亚砷酸钠对大鼠骨髓间充质干细胞的过氧化损伤

NaAsO_2 induces cytotoxicity to rat bone marrow mesenchymal stem cells

目的:探索亚砷酸钠(sodiumarsenite,NaAsO2)对原代培养的大鼠骨髓间充质干细胞(mesenchymal stem cells,MSCs)的氧化应激作用.方法:采集4周龄SD大鼠骨髓,体外培养获得大鼠骨髓MSCs,取第3代大鼠骨髓MSCs,将不同浓度NaAsO2(10、20、30、40和50mol/L)作用于大鼠骨髓MSCs,进行染毒,染毒时间为24h,采用细胞毒性试验法观察NaAsO2对大鼠MSCs的细胞毒性作用,利用2',7'-二乙酰二氯荧光素(2',7'-dichlorofluorescin diacetate,DCFH-DA)检测细胞内活性氧(reactive oxygen species,ROS)水平,并测定细胞中丙二醛(malondialdehyde,MDA)含量及超氧化物歧化酶(superoxide dismutase,SOD)活力,同时测定细胞内还原型谷胱甘肽(reduced glutathione,GSH)的含量,Caspase3试剂盒检测Caspase3酶活性.结果:不同浓度的NaAsO2作用于大鼠骨髓MSCs,用MTS法进行细胞毒性检测显示,当NaAsO2浓度≥20mol/L时,大鼠骨髓MSCs吸光度值分别为1.98±0.09、1.45±0.12、1.26±0.11和1.02±0.15,与对照组比较明显降低(P<0.01);不同浓度NaAsO2作用于大鼠骨髓MSCs,DCF荧光强度分别为1572±124,1998±97、2337±210、2878±154和3960±135,与对照组相比均明显增高(P<0.05);不同浓度NaAsO2作用于大鼠骨髓MSCs,MDA分别为1.4EU/mL±0.06EU/mL、1.9EU/mL±0.12EU/mL、2.3EU/mL±0.08EU/mL、2.6EU/mL±0.11EU/mL和3.3EU/mL±0.09EU/mL,与对照组相比均明显增高(P<0.05);不同浓度NaAsO2作用于大鼠骨髓MSCs,SOD分别为29EU/mL±0.19EU/mL、22EU/mL±0.12EU/mL、17EU/mL±0.06EU/mL、12EU/mL±0.11EU/mL和10EU/mL±0.06EU/mL,与对照组相比均明显降低(P<0.05);当NaAsO2浓度≥20mol/L时,大鼠骨髓MSCs的GSH分别为75EU/mL±0.11EU/mL、71EU/mL±0.8EU/mL、62EU/mL±0.12EU/mL和54EU/mL±0.9EU/mL,与对照组相比均明显降低(P<0.05).结论:NaAsO2可以诱导大鼠骨髓MSCs氧化损伤增加,提示氧化应激可能是NaAsO2致大鼠骨髓MSCs细胞毒作用机制之一.

AIM： To investigate the toxic effect of NaAsO2 on rat bone marrow mesenchymal stem cells （MSCs） and to explore the possible mechanism involved. METHODS： Cultured rat bone MSCs were exposed to different concentrations of NaAsO2 （10, 20, 30, 40 and 50 mol/L） for 24 h. MTT assay was used to evaluate cell viability. The level ofreactive oxygen species （ROS） was detected by staining cells with DCFH-DA. The content of malondialdehyde （MDA） and activity of super-oxide dismutase （SOD） in rat bone MSCs were also measured. RESULTS： Treatment with NaAsO2 significantly decreased cell viability, GSH content and SOD activity, increased ROS and MDA formation, and induced Caspase 3 activation in rat bone MSCs cells. CONCLUSION： Our findings suggest that NaAsO2-induced oxidative stress may cause MSC apoptosis via mitochondria-dependent signaling pathways.