中文摘要：

目的研究苯并[a]芘（B[a]P）对体外培养大鼠皮层神经元热休克蛋白70（HSP70）表达的影响。方法分离培养SD大鼠皮层神经元,用0、0.1、0.5、1、5和10μmol/L浓度的B[a]P染毒24h观察剂量-效应关系;用0、0.5和5μmol/L浓度的B[a]P染毒24、48和72h观察时效关系。培养液中均添加体积分数为3%的S9混合液。MTT法检测神经元活性,Western blot法检测神经元HSP70的表达水平,荧光免疫组化法观察神经元HSP70表达定位情况。结果 B[a]P染毒24h后,0.1μmol/L组神经元HSP70表达水平与对照组（0μmol/L组）相比差异无统计学意义（P〉0.05）,而0.5～10μmol/L组神经元HSP70表达水平则逐渐下降,与0.1μmol/L组相比,差异均具有统计学意义（P〈0.05）,其中10μmol/L组与对照组相比差异具有统计学意义（P〈0.05）。0、0.5、5μmol/L B[a]P分别染毒24、48、72h后,0.5、5μmol/L组各时间点神经元的HSP70表达水平均显著低于相应对照组（P〈0.05）,且均有随着时间延长表达水平降低的趋势,其中72h组与24h组比较差异有统计学意义（P〈0.05）。免疫组化结果显示,对照组HSP70仅在细胞浆中表达,0.1μmol/L组荧光表达增强,细胞浆和核中均有表达。随着剂量增大,核内表达逐渐增多,荧光强度逐渐减弱。结论较大剂量（0.5～10μmol/L）的B[a]P代谢产物可抑制大鼠皮层神经元HSP70表达,且存在剂量-效应关系和时效关系。随着B[a]P剂量增大,HSP70逐渐向细胞核内移位。

英文摘要：

Objective To explore the effect of benzo[a]pyrene（B[a]P） on heat shock protein 70（HSP70） expression in rat cortical neurons in vitro.Methods SD rat cortical neurons were isolated and cultured,then they were exposed 24h with the doses of B[a]P（0,0.1,0.5,1,5 and 10μmol/L）and exposed 24h,48h,72h with doses of B[a]P（0,0.5 and 5μmol/L）,observing dose-response relationship and time-dependent relationship,respectively.Including the 3% volume of S9 mixture was added in the medium.The neurons viability was detected by MTT assay.Western blot was used to measure the expression of HSP70 and immunofluorescence histochemistry was used to observe the expression and location of HSP70.Results When neurons were exposed 24h with B[a]P,compared with the control group,the expression of HSP70 was no significant difference in the group of 0.1μmol/L dose of B [a] P（P0.05）.The expression of HSP70 in groups from 0.5 to 10μmol/L doses of B[a]P gradually decreased and was significantly reduced compared with 0.1μmol/L group（P0.05）.The difference between 10μmol/L group and control group was significant（P0.05）.After neurons were exposed 24,48 and 72h with B[a]P in groups of 0,0.5 and 5μmol/L,HSP70 expression at different time-piont in 0.5,5μmol/L groups were significantly lower than the control group（P0.05）.HSP70 expression decreased with time extend in 0.5,5μmol/L groups and the difference between time points of 24h and 72h were significantly different（P0.05）.Immunohistochemistry showed that HSP70 expression was only in the cytoplasm in control group,but fluorescence expressed strongly and expressed in both cytoplasm and nuclear in 0.1μmol/L group.As the dose increased,the expression of HSP70 in nucleus gradually increased and the fluorescence intensity gradually decreased.Conclusion High doses of metabolites of B[a]P（0.5-10μmol/L） inhibit the expression of HSP70 in cultured cortical neurons in rat,and there were relationships of dose and time-dependent between the dose and the inhibition.Wit