WT1基因表达下调对K562／A02细胞阿霉素敏感性的影响

The effects of WT1 gene down regulation on the sensitivity of K562/A02 cells to adrimycin

目的探讨WT1基因表达下调对人红白血病耐药细胞系K562／A02阿霉素敏感性的影响。方法将WT1mRNA的短发夹RNA（shorthairRNA，shRNA）构建至真核表达载体后转染K562／A02细胞，流式细胞术检测转染效率；荧光定量RT—PCR和Westernblot法分析WT1基因在转染前后表达的差异；pWT1shRNA转染K562／A02细胞48h并经阿霉素作用后，MTT法检测各组细胞阿霉素IC50值；流式细胞术检测细胞阿霉素累积量及细胞凋亡率。结果与未转染对照组及空载体组相比，转染WT1shRNA质粒的K562／A02细胞WTImRNA和蛋白水平均明显降低；经阿霉素诱导24h后，其对阿霉素的敏感性明显增高，相对逆转率为71．5％，细胞内阿霉素的累积量较各对照组明显增高（P值均〈0．05），细胞凋亡率明显升高（P〈0．05）。结论靶向WT1shRNA干扰质粒可有效抑制K562／A02细胞WT1基因表达，增强K562／A02细胞对阿霉素的敏感性。

Objective To investigate the effects of WT1 gene clown regulation on the sensitivity of K562/A02 cells to aclrimycin （ADM）. Methods Short hair RNA （shRNA） plasmid vector targeting WT1 gene with enhanced green fluorescent protein （EGFP） was transfeeted into K562/A02 cells. Transfection efficiency was detected and transfected cells were sorted by flow cytometery. Expression of WT1 gene was detected by real time fluorescence RT-PCR and Western blot. The IC50 of ADM on K562/A02 cells was measured by MTT assay. Intracellular ADM accumulation and ADM-induced apoptosis of K562/A02 ceils were detected by flow cytometry. Results 48 h after transfection, the transfection efficiency was （ 68.2 _＋ 4.5 ） %. Both the mRNA and protein of WT1 were significantly decreased in K562/A02 cells transfected with WT1 shRNA plasmid than in control. After 24 h ADM induction, the relative reverse rate of sensitivity of K562/A02 cells to ADM was 71.5%. The intracellular accumulation of ADM was greatly increased （ P 〈 0.05 ） and ADM-induced apoptosis rate elevated from 10.4% to 67.3% （P 〈 0.05）. Conclusion shRNA targeting WT1 gene can efficiently inhibit the WT1 expression in K562/A02 cells and enhance the drug sensitivity of K562 cells to ADM.