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目的:研究纳米雄黄对药物敏感性白血病细胞的凋亡诱导作用。方法:采用机械研磨法制备纳米雄黄。以白血病药物敏感K562细胞为靶细胞,采用MTT法检测纳米雄黄对K562细胞的增殖抑制作用,AnnexinV/PI双染色法检测细胞凋亡,细胞流式细胞术检测Bax,Bcl-2,P-gp,BCRP,P-53蛋白的表达水平,Caspase-3活性。结果:雄黄经高能球磨机球磨10～50h,扫描电镜(SEM)和透射电镜(TEM)观察,电镜测量纳米雄黄的平均粒径为(72.72±22.18)nm,符合纳米颗粒的形貌特征。纳米雄黄对K562细胞有明显的增殖抑制作用。24,48,72 h的IC50分别为43.48,20.52,16.07 mg.L-1。20,50 mg.L-1纳米雄黄作用48 h后Annexin V/PI染色显示凋亡细胞明显增高,凋亡率分别为10.52%,73.25%。纳米雄黄作用48 h后Bax蛋白表达由对照的(75.80±2.40)%升高到(87.50±1.20)%和(99.60±0.10)%;Bcl-2蛋白表达由对照的(73.85±0.15)%升高到(94.80±2.00)%和(97.75±0.55)%。20,50 mg.L-1纳米雄黄处理K562细胞24 h后,K562细胞Caspase-3蛋白表达水平由对照的(3.14±0.24)%升高到(8.87±2.74)%和(72.55±1.45)%;细胞的BCRP,P-gp蛋白,P53蛋白表达总体呈上升趋势,共表达也呈上升趋势。结论:纳米雄黄可以显著诱导白血病药物敏感K562细胞发生凋亡。
Objective: To study the apoptosis induction effect of nano-realgar on drug-sensitive leukemia cells. Methods: Nano realgar was prepared by mechanical grinding method. To leukemia drug-sensitive K562 cells as target cells, MTT assay was used to detect the proliferation inhibition effect of nano-realgar on K562 cells, apoptosis was detected by Annexin V / PI double staining and Bax, Bcl-2 and P-gp were detected by flow cytometry , BCRP, P-53 protein expression level, Caspase-3 activity. Results: The realgar was ball-milled for 10 ~ 50h by high-energy ball mill, and observed by SEM and TEM. The average diameter of realgar was 72.72 ± 22.18 nm, which was consistent with the morphology of nanoparticles. Nano-realgar on K562 cells had a significant inhibition of proliferation. The IC50 values at 24, 48, and 72 h were 43.48, 20.52, 16.07 mg.L-1.20, 50 mg.L-1, respectively. Annexin V / PI staining showed that apoptotic cells were significantly increased Respectively 10.52% and 73.25%. The expression of Bax protein was increased from (75.80 ± 2.40)% to (87.50 ± 1.20)% and (99.60 ± 0.10)% at 48 h after nano-realgar administration. The expression of Bcl-2 protein increased from 73.85 ± 0.15% High to (94.80 ± 2.00)% and (97.75 ± 0.55)%. Caspase-3 protein expression in K562 cells increased from (3.14 ± 0.24)% to (8.87 ± 2.74)% and (72.55 ± 1.45)% in K562 cells treated with 20 and 50 mg · L- ; The expression of BCRP, P-gp and P53 protein in cells showed an upward trend, and the co-expression also showed an upward trend. Conclusion: Nano-realgar can significantly induce the apoptosis of K562 cells induced by leukemia.