DNA损伤时钠氢交换蛋白1在K562和HL-60细胞中的表达和对细胞凋亡的影响

摘要

本研究主要探讨在依托泊苷引起的DNA损伤下,BCR-ABL阳性细胞系K562中钠氢交换蛋白-1(NHE1)的表达变化情况,并初步探讨其在何种水平被调控.采用实时定量PCR技术检测NHE1在mRNA水平的表达;Western blot检测依托泊苷对细胞NHE1在蛋白水平的影响;流式细胞术测定细胞凋亡情况;构建NHE1启动子区荧光素酶报告载体,并测定不同依托泊苷浓度作用下的荧光素酶活性.结果表明:DNA损伤引起HL-60细胞NHE1在mRNA和蛋白水平升高(p<0.05),在K562细胞中未发现明显变化(P>0.05);依托泊苷对HL-60细胞有明显的促凋亡作用,阻止细胞内pH值的升高可以降低依托泊苷的促凋亡作用,依托泊苷对K562细胞凋亡率无明显影响;K562细胞在DNA损伤时,NHE1启动子区构建的荧光素酶表达载体活性升高.结论:依托泊苷造成的DNA损伤,促进HL-60细胞凋亡,并且依赖PHi的改变;在K562细胞中NHE1表达没有改变,但其转录活性升高.%This study was aimed to investigate the expression of Na +/H + exchanger 1 ( NHE1 ) in K562 and HL-60 cells undergoing DNA damage induced by etoposide and to elucidate the regulating mechanism.Real-time quantitative PCR (RQ-PCR) and Western blot methods were used to determine the expression of NHE1 in K562 cells after the treating with etoposide.Meanwhile, the flow cytometry was used to detect the apoptosis of leukemic cells.The luciferase reporter vector containing NHE1 promoter was constructed to measure relative luciferase activity after treating with different etoposide concentrations.The results showed that the mRNA and protein of NHE1 increased in accordance with apoptosis ratio in HL-60 cells after treated with etoposide (p ＜ 0.05 ), but no such obvious increase in K562 cells.Treatment with NHE1 specific inhibitor could block etoposide induced alkalization and reduce the apoptosis ratio of HL-60 cells.The expression pattern and apoptosis alteration was not simllar in K562 cells.Relative luciferase activity of reporter vector containing NHE1 promoter however increased in K562 cells after treated with etoposide.It is concluded that the expression of NHE1 is up-regulated in the process of apoptosis of HL-60 cells induced by etoposide and depends on the pHi increasing caused by NHE1 up-regulation which is not found in K562 cells although the transcriptional activity increased.