生理范围直流电场对肺癌细胞凋亡及细胞周期影响的观察

Physiological range direct-current electric fields effect on apoptosis and cell cycle of lung cancer cells

目的:研究生理范围直流电场对人肺腺癌细胞系A549细胞的凋亡及细胞周期的影响。方法:按A549肺癌细胞是否暴露于1.5 V/cm直流电场及暴露时间分为A,B,C 3组,A组为未暴露电场的对照组,B组暴露于电场12 h,C组暴露于电场24 h。Hochest33258荧光染色及流式细胞仪检测各组细胞凋亡值及凋亡比率。流式细胞仪检测各组细胞周期,Western blot检测各组细胞中ERK、AKT及两者矿磷酸化水平pERK、pAKT的量,同时检测各组细胞中细胞周期蛋白Cyclin D1与Cyclin E,细胞周期抑制蛋白p21与p27的表达量。结果:荧光染色结果显示A、B、C 3组凋亡细胞比率分别为0.89%、1.15%及0.98%,差异无统计学意义,P>0.05。流式细胞结果显示A、B、C 3组凋亡细胞比率分别为1.06%、1.18%及0.92%,差异无统计学意义,P>0.05;与A组细胞相比,B、C 2组细胞周期S期和G2期的比例升高明显B组两者和升高9.72%,C组升高20.08%。Western blot检测提示电场促进ERK与AKT蛋白磷酸化水平,同时促进细胞周期蛋白Cyclin D1与CyclinE的表达,抑制了细胞周期抑制蛋白p21与p27的表达。结论:肺癌A549细胞暴露于1.5 V/cm大小的体外直流电场后,凋亡比率无变化,细胞周期明显改变,且细胞增生更为活跃,此变化可能是由于电场激活了ERK与AKT信号通路,从而影响细胞周期相关蛋白有关。

OBJECTIVE： To study the effect of direct-current electric fields（dcEFs） on apoptosis and cell cycle of A549 lung cancer cells.METHODS： The A549 cells were divided into 3 groups according to whether exposing to dcEFs and exposing time as A,B and C.Group A unexposing to dcEFs was used as control.Group B was exposed to 1.5 V/cm dcEFs 12 hours.Group C was exposed to 1.5 V/cm dcEFs 24 hs.The apoptosis number and rate were measured by fluorescent staining by Hochest33258 and flow cytometry.Cell cycle was measured by flow cytometry too.Western blot method was performed to determine the quantity of pERK and pAKT protein,and also the cell cycle related proteins including Cyclin D1,Cyclin E,p21 and p27.RESULTS： Fluorescent staining showed that the apoptosis rate of A,B and C group were 0.89%,1.15% and 0.98%（P〉0.05）.Flow cytometry showed that the apoptosis rate of A,B and C group were 1.06%,1.18% and 0.92%（P〉0.05）.Compare with group A,the proportion of cells in phase S and phase G2 were increased in group B by 9.72% and group C by 20.08%.Western blot illustrated that phosphorylated ERK and Akt were promoted.The expression of Cyclin D1 and Cyclin E were promoted,and the expression of p21 and p27 were inhibited.CONCLUSIONS： After exposing to 1.5 V/cm dcEFs,the apoptosis rate of A549 cells was of no change.However cell cycle revealed great enhancement of cellular proliferation.This enhancement may due to the promotion of ERK and Akt signal conduction path,which affect the cell cycle related proteins of the A549 cells.