组织血型抗原Le^(y)低表达的人巨核细胞系的建立和鉴定  

Establishment and identification of a human megakaryocytic cell line with low Le^(y) expression

作　　者：朱慧君[1] 马勤勤 赵俸涌[1] 李勤[1] 陆萍[1] ZHU Huijun;MA Qinqin;ZHAO Fengyong;LI Qin;LU Ping(Shanghai Blood Center,Shanghai 200051,China)

机构地区：[1]上海市血液中心,上海200051

出　　处：《中国输血杂志》2022年第9期891-895,共5页Chinese Journal of Blood Transfusion

基　　金：中国自然青年项目(82100246);上海市卫生健康委员会卫生行业临床研究专项(202140060)。

摘　　要：目的应用CRISPR/cas9技术,建立血小板Le^(y)抗原差异化表达的人白血病巨核细胞系,为后续研究Le^(y)抗原在血小板活化中的作用奠定基础。方法选取人巨核细胞白血病细胞DAMI,用Western Blot及流式细胞法确定Le^(y)抗原的表达量;用实时定量PCR方法检测编码Le^(y)抗原合成相关的岩藻糖基转移酶(fucosyltransferase,FUT)基因的表达,确定候选敲除基因,用CRISPR/Cas 9敲除候选FUT基因,使用流式细胞仪分选出Le^(y)抗原低表达的细胞群,培养细胞并鉴定Le^(y)抗原的表达量。用活细胞染色结合流式细胞法监测细胞增殖。结果DAMI细胞上具有大量的Le^(y)抗原表达;FUT1和FUT4在DAMI中有相对较高的mRNA表达,可能对Le^(y)抗原的表达起关键作用;敲除FUT1后的DAMI细胞系Le^(y)抗原的表达量与对照相比有显著降低,细胞增殖能力与野生型细胞相比没有显著改变。结论Le^(y)抗原的生物合成涉及多种FUT基因,对其中主要的FUT进行基因敲除无法彻底阻断Le^(y)抗原的合成,只能降低Le^(y)抗原的表达,本研究应用CRISPR/cas9技术敲除FUT基因建立的稳转人白血病巨核细胞系,为研究Le^(y)抗原对血小板功能的影响和意义奠定了基础。Objective To establish a stable human megakaryocytic cell line with low expression of Le^(y) antigen to further study the role of Le^(y) on activation of platelets.Methods The expression level of the Le^(y) antigen in a human megakaryocytic cell line,DAMI,was determined using Western Blot and flow cytometry.The expression level of genes that encode fucosyltransferase(FUTs),which was involved in the biosynthesis of Le^(y) antigen,was also determined to identify the candidate genes to be knocked out.The candidate FUT gene was knocked out via a CRISPR/Cas 9 gene knockout system and cells with low Le^(y) antigen expression were sorted by flow cytometry.The sorted cell line was cultured and characterized.Results The Le^(y) was expressed intensively on DAMI cell.FUT1 and FUT4 mRNA was expressed relatively higher,both may be key enzymes for the biosynthesis of the Le^(y) antigen.In the DAMI cell line with the knockout of FUT1 gene,the expression of the Le^(y) adntigen was remarkedly reduced,while cell proliferation was not affected compared to the wildtype control cells.Conclusions Since various FUTs contributes to the biosynthesis of the Le^(y) antigen,the knockout of the primary one of them cannot totally block its biosynthesis,but only reduce its expression.In this study,a stable FUT gene knockout human megakaryocyticcell line is established using CRISPR/Cas 9 technology,which provides basis for the study of the impact of the Le^(y) antigen on platelet functions.

关 键 词：组织血型抗原 血小板功能 人巨核细胞 岩藻糖基转移酶 基因敲除 

分 类 号：R457.1[医药卫生—治疗学]