硝普钠诱导K562细胞凋亡过程中STAT3/P38MAPK活化及端粒酶hTERT-mRNA表达的研究  

Effects of Sodium Nitroprusside on P38MAPK/STAT3 Activation and Telomerase Reverse Transcriptase mRNA Expression in Inducing Apoptosis of K562 Cell Line

作　　者：周永列[1] 吕亚萍[2] 吕火祥[1] 邱莲女[1] 王文松[1] 林惠君[1] 刘建栋[1] 

机构地区：[1]浙江省人民医院中心实验室,杭州310014 [2]浙江工业大学药学院,杭州310014

出　　处：《中国实验血液学杂志》2006年第4期686-691,共6页Journal of Experimental Hematology

基　　金：浙江省医学科学研究基金资助项目;编号:2002A0010

摘　　要：为了研究外源性一氧化氮供体硝普钠(SNP)诱导K562细胞凋亡过程中STAT3及P38MAPK活化及端粒酶hTERT-mRNA表达的变化,探讨硝普钠诱导K562细胞凋亡机制,用Annexin-V/PI双标记、DNA片段原位末端标记法、DNA凝胶电泳、DNA含量及细胞周期分析等方法检测细胞凋亡。用流式细胞术测定经硝普钠干预后K562细胞磷酸化P38MAPK和磷酸化STAT3的表达,同时利用实时荧光PCR定量检测端粒酶hTERT-mRNA表达的变化。结果表明K562细胞经硝普钠作用后出现典型的细胞形态改变,DNA片段化,显现亚G1峰Ⅱ并显著增加。Annexin-V/PI和DNA片段原位末端标记表达增加。这些均证实NO能诱导K562细胞凋亡,大部分细胞阻滞于G0/G1期。SNP诱导K562细胞凋亡过程中,磷酸化P38MAPK和磷酸化STAT3的表达随SNP浓度的增加而表现为先增强后下降;K562细胞与2.0mmol/LSNP孵育不同的时间内,磷酸化P38MAPK的表达在12小时时达到高峰并持续至48小时,72小时后表达下降;磷酸化STAT3的表达在24小时时达高峰,48小时后表达即显著下降;端粒酶hTERT-mRNA的表达随SNP作用的浓度增加和时间延长而显著下调。结论SNP能诱导K562细胞凋亡,其机制可能与P38MAPK的活化、抑制端粒酶逆转录酶和STAT3的活化有关。This study was aimed to investigate the activation of P38MAPK/STAT3 and expression of telomerase reverse transcriptase during sodium nitroprusside （SNP） inducing apoptosis of human leukemia cell line K562 and to explore the molecular mechanisms of SNP-inducing apoptosis in K562 cells. The K562 cell were treated with different concentrations of SNP and were cultured for different time. Cell apoptosis was analysed by cell morphology, DNA agarose gel electrophoresis, DNA content, and Annexin-V/PI labeling method. The TdT-mediated dUTP nick end labeling （TUNEL） assay was used to quantitate the in situ cell apoptosis. The expressions of phosphorylated p38MAPK or STAT3 were analysed by flow cytometry, while the expression of hTERT mRNA in transcriptional level was measured by fluorescence quantitative RT-PCR. The results showed that SNP inhibited K562 cell growth. The K562 cell apoposis was comfirmed by typical cell morphology and DNA fragment, peak of sub-G1 phase , TUNEL and Annexin-V/PI labeling. A majority of K562 cells were arrested in G0/G1 phase. After treatment with SNP at 0. 5 - 3.0 mmol/L, the expression of phosphorylated-P38MAPK and phosphorylated-STAT3 increased first and decreased afterwards. Incubation of K562 cell with SNP （2 mmol/L） could increase the expression of phosphorylated-P38MAPK and phosphorylated- STAT3 at 12 hours and 24 hours respectively, and down-regulated at 72 hours and 48 hours. SNP could decrease the expression of hTERT-mRNA in time-and dose-dependent manner. It is concluded that SNP can significantly induce K562 cells apoptosis, its mechanism may be related to the activation of P38MAPK and suppression of phosphorylated-STAT3 and hTRET-mRNA.

关 键 词：硝普钠 一氧化氮 K562细胞 细胞凋亡 STAT3 P38MAPK 端粒酶 hTERT—mRNA 

分 类 号：R733.7[医药卫生—肿瘤] R979.1[医药卫生—临床医学]