D-氨基酸氧化酶/D-丙氨酸系统杀伤K562e细胞的作用机制探讨  

Study on mechanism of D- amino acid oxidase/D- Alanine system on killing K5 62 e cell in vitro

作　　者：翟勇平[1] 王健民[2] 张雨生[2] 吕书晴[2] 

机构地区：[1]南京军区南京总医院血液科,南京210002 [2]第二军医大学长海医院血液科,上海200433

出　　处：《第二军医大学学报》2002年第2期163-165,共3页Academic Journal of Second Military Medical University

基　　金：国家自然科学基金资助项目 (39870 71 0 );上海市卫生系统百名跨世纪优秀学科带头人培养计划资助项目 (98BR0 2 9)

摘　　要：目的 :探讨 D-氨基酸氧化酶 (DAAO) / D-丙氨酸 (D - Ala)系统用作自杀基因治疗白血病的作用机制。方法 :以 D- Ala杀伤稳定表达 DAAO和绿荧光蛋白 (GFP)的高致瘤性 K 5 6 2 e单克隆细胞 KDf Gd、KDf GC,应用 MTT法检测细胞存活率 ,并用酚红氧化法测定培养上清 H2 O2 和 L owry法测定细胞蛋白数量 ,流式细胞仪测定细胞 GFP的荧光强度。结果 :在 2 5 m mol/ L D-Ala作用下 2 4 h即可完全杀死 KDf Gd细胞 ,当 D- Ala为 2 0 mmol/ L 时延长作用时间至 4 8h,杀伤率由 6 4 %增加至 96 .8% ,而D- Ala≤ 15 mm ol/ L 时杀伤率增加不明显。低于 2 0 m mol/ L,D- Ala对 K5 6 2 e几乎无杀伤作用。培养上清的 H2 O2 变化与杀伤转基因细胞作用相一致。 结论 :DAAO/ D- Ala系统可能是通过产生 H2 O2 发挥杀伤转基因 K5 6 2Objective:To investigate the m echanism of D- amino acid oxidase/D- Alanine system in killing K5 6 2 e cells in vitro.Methods:The killing effects of D- Ala on K5 6 2 e cells stably expressing DAAO and GFP were observed.H2 O2 production by DAAO+ cells were m easured by the phenol red oxidation assay.L owry method was used to determ ine the protein quantities of cells and fluorescent intensities of GFP+ cells were assayed by flow cytom eter.Results:KDf Gd cells were killed completely after treated with 2 5 mm ol/L D- Ala for 2 4 h.The effect of D - Ala at 2 0 m mol/L on KDf Gd cells increased apparently within 4 8h,but the same effect was not observed if D - Ala was below 15 m mol/L .The cytotoxicity of D- Ala in KDf Gd cells was more sensitive than in parental K 5 6 2 e cells.The H2 O2 levels in the medium were consistent with the killing effects of D- Ala.Conclusion:The killing effects of DAAO/D- Ala system is m ediated by H2 O2 . [

关 键 词：D-氨基酸氧化酶 D-丙氨酸 过氧化氢 白血病 K562e细胞 

分 类 号：R733.7[医药卫生—肿瘤]