ASXL1缺失对人白血病HEL细胞生物学特性的影响  

Role of ASXL1 loss in biological functions of HEL cells

作　　者：蒋晓 肖方楠 刘怡宁 张明英 袁佳佳 邢文 周圆 JIANG Xiao;XIAO Fang-nan;LIU Yi-ning;ZHANG Ming-ying;YUAN Jia-jia;XING Wen;ZHOU Yuan(State Key Laboratory of Experimental Hematology,National Clinical Research Center for Blood Diseases,Institute of Hematology&Blood Diseases Hospital,Chinese Academy of Medical Sciences&Peking Union Medical College,Tianjin 300020,China)

机构地区：[1]中国医学科学院血液病医院,中国医学科学院血液学研究所,北京协和医学院血液学研究所,实验血液学国家重点实验室,国家血液系统疾病临床医学研究中心,天津300020

出　　处：《中国药理学通报》2021年第8期1057-1062,共6页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金资助项目(No 81770128,81970120)。

摘　　要：目的本研究以携带JAK2 V617F纯合突变的人红白血病细胞HEL为基础,建立ASXL1和JAK2 V617F双突变模型,为研究JAK2 V617F与ASXL1协同参与MPN疾病进展的机制以及寻找新的药物治疗靶点奠定基础。方法利用CRISPR/Cas9介导的基因编辑技术建立ASXL1基因敲除的HEL细胞系,并进行一系列细胞功能实验来验证ASXL1敲除对HEL细胞增殖、克隆形成以及对化疗药物的敏感性的影响。结果成功构建ASXL1基因完全敲除的HEL细胞系(HEL-AKO),模拟JAK2 V617F突变与ASXL1基因功能缺失性突变并存。细胞功能实验发现,HEL-AKO细胞较HEL细胞迁移能力变强,增殖能力变慢,细胞周期G 2/M期的进程延长,且HEL-AKO细胞的克隆形成能力减弱,药物敏感实验表明,与HEL细胞相比,HEL-AKO细胞对JAK抑制剂芦可替尼产生一定耐受。结论运用CRISPR-Cas9基因编辑技术能够实现对JAK2 V617F突变的人红白血病细胞系HEL细胞系的特定编辑,且ASXL1基因敲除之后会对HEL细胞的生物学功能产生明显影响。Aim To reveal the underlying mechanisms of the co-occurrence of ASXL1 and JAK2 V617F mutation by using human leukemia cell line HEL that carried homozygous JAK2 V617F mutation in the elucidation of the role of ASXL1 loss of function mutation in myeloproliferative neoplasms,so as to provide an important model for investigating the role of ASXL1 mutation in myeloproliferative neoplasms at the cellular level.Methods HEL cell line with ASXL1 knockout(HEL-AKO)was established by using CRISPR/Cas9-mediated gene editing.And a series experiments based were preformed to verify the effect of ASXL1 on HEL cell proliferation,clone formation and chemosensitivity.Results HEL-AKO cell line was successfully established,confirmed by sequencing results.We found that the loss of ASXL1 could inhibit proliferation and induce cell cycle arrest at the G 2/M phase.The colony-forming capacity of HEL-AKO cells was also markedly inhibited.Moreover,the HEL-AKO had higher cloning efficiency than HEL Control after ruxolitinib treatment.Conclusions Loss of function of ASXL1 has an impact on cell biological function of HEL.Therefore,HEL-AKO cell line can be used to further explore the biological contribution of concomitant ASXL1 in JAK2 V617F mutated MPN.

关 键 词：ASXL1 JAK2 V617F 骨髓增殖性肿瘤 HEL细胞 CRISPR/Cas9 芦可替尼 

分 类 号：R329.28[医药卫生—人体解剖和组织胚胎学] R345.57[医药卫生—基础医学]