摘要
目的探讨高迁移率族蛋白1（HMGB1）基因沉默对白血病细胞耐药逆转的作用。方法将HMGB1基因特异性干扰RNA（HMGB1 siRNA）导入K562／A02细胞中，通过Western blot和RT—PCR方法检测HMGB1基因在转染前后的表达；WST8法检测阿霉素（ADM）对转染前后K562／A02细胞的半数抑制浓度（IC50）；流式细胞术检测凋亡细胞百分率；Western blot检测线粒体促凋亡蛋白Smac／DIABLO的释放：采用caspase活性定量检测试剂盒分析caspase-3的活性。结果①与未经处理的K562／A02细胞组相比，转染HMGB1 siRNA的K562／A02细胞组HMGB1 mRNA和蛋白水平分别下降86％和71％；②HMGB1基因沉默使K562／A02细胞对ADM的药物敏感性增强，其IC50值从转染前的（4．83±0.08）μg／ml降低到（1．33±0．10）μg／ml，并在ADM浓度为1μg／ml和5μg／ml时，细胞凋亡百分率分别增加27％、32％；③HMGB1基因沉默可促进ADM所致Smac／DIABLO从线粒体向胞浆释放。并增加caspase-3的活性。结论HMGB1基因沉默能明显增加K562／A02细胞对ADM的敏感性，逆转K562／A02细胞对ADM耐药。
Objective To investigate the effect of high mobility group boxl （ HMGB1 ） gene silence on adriamycin （ADM）-induced apoptosis in K562/A02 drug resistance leukemia cells. Methods K562/ A02 cells were transient transfected with HMGB1- small interference RNA（siRNA） vector, and the levels of HMGB1 gene differential expression pre-and post-transfection were measured by RT-PCR and Western blotting. 50% inhibition concentration （IC50） of ADM on K562/A02 was determined by WST-8 assay. Cell apoptosis was assessed by flow cytometry. The release of Smac/DIABLO from the mitochondria to the cytoplasm was assayed by Western blotting. Activity of Caspase-3 was assayed with a Caspase Colorimetric Assay Kit. Results ①The HMGB1 expression at mRNA and protein levels in HMGB1 siRNA transfected K562/A02 cells were decreased by 86% and 71% respectively compared with control. ②Suppression of HMGB1 by siRNA in K562/A02 cells resulted in a reversal of the resistance to ADM, and decreased IC50 from （4.83 ± 0.08 ） μg/ml to （ 1.33 ± 0.10 ） μg/ml. 1 μg/ml and 5 μg/ml of ADM treatment increased cell apoptotic rate by 27% and 32% respectively.③HMGB1 suppression in K562/A02 cells significantly promoted ADM- induced Smac/DIABLO release from the mitochondria to the cytoplasm, and increased the activities of Caspase-3. Conclusion HMGB1 gene silence can enhance sensitivity of K562/A02 cells to ADM and reverse cell resistant to ADM.
出处
《中华血液学杂志》
CAS
CSCD
北大核心
2008年第8期549-552,共4页
Chinese Journal of Hematology
基金
国家自然科学基金（30571982、30772353）
