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下调MLAA-22基因对U937细胞增殖与分化的影响 预览

Effect of down-regulation of MLAA-22 gene on proliferation and differentiation of U937 cells
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摘要 目的研究MLAA-22基因的表达下调对U937细胞增殖及分化的影响。方法采用规律成簇间隔短回文重复序列(CRISPR)/CRISPR相关核酸酶9(Cas9)系统下调U937细胞MLAA-22基因的表达;CruiserTM酶切法检测单链引导RNA(single guide RNA,sgRNA)的活性;基因突变区PCR产物的TA克隆及测序法检测MLAA-22基因的突变率;细胞计数试剂盒-8(cell counting kit-8,CCK-8)检测细胞增殖;流式细胞仪检测CD11b的表达。结果CruiserTM酶切结果显示:所采用的CRISPR/Cas9系统的sgRNA序列能够高效地识别基因编辑靶点;TA克隆及测序结果显示:MLAA-22基因的突变率为61.3%;CCK-8检测结果显示:MLAA-22基因下调后,U937细胞的增殖明显受到抑制;流式细胞仪检测结果显示:MLAA-22基因下调后,CD11b阳性细胞率显著升高。结论 MLAA-22基因可能通过调控细胞分化而影响U937细胞增殖,从而促进急性单核细胞白血病的发生与发展。 Objective To explore the effect of down-regulation of MLAA-22 gene on proliferation and differentiation of U937 cells.Methods MLAA-22 gene was down-regulated by clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated9(Cas9)system in U937 cells.The activity of single guide RNA(sgRNA)was detected by CruiserTM enzyme digestion assay.The mutation rate of MLAA-22 gene was analyzed by TA cloning and sequencing assay of PCR products of the gene mutation region.Cell proliferation was evaluated by CCK-8 assay.Expression of CD11 b was tested by flow cytometry to evaluate cell differentiation.Results CruiserTMenzyme digestion assay showed the sgRNA of the CRISPR/Cas9 system identified the target spot efficiently.TA cloning and sequencing assay displayed the mutation rate of MLAA-22 gene was 61.3%.CCK-8 assay demonstrated that the proliferation was obviously inhibited in MLAA-22-knockdown U937 cells.In addition,flow cytometry assay indicated CD11 b-positive cells significantly increased in MLAA-22-knockdown U937 cells.Conclusion MLAA-22 gene regulates the proliferation of U937 cells probably by regulating their differentiation,thus promoting the occurrence and development of acute monocytic leukemia.
作者 崔鹤 张王刚 CUI He 1,2 , ZHANG Wang-gang 2(1. Department of Clinical Hematology, Affiliated Hospital of Inner Mongolia Medical University, Hohhot 010059; 2. Department of Clinical Hematology,the Second Affiliated Hospital of Xi'an Jiaotong University, Xi'an 710004, China)
出处 《西安交通大学学报:医学版》 CSCD 北大核心 2018年第4期519-524,共6页 Journal of Xi‘an Jiaotong University:Medical Sciences
基金 国家自然科学基金资助项目(No.81100374)
关键词 MLAA-22基因 细胞增殖 细胞分化 急性单核细胞白血病 MLAA-22 gene cell proliferation cell differentiation acute monocytic leukemia
作者简介 通信作者:张王刚,教授,主任医师,博士生导师. E-mail:zhangwanggang2003@yahoo.com
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