一氧化氮增强环磷酰胺对白血病细胞损伤效应的实验研究  

Experimental study of the nitric oxide-enhanced damaging effect of cyclophosphamide therapy on leukemia cells

作　　者：姚仁南[1] 朱培元[2] 张春雷[1] 王涛[1] 

机构地区：[1]解放军第九七医院输血科,江苏徐州221004 [2]南京军区南京总医院输血科,江苏南京210002

出　　处：《医学研究生学报》2006年第4期315-318,共4页Journal of Medical Postgraduates

基　　金：南京军区南京总医院科研基金资助项目(批准号:2004072)

摘　　要：目的:观察体外培养条件下一氧化氮(NO)能否增强环磷酰胺对L1210细胞的损伤效应,并探讨其作用机制。方法:将不同的转染3T3细胞与L1210细胞进行共培养,DMEM培养液中加入终浓度为400μg/ml的环磷酰胺。根据接种的转染细胞不同分为三组:第1组为pcDNA3.0iNOS转染的3T3细胞,第2组为pcDNA3.0转染的3T3细胞,第3组为pcDNA3.0iNOS转染3T3细胞,并添加终浓度100μmmol/L的Caspase-3特异性抑制剂DEVDCHO。分别采用锥虫蓝拒染试验和TUNEL试验,检测各组L1210细胞在不同培养时间的细胞成活率和凋亡率,同时利用流式细胞术分析各组L1210细胞的增殖状态。结果:①各组L1210细胞成活率随培养时间的延长均逐渐下降,第1组较第2组下降更快,从12h开始两组差异已有显著性意义(P<0.05);第3组的细胞成活率明显高于第1组,培养24h开始差异有显著性意义(P<0.05)。②各组L1210细胞的凋亡率持续上升,第1组较第2组升高更快,从培养12h开始即差异有显著性意义(P<0.05);第3组细胞凋亡率明显下降,从12h开始即显著低于第1组(P<0.05)。③培养4h时,各组L1210细胞中的G1期细胞百分率(G1%)显著升高,S期细胞百分率(S%)明显下降,第2、3组的G1%显著低于第1组(P<0.05),S%显著高于第1组(P<0.01、P<0.05);培养8、12h时,三组之间的G1%和S%均无明显差异;培养24h后,第2、3组G1%开始下降,S%上升较快,第1组的G1%保持较高水平,S%持续较低(P均<0.05)。结论:NO可增强环磷酰胺对L1210细胞的直接损伤作用,促进细胞凋亡,使环磷酰胺对G1期细胞的阻滞作用更敏感、持续时间更长,上述作用可能与Caspase3蛋白的活化有关。Objective ： To observe whether nitric oxide （NO） could enhance the damaging effect of cyclophosphamide on L1210 cells cultured in vitro, and to investigate the mechanism of this action. Methods：L1210 cells were co-cultured with 3T3 cells in DMEM medium supplemented with cyclophosphamide （400μg/ml）. The L1210 cells were divided into three groups based on different transfected 3T3 cells ： pcDNA3.0-iNOS plasmid transfected 3T3 cells （ Group 1 ）, pcDNA3.0 plasmid transfected 3T3 cells （ Group 2）, pcDNA3.0-iNOS plasmid transfected 3T3 cells plus DEVD-CHO（ Group 3 ）. The viability and apoptosis rate of L1210 cells at different culture periods were determined by trypan blue exclusion and TUNNEL method, respectively. And the cell cycles at GI and S phase were detected by flow cytometry. Results ：①After cyclophosphamide treatment, the viability of L1210 cells was significantly lower in Group 1 than that in Group 2 during 12 - 72 h （ P 〈 0.05 ）, and there was also significant difference between Group 1 and Group 3 during 24 - 72 h （ P 〈 0.05 ）. ②Mter cyclophosphamide treatment, the apoptosis rate of L1210 cells in Group 1 was significantly higher than that in Group 2 during 12 -72 h （P 〈 0.05）, and there was also significant difference between Group 1 and Group 3 during 12 -72 h （P 〈 0.05 ）. ③After cyclophosphamide treatment, cells at G1 phase were more at 4 h and during 24 - 72 h in Group 1 than those in Group 2 （P 〈 0.05 ）. Cells at S phase were less at 4 h and during 24 - 72 h in Group 1 than those in Group 2 （P 〈 0.05 ）. In Group 3, cells at G1 phase were less at 4 h and during 24 - 72 h than those in Group 1 （ P 〈 0.05 ） ; cells at S phase were more at 4 h and during 24 - 48 h than those in Group 1 （ P 〈 0.05 ）. Conclusion ： NO can enhance the direct damaging effect of cyclophosphamide on L1210 cells, through enhancement of apoptosis and blockage of G1 phase, which may depend on Caspase-3 activation.

关 键 词：一氧化氮 白血病 损伤效应 细胞凋亡 CASPASE-3 

分 类 号：R733.7[医药卫生—肿瘤]