利用基因芯片分析二氢青蒿素的抗肿瘤作用机制

Analyzing anti-cancer action mechanisms of dihydroartemisinin using gene chip

目的:通过基因芯片对二氢青蒿素处理的K562细胞基因表达情况的检测,探讨二氢青蒿素抑制K562增殖的作用机制。方法:配制1×10^(-5),4×10^(-5),16×10^(-5),64×10^(-5),256×10^(-5)mol·L^(-1)二氢青蒿素处理K562细胞24h,倒置光显微镜和荧光显微镜观察细胞变化,流式细胞仪检测细胞周期,提取总RNA,逆转录成cDNA与基因芯片杂交,扫描仪检测杂交结果。结果:倒置光显微镜下细胞出现不同程度的皱缩,核分裂相减少,细胞密度下降。荧光显微镜下染色质高度浓缩、边缘化,凝聚成明亮的团块,即凋亡小体。流式细胞仪G_2期细胞的比例增加。扫描杂交结果显示有13条基因表达有差异,其中chk1表达上调,PCNA,cyclinB1,cyclinD1,cyclinE1,cdk4,cdk2,E2F1,DNA-PK,DNA-TopoⅠ,mcl-1,jNK,VEGF表达下调。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 understand the action mechanisms of artesunate on inhibiting leukaemia cell line K562 on the molecular level. Method： The gene chip was used to detect the expression panel of genes of leukaemia cell line K562 treated by dihydroartemisinin. K562 cells were treated with 1 × 10^-5, 4× 10^-5, 16× 10^-5, 64 × 10^-5, 256× 10^-5 mol·L^-1 dihydroartemisinin for 24 h, and then studied the modality changes by invert microscope. The morphological changes of the nucleons were observed by Hoechst33342/PI staining. The cell cycle were examined by flow cytometry analysis（FCM）. Total RNA samples were obtained by TRIzol and were reverse transcribed to the cDNA. The cDNA samples were hybridized to our gene chips. Hybridization signal were collected and analyzed following scanning by Gene Pix 4100A. Result： The numbers of drift cells 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 cell apoptosis such as karyopyknosis and conglomeration were observed by Hoechst 33342/PI staining. Flow cytometric analysis showed that cells were arrested in G2 phase. There were 13 differentially expressed genes identified. Hybridization analysis showed up-regulation of chkl and down-regulation of PCNA, cyclinB1, cyclinD1, cyclinE1, cdk4,cdk2, E2F1, DNA-PK, DNA-Topo I, mcl-1, jNK, VEGF in the dihydroartemisinin-treated K562 cells. Conclusion： Dihydroartemisinin can Inhibit the leukaemia cell line K562 and exert its anticancer effect by altering the expression of these genes involved in cell cycle; dihydroartemisinin may act via apoptosis pathway.