1,4-苯醌致人胚肺成纤维细胞的凋亡效应与调控机制

Apoptotic effect and regulatory mechanism of 1,4-benzoquinone on HELF cells

目的:研究1,4-苯醌(1,4-benzoquinone,PBQ)对人胚肺成纤维细胞(human embryonic lung fibroblast,HELF)的凋亡效应与调控机制。方法:分别用不同浓度(10、20、40、60、80μmol/L)的PBQ处理HELF细胞24 h和40μmol/L PBQ处理HELF细胞24、48、72 h后,应用噻唑蓝(MTT)比色法检测PBQ对HELF细胞增殖的抑制作用,以PI单染法检测细胞周期的改变,用Annexin-Ⅴ/PI双染法检测细胞凋亡,以实时荧光PCR法检测Bax、Bcl-2、p53 mRNA表达水平的改变。结果:与对照组相比,不同PBQ染毒浓度下作用24 h后,随PBQ浓度增加,细胞相对增殖率显著下降(P<0.05),G0/G 1期细胞下降(在40和60μmol/L时,P<0.05),S期细胞增加(P<0.05),以40μmol/L PBQ浓度作用于HELF细胞不同时间,随着染毒时间的增加其相对增殖率和S期细胞均下降(P<0.05),而G0/G1期细胞随着染毒时间的增加呈上升趋势(P<0.05);当染毒浓度大于20μmol/L时,染毒24 h可诱导HELF细胞产生凋亡,凋亡率随着染毒时间的增加而上升,与对照组相比差异具有统计学意义(P<0.05);不同浓度的PBQ染毒24 h,细胞的Bax、Bcl-2、p53 mRNA表达水平均上调。40μmol/L PBQ作用不同时间后,Bax、p53 mRNA水平随着染毒时间的增加而上升,与对照组相比差异均具有统计学意义(P均<0.05);Bcl-2随染毒时间增加而下降,48和72 h时与对照组相比差异具有统计学意义(P<0.05);Bax/Bcl-2比值随染毒时间增加而增加,与对照组相比差异具有统计学意义(P<0.05)。结论:PBQ能抑制HELF细胞增殖,影响HELF细胞周期分布,诱导细胞发生凋亡,其凋亡机制可能与Bax/Bcl-2比值的上升,及p53 mRNA表达的上调有关。

OBJECTIVE：To study the apoptotic effect and regulation of 1,4-benzoquinone（PBQ） on human embryonic lung fibroblast（HELF）,cells.METHODS：MTT assay was used to detect the relative growth rate （RGR）,cell cycle was determined by single-stained assay with PI and apoptosis was determined by double-stained assay with Annexin-Vand PI using flow cytometry.Bax,Bcl-2,p53 mRNA levels were determined with real-time PCR. RESULTS：The RGRs of HELF cells exposed to different PBQ concentrations after 24 h decreased（P 0.05）,cell cycle distribution was changed at 40 and 60μmol/L after 24 h（P 0.05）.The RGRs of HELF cells exposed to 40μmol/L PBQ after different times decreased（P 0.05）,the rates of G0/G1 phase increased with time（.P0.05）.Apoptosis was induced with PBQ higher than 20μmol/L,and increased with PBQ concentration（P0.05）.The relative expression levels of Bax,Bcl-2,p53 increased with concentration after exposure for 24 h（P0.05）.The relative expression levels of Bax and p53 exposed at 40μmol/L increased with time（P0.05）,while bcl-2 decreased with time（P 0.05）,with Bax/Bcl-2 ratio increased with time（P 0.05）.CONCLUSION：PBQ was able to change cell cycle distribution of HELF cells,and induce apoptosis,with dose-response and time-response effects.The up-regulation of mRNA expression level of p53 and the elevated Bax/Bcl-2 ratio may be the mechanisms for apoptosis of HELF cells after PBQ exposure.