基因芯片技术分析二氢青蒿素抑制K562细胞增殖的作用机制

Analyzing the anti-K562 cell mechanisms of dihydroartemisinin with gene chip

【目的】利用基因芯片检测二氢青蒿素作用于K562细胞后的基因表达情况,从基因水平探讨二氢青蒿素抑制K562细胞增殖的作用机制。【方法】K562细胞经不同浓度二氢青蒿素处理24h,倒置光显微镜和荧光显微镜观察细胞形态学变化,流式细胞仪检测细胞周期变化。提取总RNA,逆转录生成cDNA,同时Cy3标记。标记好的cDNA与基因芯片杂交,用扫描仪检测杂交结果。【结果】倒置光显微镜可见细胞出现不同程度的皱缩,核分裂相减少,细胞密度下降,漂浮细胞增多。荧光显微镜下可观察到染色质高度浓缩、边缘化,凝聚成明亮的团块,即凋亡小体,同时有死亡细胞。流式细胞仪检测显示G2期细胞的比例明显增加。扫描杂交结果数据显示其中13条基因表达有差异。表达上调的基因有chk1,表达下调的基因有:PCNA、cyclinB1、cyclinD1、cyclinE1、cdk4、cdk2、E2F1、DNA-PK、DNA-TopoⅠ、mcl-1、jnk、VEGF。【结论】二氢青蒿素可以抑制K562细胞增殖,作用机制与改变细胞周期某些调控物质的基因表达、诱导K562细胞凋亡等有关。

[ Objective] To detect the expression panel of genes of leukemia cell line K562 treated with dihydroartemisinin and to explore the anti-K562 cell mechanism of dihydroartemisinin on molecular level. [Methods] K562 cells were treated with dihydroartemisinin for 24 h. Morphological changes of K562 cells were studies by invert microscope and Hoechst33342/PI staining. The cell cycle state were examined by flow cytometry analysis （FCM）. Total RNA samples were obtained by TRIzol and reverse transcribed to the cDNA, cDNA samples were hybridized with gene chips and hybridization signal were collected and analyzed following scanning by Gene Pix 4100A. [Results] The numbers of drift cell were increased and the density of cells was decreased under invert microscope after K562 cells were treated with dihydroartemisinin for 24 h. Morphological changes of apoptotic cell such as karyopyknosis and conglomeration were observed. Flow cytometric analysis showed that cells were arrested at G2 phase. There were 13 differentially expressed genes identified. Hybridization analysis showed up-regtdation of chkl and down-regulation of PCNA, cyclinBl, cyclinD1, cychnE1, cdk4, cdk2, E2F1, DNA-PK, DNA-Topo Ⅰ , mcl-1, jnk and VEGF in the dihydroartemisinin-treated K562 cells. [ Conclusions] Dihydroartemisinin may inhibite leukaemia cell line K562. Dihydroartemisinin exert its anti K562 cell effect by altering the expression of these genes involved in cell cycle and its action may be related with apoptosis pathway.