5-Aza-2'-dc预处理致敏百草枯对V79细胞活性氧及Bcl-2/Bax表达的影响

Influences of Pretreating with 5-Aza-2'-dc on the Reactive Oxygen Level and the Expression of Protein Bcl-2 and Bax in Paraquat Sensitized V79 Cells

目的研究DNA甲基化酶抑制剂5-氮杂-2'-脱氧胞苷(5-Aza-2'-dc)与百草枯联合对V79细胞作用后的活性氧含量及抗凋亡Bcl-2蛋白和促凋亡Bax蛋白表达的影响.方法 V79细胞经传代培养后分为5-Aza-2'-dc作用组(A组)、百草枯作用组(B组)、5-Aza-2'-dc加百草枯作用组(C组,即先应用5-Aza-2'-dc对V79细胞进行预处理12h,然后给予百草枯作用12h)、对照组(D组).采用2,7二氯荧光黄双乙酸盐(DCFH-DA)荧光探针装载,流式细胞仪测定各实验组V79细胞内活性氧含量,Western blot检测各实验组V79细胞内抗凋亡Bcl-2蛋白和促凋亡Bax蛋白的表达情况.结果 5-Aza-2'-dc与百草枯联合作用组(C组)V79细胞内活性氧含量、抗凋亡Bcl-2蛋白、促凋亡Bax蛋白表达与5-Aza-2'-dc组(A组)、百草枯组(B组)和对照组(D组)比较有统计学差异(P<0.05);C组V79细胞内抗凋亡Bcl-2蛋白表达、抗凋亡Bcl-2蛋白/促凋亡Bax蛋白比值降低,活性氧含量和促凋亡Bax蛋白表达升高.结论 5-Aza-2'-dc调控DNA甲基化可能通过活性氧代谢失衡、细胞调亡来促进百草枯对V79细胞的毒性损伤.

Objective To study the reactive oxygen level and the expression of anti-apoptotic protein Bcl-2and pro-apoptotic protein Bax after treatment of DNA methyltransferase inhibitor5-aza-2'-deoxycytidine(5-Aza-2'-dc)and paraquat in V79cells.Methods Cultured V79cells were divided into5-Aza-2'-dctreatment group(group A),paraquat treatment group(group B),5-Aza-2'-dc and paraquat treatment group(group C,V79cells were pretreated with5-Aza-2'-dc for12h followed by exposure to paraquat for12h)andcontrol group(group D).Reactive oxygen level in V79cells was measured by DCFH-DA flow cytometry andexpression of Bcl-2and Bax was detected by Western blot.Results Reactive oxygen levels and expression levelsof Bcl-2and Bax in V79cells were significantly different(<0.05)in5-Aza-2'-dc and paraquat treatment group(group C),compared with5-Aza-2'-dc treatment group(group A),paraquat treatment group(group B)andcontrol group(group D).Expression levels of Bcl-2and the ratio of Bcl-2and Bax were lower while reactiveoxygen levels and expression levels of Bax were higher in group C than in groups A,B and D.Conclusion5-Aza-2'-dc regulates DNA methylation by the imbalancing the reactive oxygen metabolism and apoptosis,thus up-regulating the toxic effect of paraquat on V79cells.