AFT024细胞对人胚胎干细胞来源造血祖细胞的体外维持作用研究  

In vitro maintenance of hematopoietic progenitor cells derived from human embryonic stem cells via co-culture of AFT024 cells

作　　者：朱美琪 张博文 吴旭敏 李基晟 宋妃灵 范增 何丽娟 裴雪涛 李艳华 ZHU Mei-qi;ZHANG Bo-wen;WU Xu-min;LI Ji-sheng;SONG Fei-ling;FAN Zeng;HE Li-juan;PEI Xue-tao;LI Yan-hua(Institute of Radiation Medicine,Academy of Military Medical Sciences,Academy of Military Sciences,Beijing 100850,China;South China Research Center for Stem Cell&Regenerative Medicine,South China Institute of Biomedicine,Guangzhou 510005,China;College of Pharmacy,Guizhou University,Guiyang 550000,China;Institute of Health Service and Transfusion Medicine,Academy of Military Medical Sciences,Academy of Military Sciences,Beijing 100850,China)

机构地区：[1]军事科学院军事医学研究院辐射医学研究所,北京100850 [2]华南生物医药研究院华南干细胞与再生医学研究中心,广州510005 [3]贵州大学药学院,贵阳550000 [4]军事科学院军事医学研究院卫生勤务与血液研究所,北京100850

出　　处：《军事医学》2022年第8期561-568,共8页Military Medical Sciences

基　　金：国家重点研发计划(2017YFA0103100,2017YFA0103103,2017YFA0103104);国家自然科学基金(81872553)。

摘　　要：目的建立小鼠胎肝基质细胞系AFT024细胞与人胚胎干细胞(hESC)来源造血祖细胞共培养体系,探究AFT024细胞对hESC来源造血祖细胞体外特性维持和扩增作用。方法建立hESC向造血祖细胞定向诱导分化的培养体系,采用拟胚体诱导方法,分三阶段添加不同细胞因子获得造血祖细胞,将hESC来源造血祖细胞与AFT024细胞进行共培养,对照组为无滋养层培养组。培养4 d后,通过细胞计数、流式细胞术分析、集落培养实验等检测造血祖细胞数量、比例及造血集落形成能力的变化;通过实时荧光定量PCR(qRT-PCR)对造血干/祖细胞增殖和分化相关基因的表达水平进行检测,以确定AFT024是否具有体外维持造血祖细胞特性的能力。结果hESC经三阶段诱导培养14后,可分化并产生大量悬浮的造血祖细胞,CD34^(+)CD43^(+)细胞比例为(22.63±2.65)%,CD34^(+)CD45^(+)细胞比例为(23.67±2.15)%;免疫荧光染色结果显示,AFT024细胞中有α-平没肌肌动蛋白(α-SMA)及Dlk-1蛋白的表达,以其为滋养层与造血祖细胞共培养4 d后,造血祖细胞比例显著高于无滋养层培养组。造血集落形成实验结果显示,滋养层培养组产生的造血集落总数显著高于无滋养层组。结论AFT024对hESC来源造血祖细胞的体外特性具有一定的维持作用。Objective To explore the role of AFT024 cells in maintaining the characteristics of human embryonic stemcells(hESCs)-derived hematopoietic progenitor cells in vitro.Methods A culture system was established to inducehESCs to differentiate into hematopoietic progenitor cells.Hematopoietic progenitor cells were generated using a three-stepprotocol by adding different cytokine in the form of an embryoid body(EB).hESCs-derived hematopoietic progenitor cellswere co-cultured with AFT024 cells,while the control group was cultured without these feeder cells.After four days of co-culture,the number and proportion of hematopoietic progenitor cells and the number of hematopoietic colonies formedwere detected by counting the number of total cells and via flow cytometry analysis and colony forming unit(CFU)assay.The expression levels of genes related to proliferation and differentiation of hematopoietic stem/progenitor cells were detected by quantitative real-time polymerase chain reaction(qRT-PCR)to find out whether AFT024 had the ability to maintainthe characteristics of hematopoietic progenitor cells in vitro.Results After fourteen days of three-stage induction,hESCscould produce a large number of hematopoietic progenitor cells,and the proportion of CD34^(+)CD43^(+)cells was about(22.63±2.65)%,compared with about(23.67±2.15)%for CD34^(+)CD45^(+)cells.Immunofluorescence staining showed thatα-SMA and Dlk-1 proteins were expressed in AFT024 cells.The proportion of hematopoietic progenitor cells coculturedwith AFT024 cells for four days was significantly larger than that of non-feeder culture group.The results of hematopoieticCFU assay showed that the total number of hematopoietic colonies produced in the co-culture group was significantly higherthan that in the control group.Conclusion AFT024 can maintain the characteristic of hESCs-derived hematopoieticprogenitor cells in vitro.

关 键 词：人胚胎干细胞 造血祖细胞 AFT024细胞 体外特性维持 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]