CAJ | 학술논문

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.
목적 건립고제RNA선감탈안매1(ADAR1)적소서MLL-AF9융합기인급성수계백혈병(AML)모형,초보탐토ADAR1대AML발병적영향.방법 채용면역자주법부집개도자격소수체-중조매Cre(ER-Cre)적ADAR1lox/lox급기대조ADAR1lox/lox소서골수Lineage-(Lin-)세포,용휴대MSCV-MLL/AF9-IRES-GFP적역전록병독감염상술Lin-세포,류식세포술검측감염효솔,이식상동수량세포지치사제량화반치사제량조사수체소서중,건립MLL-AF9유도적AML모형.이식48 h후유도ADAR1고제,체내실험분위실험조(ER-Cre;ADAR1lox/lox+타막석분)화대조조(①ER-Cre;ADAR1lox/lox+공재체、②ADAR1lox/lox+타막석분、③ADAR1lox/lox+공재체),제10、15、20천분별검측소서외주혈GFP+세포비례,관찰각조소서적존활정황.체외실험분조동상,장타막석분개위4-간기타막석분,관찰각조AML세포병검측기조망정황.결과 성공건립고제ADAR1적MLL-AF9융합기인AML소서모형.여대조조비교,체내실험중실험조AML소서재각시간점외주혈GFP+세포비례균강저,존활시간명현연장,차이균유통계학의의(P치균＜0.05);체외실험중실험조세포총수、GFP+세포비례균강저,Annexin Ⅴ+7-AAD+화Annexin Ⅴ+세포비례균승고,차이균유통계학의의(P치균＜0.05).결론 고제ADAR1가감완AML적발병,증가AML세포조망.ADAR1재MLL-AF9유도적AML발생화유지과정중기관건작용.
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.