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本研究探讨5-氮杂胞苷(5-aza-2′-deoxycytidine,5-aza-CdR)对K562细胞中抑癌基因SHP-1的转录调控作用及对K562细胞增殖凋亡的生物学影响,为寻找肿瘤治疗新靶点提供实验依据。采用MSP方法检测SHP-1基因甲基化状态,MTT法检测不同剂量(0.5,1,2μmol/L)5-aza-CdR作用K562细胞1,3,5天时对K562细胞存活率的影响,流式细胞术(FCM)分析5-aza-CdR作用1,3,5天时细胞周期及凋亡率的变化,实时定量PCR(FQ-PCR)和Westernblot方法检测5-aza-CdR处理前后SHP-1mRNA及蛋白表达水平、p-STAT5蛋白表达变化。结果表明,K562细胞中存在SHP-1基因的甲基化,5-aza-CdR作用使K562细胞中SHP-1mRNA和蛋白重新表达,而p-JAK2蛋白表达随之呈下降趋势;5-aza-CdR明显抑制K562细胞增殖,并与药物浓度及作用时间呈正相关。JAK2特异性抑制剂AG490明显抑制K562细胞增殖;5-aza-CdR可使K562细胞凋亡率增加,且作用也呈时间依赖性,用药1,3,5天细胞凋亡率分别为9.3%,24.2%,37.7%。2μmol/L的5-aza-CdR处理24小时后,G0/G1期细胞逐渐增多,G2/M期细胞逐渐减少,细胞阻滞在G0/G1期。5-aza-CdR作用1,3,5天时G2/M期细胞比例分别为30.7%,23.4%,19.3%。结论:特异性甲基化转移酶抑制剂5-aza-CdR能使K562细胞中沉默的SHP-1基因重新表达,并可能通过JAK/STAT路径活化抑制白血病细胞增殖并诱导其凋亡。
This study was aimed to investigate the biological effects of 5-aza-2’-deoxycytidine (5-aza-CdR) on the transcriptional regulation of tumor suppressor gene SHP-1 in K562 cells and on the proliferation and apoptosis of K562 cells , To provide experimental evidence for finding a new target for tumor therapy. The methylation status of SHP-1 gene was detected by MSP method. The effect of different doses (0.5, 1, and 2μmol / L) of 5-aza-CdR on the survival rate of K562 cells at 1, The changes of cell cycle and apoptosis rate after 5-aza-CdR treatment at 1, 3 and 5 days were analyzed by flow cytometry (FCM). The expression of SHP-1 mRNA was detected by FQ-PCR and Westernblot And protein expression, p-STAT5 protein expression changes. The results showed that the methylation of SHP-1 gene was present in K562 cells and the expression of SHP-1 mRNA and protein in K562 cells was re-expressed by 5-aza-CdR while the expression of p-JAK2 protein was decreased. CdR significantly inhibited the proliferation of K562 cells, and was positively correlated with drug concentration and duration of action. AG490, a specific inhibitor of JAK2, significantly inhibited the proliferation of K562 cells. 5-aza-CdR increased the apoptosis rate of K562 cells in a time-dependent manner. The apoptotic rates of the cells treated with AG490 for 9 and 5 days were 9.3% 24.2%, 37.7%. After treated with 2μmol / L 5-aza-CdR for 24 hours, the number of G0 / G1 phase cells gradually increased, the number of G2 / M phase cells gradually decreased, and the cells arrested in G0 / G1 phase. The proportion of cells in G2 / M phase was 30.7%, 23.4% and 19.3% when treated with 5-aza-CdR for 1, 3 and 5 days, respectively. CONCLUSIONS: 5-aza-CdR, a specific inhibitor of methylated transferase, can re-express the silenced SHP-1 gene in K562 cells and possibly inhibit the proliferation and induce the apoptosis of leukemic cells through activation of JAK / STAT pathway.