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目的研究尼洛替尼(nilotinib,AMN107)诱导K562/A02细胞凋亡及对血红素加氧酶-1(HO-1)基因表达的影响。方法采用荧光原位杂交(FISH)法检测K562/A02细胞中的BCR-ABL融合基因;用不同浓度的AMN107分别处理K562/A02细胞24 h后,通过实时荧光定量PCR(RQ-PCR)法检测BCR-ABL融合基因mRNA的表达水平;采用MTT法观察细胞增殖变化;通过An-nexin V/PI双染色法检测细胞凋亡和细胞周期;采用RT-PCR法和Western Blot法检测HO-1基因的表达。结果 FISH法分析结果显示,K562/A02细胞BCR-ABL融合基因阳性细胞占细胞总数98%;RQ-PCR法检测结果显示,0,5,10,20μmol.L-1AMN107作用于K562/A02细胞24 h后BCR-ABL融合基因表达随AMN107浓度增加而逐渐下降;MTT法和Annexin V/PI法显示与对照组相比,细胞存活率随AMN107浓度升高而下降,凋亡率逐渐增加;细胞周期分析显示,经AMN107处理的细胞,G0/G1期和S期细胞明显减少,细胞阻滞在G2/M期;RT-PCR法和Western blot法均检测到HO-1基因表达随AMN107浓度升高而降低。结论 AMN107可抑制BCR-ABL融合基因和HO-1基因表达,从而诱导慢性粒细胞白血病(CML)耐药细胞的凋亡,说明HO-1是CML细胞生长以及BCR-ABL基因存在的一个相关因子,HO-1基因是克服慢性粒细胞白血病耐药的一个潜在靶向。
Objective To investigate the effect of nilotinib (AMN107) on the apoptosis of K562 / A02 cells and on the expression of heme oxygenase-1 (HO-1) gene. Methods The BCR-ABL fusion gene in K562 / A02 cells was detected by fluorescence in situ hybridization (FISH). K562 / A02 cells were treated with different concentrations of AMN107 for 24 h, and then detected by real-time quantitative PCR (RQ- BCR-ABL fusion gene mRNA expression was detected by MTT assay; cell proliferation was observed by MTT assay; apoptosis and cell cycle were detected by An-nexin V / PI double staining; HO-1 gene was detected by RT-PCR and Western Blot expression. Results FISH analysis showed that the positive cells of BCR-ABL fusion gene in K562 / A02 cells accounted for 98% of the total number of cells. The results of RQ-PCR assay showed that 0, 5, 10, 20μmol.L-1AMN107 acted on K562 / A02 cells 24 The expression of BCR-ABL fusion gene decreased gradually with the increase of AMN107 concentration. The MTT assay and Annexin V / PI assay showed that the cell viability decreased with the increase of AMN107 concentration and the apoptosis rate increased gradually. The cell cycle The results showed that the cells treated with AMN107 significantly decreased the number of cells in G0 / G1 and S phase and arrested the cells in G2 / M phase. The expression of HO-1 gene was detected by RT-PCR and Western blot with the increase of AMN107 Lower. Conclusion AMN107 can inhibit the expression of BCR-ABL fusion gene and HO-1 gene and induce the apoptosis of CML-resistant cells, indicating that HO-1 is a related factor of CML cell growth and BCR-ABL gene expression , HO-1 gene is a potential target to overcome the drug resistance of chronic myeloid leukemia.