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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。
In this study, we investigated the expression changes of sodium hydrogen-exchange protein-1 (NHE1) in BCR-ABL positive cell line K562 under etoposide-induced DNA damage and determined the level at which it is regulated. The expression of NHE1 at the mRNA level was detected by real-time quantitative PCR. The effect of etoposide on the protein level of NHE1 was detected by Western blot. The apoptosis of NHE1 was detected by flow cytometry. The luciferase reporter vector of NHE1 promoter region was constructed. The luciferase activity under different concentrations of etoposide was determined. The results showed that DNA damage caused NHE1 mRNA and protein levels in HL-60 cells (p <0.05), and no significant changes in K562 cells (p> 0.05). Etoposide significantly promoted the expression of NHE1 in HL-60 cells The apoptosis of K562 cells was inhibited by etoposide and the apoptosis of K562 cells was inhibited by the increase of pH value. The fluorescence of NHE1 promoter in K562 cells Enzyme expression vector activity increased. CONCLUSION: The DNA damage induced by etoposide can promote the apoptosis of HL-60 cells and depend on the change of pHi. The expression of NHE1 in K562 cells has no change, but the transcriptional activity is increased.