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目的研究去甲斑蝥素(DMC)对慢性粒细胞白血病细胞株K562细胞凋亡的影响,探讨DMC致细胞凋亡后基因表达的变化。方法 20 mg.L-1DMC作用细胞36 h后,倒置显微镜观察细胞形态变化;提取细胞DNA,琼脂糖凝胶电泳检测凋亡细胞DNA改变;提取细胞总RNA,基因芯片技术检测基因表达谱变化;通过反转录(RT)-PCR、ELISA验证芯片部分基因表达变化。采用SPSS13.0软件进行统计学分析。结果 DMC处理K562细胞后,倒置显微镜下可见细胞状态变差,活细胞数目明显减少;DNA电泳后可见明显的阶梯状变化;在利用基因芯片技术检测的21 522个基因转录本中,共筛选出差异表达基因约2 300个,其中超过500个基因的表达明显升高,有350个基因表达显著降低;RT-PCR和ELISA结果显示,Bcl-2、E2F1和E2F3基因表达显著降低,RB1和P53表达显著增高,均与芯片结果一致。结论 DMC可致K562细胞凋亡,并可引起凋亡细胞相关基因表达变化。
Objective To study the effect of norcantharidin (DMC) on the apoptosis of chronic myeloid leukemia cell line K562 and the change of gene expression after DMC induced apoptosis. Methods The morphological changes of the cells were observed under inverted microscope after 36 hours of treatment with 20 mg.L-1DMC. The DNA of the cells was extracted by DNA and the DNA of the apoptotic cells was detected by agarose gel electrophoresis. The total RNA was extracted and the gene expression profile was detected by microarray. The changes of the gene expression of the chip were verified by reverse transcription (RT) -PCR. SPSS13.0 software was used for statistical analysis. Results After K562 cells were treated with DMC, the cell morphology was deteriorated under inverted microscope and the number of viable cells was significantly reduced. The obvious step-like changes were observed after DNA electrophoresis. Among 21 522 gene transcripts detected by gene chip technology, The number of differentially expressed genes was about 2 300, of which over 500 genes were significantly increased and 350 genes were significantly decreased. RT-PCR and ELISA showed that the expression of Bcl-2, E2F1 and E2F3 genes were significantly decreased, while RB1 and P53 Significantly increased expression, consistent with the chip results. Conclusion DMC can induce apoptosis in K562 cells and induce changes in the expression of related genes in apoptotic cells.