RNA腺苷脱氨酶在MLL-AF9诱导的小鼠急性髓系白血病发病中的作用  

Effect of ADAR1 on the development of MLL-AF9 induced murine AML

作　　者：彭路芸 杨鑫[2] 张英驰 胡甜园[1] 王伟丽[1] 汪晓敏[1] 许静[1] 程涛[1] 袁卫平[1] 高瀛岱[1] 

机构地区：[1]中国医学科学院、北京协和医学院血液学研究所、血液病医院 实验血液学国家重点实验室,天津300020 [2]山东省德州市人民医院

出　　处：《中华血液学杂志》2015年第5期383-388,共6页Chinese Journal of Hematology

基　　金：科技部国家973项目（2012CB966601、2013BAl01809）;国家自然科学基金（81090413、81470280、81328003）

摘　　要：目的 建立敲除RNA腺苷脱氨酶1（ADAR1）的小鼠MLL-AF9融合基因急性髓系白血病（AML）模型,初步探讨ADAR1对AML发病的影响.方法 采用免疫磁珠法富集介导雌激素受体-重组酶Cre（ER-Cre）的ADAR1lox/lox及其对照ADAR1lox/lox小鼠骨髓Lineage-（Lin-）细胞,用携带MSCV-MLL/AF9-IRES-GFP的逆转录病毒感染上述Lin-细胞,流式细胞术检测感染效率,移植相同数量细胞至致死剂量和半致死剂量照射受体小鼠中,建立MLL-AF9诱导的AML模型.移植48 h后诱导ADAR1敲除,体内实验分为实验组（ER-Cre;ADAR1lox/lox＋他莫昔芬）和对照组（①ER-Cre;ADAR1lox/lox＋空载体、②ADAR1lox/lox＋他莫昔芬、③ADAR1lox/lox＋空载体）,第10、15、20天分别检测小鼠外周血GFP＋细胞比例,观察各组小鼠的存活情况.体外实验分组同上,将他莫昔芬改为4-羟基他莫昔芬,观察各组AML细胞并检测其凋亡情况.结果 成功建立敲除ADAR1的MLL-AF9融合基因AML小鼠模型.与对照组比较,体内实验中实验组AML小鼠在各时间点外周血GFP＋细胞比例均降低,存活时间明显延长,差异均有统计学意义（P值均＜0.05）;体外实验中实验组细胞总数、GFP＋细胞比例均降低,Annexin Ⅴ＋7-AAD＋和Annexin Ⅴ＋细胞比例均升高,差异均有统计学意义（P值均＜0.05）.结论 敲除ADAR1可减缓AML的发病,增加AML细胞凋亡.ADAR1在MLL-AF9诱导的AML发生和维持过程中起关键作用.Objective To establish the ADAR1 （adenosine deaminase that act on RNA 1） knockout MLL-AF9 acute myeloid leukemia （AML） mouse model,and to preliminarily investigate the effects of ADAR1 deletion on the development of AML.Methods The lineage-（Lin） cells of ERCreADAR1lox/lox mice and their ADAR1loxlox counterparts were enriched by magnetic activated cell sorting （MACS） and then transduced with retrovirus carrying MSCV-MLL/AF9-IRES-GFP fusion gene.The efficiency of transduction was detected by flow cytometry,and equal number of GFP ＋ cells were transplanted into lethally irradiated recipient mice.The recipient mice were treated with tamoxifen at 48 hours after transplantation to induce ADAR1 knockout and divided into following groups：experimental group （ER-Cre;ADAR1lox/lox＋tamoxifen）,control groups （①ER-Cre;ADAR1 lox/lox＋vechile,②ADAR1lox/lox＋tamoxifen,③ADAR1lox/lox＋vechile）.The percentage of GFP＋ cells in peripheral blood was examined at 10,15 and 20 days respectively after transplantation and the survival of the recipient mice was observed.In vitro study,ER-Cre;ADAR1lox/lox and ADAR1lox/lox AML cells were cultured and the apoptosis rates of these cells 48 hours after 4-hydroxytamoxifen treatment were examined.Results The ADAR1 deletion MLL-AF9 AML mouse model was successfully established.Deletion of ADAR1 could decrease the percentage of GFP＋ cells in the peripheral blood and significantly prolong the survival rate of recipient mice （P〈0.05）.In vitro study showed that the cultured total cell number,percentage of GFP ＋ cells decreased and the apoptosis rate of AML cells increased.Conclusion Ablation of ADAR1 could delay the progression of AML in recipient mice.ADAR1 plays a critical role in the development and maintenance of murine MLL-AF9 AML.

关 键 词：腺苷脱氨酶 重组融合蛋白质类 白血病 髓样 急性 基因敲除技术 小鼠 转基因 

分 类 号：R733.71[医药卫生—肿瘤]