苯并(a)芘代谢产物作用下人胚肺细胞热应激蛋白70表达与DNA损伤

Study on the relationship between HSP70 expression and DNA damage in human embryonic lung(HEL)cells by benzo(a)pyrene metabolites

目的 探讨苯并 (a)芘 (BaP)代谢产物作用下 ,人胚肺细胞HSP70表达与DNA损伤的关系。方法 选用正常人胚肺 (HEL)二倍体细胞 ,进行如下处理 :一般处理组 ,以 0、10、5 0、10 0、2 0 0μmol/LBaP染毒 3h(体外经大鼠肝S9 mix活化诱导 ) ,单细胞凝胶电泳技术检测DNA损伤情况 ;热应激组 ,细胞预热应激 (4 1℃ 1h ,37℃ 2h) ,再以同样浓度的BaP染毒 3h ,采用Westernblot和单细胞凝胶电泳技术检测HSP70表达与DNA损伤情况。结果 一般处理组BaP在 5 0 μmol/L时可引起HEL细胞DNA的明显损伤 ,且随BaP染毒剂量增加 ,DNA损伤级别加重 ;接受预热应激的细胞经不同浓度BaP染毒 3h后 ,与对照组比 ,HSP70表达水平降低 ,BaP在 10 μmol/L时可见明显的DNA损伤 ,随染毒剂量的增加 ,DNA损伤级别加重 ,但在最高剂量组 2 0 0 μmol/L时 ,损伤减轻。与一般处理组比 ,发生“ + +”级DNA损伤的细胞减少 ,差异有显著性 (P <0 .0 5 ) ;“ + + +”级DNA损伤程度差异无显著性 (P>0 .0 5 )。结论 HSP70对BaP引起的HEL细胞DNA损伤的保护作用不明显 ,可能与预热应激后BaP染毒使HSP70表达受到抑制有关。

Objective To investigate the relationship between HSP70 expression and DNA damage in human embryonic lung(HEL) cells treated by benzo(a)pyrene(BaP) metabolites in vitro. Methods HEL cells of unheated group were stimulated by 0,10,50,100,200 μmol/L BaP for 3 h incubation(induced by S9 mix in vitro).HEL cells of heated group were treated at 41 ℃ for 1 h,recovered 2 h at 37 ℃,then were stimulated by BaP at the same conditions as the untreated group.The DNA damage and HSP70 expression were determined by single cell gel assay and Western blot respectively. Results DNA damage in HEL cells induced by BaP was detectable at 50 μmol/L in untreated group,while DNA damage in heated group was detectable at 10 μmol/L.The level of HSP70 expression decreased,but DNA damage of HEL cells reduced at the highest dose(200 μmol/L BaP) compared with the control.Compared with the untreated group,the number of cells with “++” level DNA damage reduced( P < 0.05) ,while that with “+++” level DNA damage was not significantly different(P>0.05). Conclusion The roles of HSP70 in the protection from DNA damage induced by BaP are not significant,which may be related to the inhibition of HSP70 expression by BaP.