钠氢交换蛋白在依托泊苷诱导HL-60细胞凋亡中的作用及其机制的初步研究

本研究探讨钠氢交换蛋白(Na+/H+ exchanger-1,NHE1)在依托泊苷诱导HL-60细胞凋亡过程中的表达变化及其对凋亡过程的调控机制。应用DNA片段化检测和远末端缺口标记(TUNEL)法分析细胞凋亡;实时定量聚合酶链反应检测NHE1 mRNA表达量,Western blot法检测NHE1蛋白表达水平,并应用激光共聚焦显微镜分析细胞内pH值(pHi)的变化。同时应用NHE1特异抑制剂卡立泊来德(cariporide)作用于细胞观察凋亡及pHi的变化情况。结果表明:依托泊苷作用24小时后能有效诱导HL-60细胞凋亡,作用12小时后NHE1 mRNA表达增高了2.848±0.886倍(p0.01),NHE1蛋白表达水平也升高(p0.01)。依托泊苷作用24小时后细胞内pHi从7.11升高到7.46,而卡立泊来德预处理组的细胞在依托泊苷处理24小时后pHi没有明显升高并且凋亡率明显降低。结论:依托泊苷诱导的HL-60细胞凋亡过程中会出现NHE1表达上调,凋亡结果依赖于NHE1表达量升高导致的细胞内pH值升高。

Role of Na+/H+ Exchanger 1 in Apoptosis of HL-60 Cells Induced by Etoposide and Its Mechanism

This study was aimed to investigate the role of Na ^＋/H ^＋ exchanger 1 （ NHE1 ） in apoptosis of HL-60 cells induced by etoposide. Real-time quantitative PCR （RQ-PCR） and Western blot methods were used to determine the expression of NHE1 in HL-60 cells after the treatment with etoposide. Meanwhile, laser scanning confocal microscopy was used to test the intracellular pH （pHi） of HL-60 cells. Cell apoptosis was measured by DNA fragmentation and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling （TUNEL） assay. The results showed that etoposide induced cell apoptosis after treatment for 24 hours. The expression level of NHE1 mRNA increased by 2. 848 ± 0. 886 times after treatment with etoposide for 12 hours （p 〈 0.01 ）, and the expression of NHE1 protein was also upregulated （p 〈 0.01 ）. The pHi of HL-60 cells increased from 7.11 to 7.46 after treatment with etoposide for 24 hours. Treatment with cariporid could block etoposide-induced alkalinisation and enhance the apoptosis HL-60 cells. It is concluded that the expression of NHE1 is up-regulated in process of apoptosis of HL-60 cells induced by etoposide and the apoptosis depends on the pH increase caused by NHE1 higher expression.