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;ZHANG Wang-gang(Department of Clinical Hematology,Affiliated Hospital of Inner Mongolia Medical University,Hohhot 010059;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）》
CAS
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)~~
