Antizyme1基因转染对K562细胞增殖与凋亡的影响

研究鸟氨酸脱羧酶抗酶蛋白对人红白血病K562细胞增殖、三氧化二砷（ As2O3）诱导凋亡时的影响。方法: 定点突变技术构建缺失frameshift位点的pEGFP-N1-AZ1-mutation重组表达载体。脂质体法转染K562细胞，通过G418筛选获得稳定表达antizyme1的K562pAZ1m细胞系。采用不同浓度的As2O3处理细胞，通过MTT法检测细胞增殖，流式细胞术分析细胞周期及凋亡变化。并通过RT-PCR方法检测antiyme1转染对cyclin D1和survivin基因表达的影响。结果：获得稳定表达antizyme1的K562-AZ1m细胞株后，其增殖能力明显减慢。CyclinD1基因表达降低，细胞主要停滞于G0/G1期。在 As2O3的诱导作用下，细胞凋亡增多，survivin基因表达降低。结论：AZ1基因能够抑制K562细胞增殖，通过对cyclinD1的负调控使细胞周期停滞于G0/G1期。并可能通过下调survivin表达来加强 As2O3对其的诱导凋亡作用

The effect of antizyme1 transfection on cell proliferation and cell apoptosis in K562 cells

Objecive: To study the effect of antizyme1 on the cell proliferation and cell apoptosis induced by As2O3 in human leukemia K562 cells. Methods: The base of frameshift was deleted by site-directed mutagenesis assay and the mutation antizyme1 gene was then cloned into vector of pEGFP-N1. Then the recombination DNA was transfected into K562 cells and stable transferctants were isolated after selection with G418. Cell proliferation was checked by MTT assay, and the cell cycle was detected by FCM. The level of cyclin D1 mRNA was measured by RT-PCR assay. After treated with various concentration of As2O3, apoptosis rate of K562pAZ1m was detected by FCM.The level of survivin mRNA was measured by RT-PCR assay also. Results: The mutation antizyme1 gene was successful cloned into pEGFP-N1. When transfected into K562 cells, the cell proliferation was inhibited and cell cycle was arrested at G0/G1 stage according with down regulation of cyclin D1 expression. After treated with As2O3, apoptosis rate of K562pAZ1m rising is coincident with the decrease of survivin expression. Conclussion: antizyme1 can inhibite proliferation in K562 cells and arrest cell cycle by down regulating cyclin D1 expression. And antizymes also enhance the apoptosis rate induced by As2O3 partly via decreasing survivin expression