⅲ tubulin positive hippocampal neurons were both decreased significantly(P<0.05),and cell survival and viability reduced after CPF withdrawal. CONCLUSION CPF exposure withdrawal can induce more seriously delayed cytotoxicity than continuous exposure in rat primary hippocampal neurons.