中文摘要：

背景与目的：高迁移率族蛋白1（high mobility group box 1，HMGB1）作为核DNA结合蛋白参与稳定染色质结构与功能和基因转录调控。近年研究发现，细胞内外HMGB1与多种肿瘤（例如，乳腺癌、结肠癌、黑素瘤）增殖和转移有密切关系，在多种实体瘤组织和未成熟细胞中表达丰富。本研究的目的是探讨转染HMGB1基因对阿霉素（adriamycin，ADS）诱导的白血病K562细胞凋亡的影响。旨在进一步阐明HMGB1在儿童白血病中的分子作用机制。方法：将HMGB1基因真核表达载体pcDNA3．1．HMGB1转染至K562细胞，构建HMGB1基因高表达的l（562细胞。Western blot和RT-PCR（reverse transcdption-polymerase chain raction）法检测基因转染前后K562细胞中HMGB1蛋白及mRNA的表达水平；WST8法检测ADM对转染前后细胞的半数抑制浓度（IC50）；流式细胞仪检测和计算凋亡细胞百分率；Western blot检测抗凋亡蛋白Bcl-2的蛋白表达；采用Caspase活性定量检测试剂盒分析Caspase-3和Caspase-9的活性。结果：与转染空载体的K562细胞相比。转染HMGB1基因的K562细胞HMGB1mRNA表达水平增加85％．蛋白表达水平增加56％。HMGB1基因过表达降低了K562细胞对ADM的药物敏感性，使ADM的IC50从转染前的（0．06±0．00）ug/mL增加到（3．46±0．06）ug／mL，并在ADM浓度为1ug／mL时凋亡细胞百分率下降31％。HMGB1基因过表达抑制了ADM所致K562细胞Bd．2蛋白表达水平下降。ADM处理细胞12h和24h后，转染HMGB1基因的K562细胞的Caspase-3和Caspase-9活化明显受到抑制（1．55±0．06vs 2．55±0．06，1．86±0．10vs．2．85±0．06；1．40±0．08vs．2．03±0．05。1．55±0．06 vs．2．22±0．05，P值均≤0．05）。结论：HMGB1高表达可通过调节Bcl-2蛋白水平和Caspase-3及Caslmse-9活性来抑制阿霉素诱导的白血病K562细胞凋亡。

英文摘要：

BACKGROUND ＆ OBJECTIVE： High mobility group box 1 （HMGB1）, a nuclear DNA-binding protein, stabilizes the structure and function of chromatin, regulates gene transcription. Recent studies found that HMGB1 is associated with the proliferation and metastasis of many tumors, including breast cancer, colon carcinoma, and melanoma, and is rich in various solid cancer tissues and immature cells. This study was to explore the role of HMGB1 in adriamycin （ADM）-induced apoptosis in leukemia K562 cells. METHODS.. K562 cells were transiently transfected with recombinant plasmid pcDNA3.1-HMGB1. The expression of HMGB1 in K562 cells were detected by Western blot and reverse transcription-polymerase chain reaction （RT-PCR）. The 50% inhibition concentration （IC50） of ADM for K562 cells was determined by WST-8 assay. Cell apoptosis was assessed by flow cytometry. The protein level of Bcl-2 was detected by Western blot. The activities of Caspase-3 and Caspase-9 were assayed with Caspase Colorimetric Assay Kit. RESULTS, The mRNA and protein levels of HMGB1 in K562 cells transfected with pcDNA3.1-HMGB1 were increased by about 85% and 56% respectively as compared with those in K562 cells transfected with pcDNA3.1. Overexpression of HMGB1 in K562 cells by transient transfection significantly increased the resistance to ADM; the IC50 of ADM was increased from （0.06±0.00） ug/mL to （3.46±0.06） ug/mL. When treated with 1ug/mL ADM, the apoptosis rate was significantly lower in HMGBl-transfected K562 cells than in pcDNA3.1- transfected K562 cells [（12.00±1.26）% vs. （44.50±1.87）%, P〈0.05]. Overexpression of HMGB1 in K562 cells significantly inhibited ADM-induced down-regulation of Bcl-2 protein. After treatment of ADM, the activities of Caspase-3 and Caspase-9 in HMGBl-transfected K562 cells were inhibited as compared with those in pcDNA3.1-transfected K562 cells （Caspase-3. 1.55± 0.06 vs. 2.55±0.06 at 12 h, 1.86±0.10 vs. 2.85±0.06 at 24 h, P〈0.05; Caspase-9. 1.40±0.08 vs. 2.03±0.05 at 12 h