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目的:通过对中华眼镜蛇毒进行分离和各组分的初筛,寻找逆转K562对阿霉素耐药的活性成分KD-Ⅲ-1,为今后研究肿瘤耐药性奠定工作基础。方法:通过凝胶分离得到的蛇毒组分分别作用于K562对阿霉素耐药株K562/A和敏感株K562/S,筛选有效活性组分;通过荧光探针Rh123测定P-gp蛋白活性和PI染色进一步确定该组分的逆转K562/A的耐药活性。结果:分别给予2μg/mL阿霉素(Adr)和各浓度蛇毒组分处理24 h后,可以发现蛇毒组分对阿霉素敏感株K562/S和耐药株K562/A都有明显的抑制作用,并呈现出剂量-效应关系。1μg/mL蛇毒组分处理组与2μg/mL蛇毒处理组抑制作用明显;在药物持续作用48 h后对K562/A的活性仍有抑制作用在0.5、1、2μg/mL的蛇毒组分组表现的更加明显。通过Rho外排实验发现蛇毒粗毒组平均荧光强度(MFI)与阴性对照组没有明显差异,而2.5μg/mL蛇毒组分组的MFI明显降低,与对照组相比具有统计学意义(P<0.05)。2.5μg/mL蛇毒粗毒和分离组分分别对K562/S敏感株和K562/A耐药株作用3 h后,K562/S的PI染色阳性率明显升高,而K562/A的PI染色阳性率并没有明显升高。结论:蛇毒组分KD-Ⅲ-1对K562/A和K562/S细胞均有明显抑制的作用,抑制作用可能与诱导凋亡有关。
Abstract：Objective:To explore the active ingredient,KD-Ⅲ-1,for reversal of K562 resistance to adriamycin(Adr) via separation and primary screening of individual components of cobra venom,thus offering the sound basis for future study on tumor drug resistance.Methods:The K562 Adr-resistant strains,K562/A,and-sensitive strains,K562/S,were incubated with isolated snake venom components derived from gel separation,for exploration of the effective component.The P-gp protein activity was measured by the fluorescent probe Rhl23,and the PI staining was applied to determine the resistance reversal capacity of the components of K562/A.Results:Following incubation with 2 μg/mL Adr and cobra venom toxin of various concentrations,we found that the latter dose-dependently inhibited the growth of K562/A and K562/S strains at hour 24.The 1 μg/mL and 2 μg/mL cobra venom toxin yielded a marked inhibitory effect,which persisted for 48 hours on K562/A strains.This effect became more apparent when compared with the 0.5 μg/ml cobra venom toxin group.Rho efflux experiments failed to show significantly different mean fluorescence intensity(MFI) ratios between the venom crude toxin group and negative control group.The MFI of 2.5μg/mL cobra venom group was significantly lower than that in control group(P < 0.05).Incubation with 2.5 μg/mL crude toxin and isolated toxin yielded a higher rate of Pi-positive staining K562/S,but not K562/A,strains.Conclusion;The venom toxin component,KD-Ⅲ-1,significantly suppresses the growth of K562/A and K562/S cell lines possibly via induction of apoptosis.
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基本信息:
中图分类号:R446.5
引用信息:
[1]杨昌山,何慧华,董伟华.蛇毒组分对K562阿霉素敏感株和耐药株的抑制作用[J].广州医学院学报,2013,41(06):6-9.
基金信息:
广州医学院校内课题(04-k-37,2006ZR021)