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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。
Objective To investigate the effect of benzo [a] pyrene (B [a] P) on the expression of heat shock protein 70 (HSP70) in cultured rat cortical neurons. Methods Cortical neurons were isolated and cultured in vitro. The dose-response relationship was observed by exposure to 0, 0.1, 0.5, 1, 5 and 10 μmol / L B [a] B [a] P exposure 24,48 and 72h observed the aging relationship. The medium was added to the volume fraction of 3% S9 mixture. The activity of neurons was detected by MTT assay. The expression of HSP70 was detected by Western blot. The expression of HSP70 was detected by fluorescence immunohistochemistry. Results Compared with the control group (0μmol / L group), HSP70 expression in neurons in the 0.1μmol / L group was not significantly different (P> 0.05) after 24h exposure to B [a] P, Compared with 0.1μmol / L group, the difference was statistically significant (P <0.05). The difference between the 10μmol / L group and the control group was statistically significant (P <0.05) . After being exposed to 0, 0.5, 5μmol / L [a] P for 24,48 and 72h, the expression of HSP70 in neurons at 0.5 and 5μmol / L groups were significantly lower than those in the corresponding control groups (P <0.05) There was a tendency that the expression level decreased with the prolongation of time. The difference between 72h group and 24h group was statistically significant (P <0.05). The results of immunohistochemistry showed that the expression of HSP70 in the control group was only expressed in the cytoplasm, the fluorescence in the 0.1μmol / L group was enhanced, and both the cytoplasm and the nucleus were expressed. With the increase of dose, the expression in the nucleus gradually increased, and the fluorescence intensity gradually weakened. Conclusion B [a] P metabolites at higher doses (0.5 ~ 10μmol / L) can inhibit HSP70 expression in rat cortical neurons with dose-response and time-dependent effects. As B [a] P dose increased, HSP70 gradually shifted into the nucleus.