舟山眼镜蛇细胞毒素1对K562细胞的抑制作用

Inhibitory effect of cytotoxin 1 from Naja atra Cantor venom on K562 cells

目的:研究舟山眼镜蛇细胞毒素1(CTX1)对人慢性粒细胞白血病细胞系K562的抑制作用。方法:应用MTS方法和细胞计数法分别检测不同浓度CTX1作用K562后的细胞相对活力和细胞数;PI染色后用倒置荧光显微镜观察活细胞培养体系中K562细胞死亡情况;流式细胞术检测Annexin V-FITC和PI双染色后细胞凋亡情况。结果:不同浓度的CTX1(2、5、10 mg/L)作用于K562细胞24 h,细胞相对活力分别为(90.50±3.07)%、(58.33±3.08)%和(27.43±1.99)%(与阴性对照组相比,下同,P<0.05),作用24 h时CTX1对K562细胞的半数抑制浓度为5.77 mg/L。CTX1(8 mg/L)作用于K562 6 h、12 h、24 h,细胞数分别占对照组的(85.01±3.54)%、(56.65±3.59)%和(43.24±4.15)%,P<0.05。随CTX1浓度增加和作用时间延长,荧光显微镜下观察到被PI染色的细胞数逐渐增多。CTX1(8 mg/L)作用于K562细胞6 h、12 h、24 h,细胞坏死率分别为(0.73±0.06)%、(13.90±0.46)%和(23.33±0.86)%;晚期凋亡率分别为(16.27±0.21)%、(26.90±1.23)%和(18.77±0.81)%。结论:CTX1对K562细胞有明显抑制作用,主要引起K562细胞晚期凋亡和坏死。

AIM： To investigate the inhibitory effect of cytotoxin 1（CTX1） from Naja atra Cantor venom on human chronic myeloid leukemia cell line K562.METHODS： The MTS and cell counting methods were used to detect cell relative viability and cell numbers of K562 cells treated with CTX1 at different concentrations.In the living culture system,the dead and dying cells stained by PI were observed by inverted fluorescence microscope.After treated with CTX1,the apoptotic cells were detected by flow cytometry with annexinV-FITC and PI double staining.RESULTS： The relative viabilities of K562 cells were（90.50±3.07）%,（58.33±3.08）% and（27.43±1.99）% when the cells were treated with CTX1 for 24 h at the concentrations of 2,5 and 10 mg/L,respectively.The median inhibitory concentration of CTX1 on K562 cells was 5.77 mg/L after 24-hour treatment.Comparment with control group,the percentages of K562 cells by cell counting were（85.01±3.54）%,（56.65±3.59）% and（43.24±4.15）% after treatment with 8 mg/L of CTX1 for 6 h,12 h,24 h,respectively.As treatment concentration of CTX1 was elevated and treatment time was prolonged,the cells stained by PI were remarkably observed under inverted fluorescence microscope.After treatment with CTX1 at 8 mg/L for 6 h,12 h and 24 h,the incidences of cell necrosis were（0.73±0.06）%,（13.90±0.46）% and（23.33±0.86）%,respectively,and the incidences of late apoptosis were（16.27±0.21）%,（26.90±1.23）% and（18.77±0.81）%,respectively.CONCLUSION： CTX1 possesses obvious inhibitory effect on K562 cells and it mainly causes the late phase of apoptosis and necrosis.