按造血发育程序诱导小鼠胚胎干细胞为造血干细胞重建体内造血的实验研究  

Sequentially Inducting Murine Embryonic Stem Cells into Hematopoietic Stem Cells in vitro by Hematopoietic Development Procedure for Reconstitution of Hematopoiesis in vivo

作　　者：蔡耘[1,2] 张绪超[2,3] 黄绍良[2] 陈惠芹[1,2] 吴北燕[2] 

机构地区：[1]中山大学附属第三医院儿科,广东广州510630 [2]中山大学附属第二医院干细胞研究中心,广东广州510120 [3]广东省人民医院医学研究中心,广东广州510080

出　　处：《中国实验血液学杂志》2011年第5期1189-1194,共6页Journal of Experimental Hematology

基　　金：国家自然科学基金资助项目(编号30300377);广东省科技计划项目(编号2008B030301046)

摘　　要：为研究小鼠胚胎干细胞(ESC)定向诱导分化为造血干细胞(HSC)在体内重建造血的功能,将小鼠E14ESC诱导为拟胚体(EB),EB细胞利用Transwell非接触共培养体系在人主动脉-性腺-中肾(AGM)区、胎肝(FL)及骨髓(BM)基质细胞饲养层上依次诱导,收获各阶段EB细胞,以流式细胞仪检测Sca-1+c-Kit+细胞含量,并接种于NOD-SCID小鼠以检测体内致瘤性。再将不同诱导阶段的EB来源细胞移植到经致死量60Coγ射线照射的BALB/c雌鼠,将受鼠随机分为5组:①AGM组,②AGM+FL组,③AGM+FL+BM组,④照射对照组,⑤正常对照组。观察各组生存率、造血重建和植入状况。结果显示:EB细胞经人AGM区和FL基质细胞共培养后Sca-1+c-Kit+细胞达到峰值(21.96±2.54)%;NOD-SCID小鼠在接种经人AGM区基质细胞诱导的EB细胞后可出现畸胎瘤,而接种经人AGM区+FL基质细胞诱导EB细胞后未见肿瘤形成;AGM组及照射对照组动物全部死亡,而AGM+FL组及AGM+FL+BM组生存率分别为77.8%、66.7%,移植后21天外周血象基本恢复,在存活受鼠检测到供体来源Sry基因。结论:按胚胎造血发育程序,体外经人AGM区、FL及BM基质细胞连续诱导小鼠ESC分化的HSC可安全、有效地重建体内造血。This study was purposed to directly induce murine embryonic stem cells（ESC） into hematopoietic stem cells（HSC） by simulating the spatial and temporal hematopoietic microenvironment changes in embryonic development,and to investigate the function of in vivo hematopoietic reconstitution of these HSC.E14 ESC were induced into embryoid body（EB） firstly.Then the cells from EB were further co-cultured with human aorta-gonad-mesonephros（AGM） region,fetal liver（FL） and bone marrow（BM） stromal cells in Transwell non-contact system in sequential orders.After 6 days of each co-cultured stage,the induced cells derived from EB were collected to analyze the Sca-1＋c-Kit＋ cells by flow cytometry,check teratoma formation and transplant to BALB/C female mice exposed to lethal dose 60Co γ-ray.The recipient mice were divided randomly into 5 groups： AGM,AGM＋FL,AGM＋FL＋BM,irradiation control and normal control groups.The survival rates,hematopoietic reconstitution and engraftment of donor cells in the different groups were monitored.The results showed that Sca-1＋c-Kit＋ cell level in EB cells co-cultured with human AGM region and FL stromal cells reached to peak value（21.96±2.54）%.Teratomas could be found in NOD-SCID mice after subcutaneous injection of EB cells co-cultured with human AGM region stromal cells,while there was no teratoma in the mice after subcutaneous injection of EB cells induced by human AGM region and FL stromal cells.The recipients in AGM group and irradiation control group all died.The survival rate was 77.8% in AGM＋FL group,and 66.7% in AGM ＋ FL ＋ BM group.The peripheral blood cell count was near normal at day 21 after transplantation,andSry gene copies from donor could be detected in recipient mice of these two groups.It is concluded that sequentiallyinductive system with feeder cells from human AGM region,fetal liver and bone marrow simulating embryonic definedhematopoiesis procedures can enhance the directed differentiation of ESC into HSC which can safely reco

关 键 词：主动脉-性腺-中肾区 胚胎干细胞 造血干细胞 造血发育程序 

分 类 号：R331.2[医药卫生—人体生理学]