胚胎干细胞诱导分化为造血干细胞重建造血功能的作用(英文)  

In Vitro Generation of Hematopoietic Stem Cells from Embryonic Stem Cell to Reconstruct Hematopoiesis

作　　者：何志旭[1] 黄绍良[2] 米蔷[1] 黄景[1] 

机构地区：[1]贵阳医学院附属医院儿科／贵阳医学院干细胞研究中心,贵阳550004 [2]中山大学干细胞研究中心,广州510120

出　　处：《实用儿科临床杂志》2008年第15期1213-1218,共6页Journal of Applied Clinical Pediatrics

基　　金：the Key International Science and Technology Cooperation Projects of Ministry of Science and Technology of China(2004DFA03400);the Key Science and Technology Projects of Ministry of Education of China(205142);the Chunhui Project Scheme of Ministry of Education of China(Z2005-1-52003)

摘　　要：目的探讨胚胎干细胞(ESC)定向分化来源的造血干细胞(HSC)体内重建造血功能的作用。方法将小鼠E14.1胚胎干细胞采用三步诱导法在体外分化发育为HSC,流式细胞仪检测HSC表面标志性抗原CD34+/Sca-1+的表达,体内畸胎瘤形成实验检测其致瘤性,造血克隆形成(CFU)实验观察其体外造血集落形成情况,免疫磁珠分选纯化HSC移植给经亚致死剂量γ射线照射的雌性小鼠观察其体内重建造血的功能。结果多种造血刺激因子联合应用能有效促进ESC发育为含丰富造血前体细胞的胚胎体(EB),诱导14 d后,EB中CD34+/Sca-1+细胞数达峰值,为(13.72±2.07)%。收获此阶段的EB行第二步诱导分化16 d后,CD34+/Sca-1+细胞数可增至(24.62±2.50)%,CFU培养出现红系和粒系造血克隆;在骨髓基质细胞加胎肝基质细胞上清液培养体系中进行第三步诱导分化15 d后,CD34+/Sca-1+细胞达峰值,为(58.64±4.20)%,CFU培养能形成较多的红系、粒系/巨噬细胞系及混合细胞集落,W right-G iemsa染色显示为原始的造血细胞。此阶段的HSC经分选纯化后移植给经γ射线照射后的小鼠,移植组小鼠+10 d造血功能开始恢复,观察40 d后除血小板恢复较慢外,白细胞、红细胞、血红蛋白等指标已接近正常,植入率为71.4%,存活率为43.0%,染色体检测证实已由受体鼠的XX转为供体鼠的XY。结论采用分阶段诱导的方法,可在体外定向诱导小鼠ESC分化发育为HSC,此来源的HSC较安全,无致瘤性,并具备体内重建造血功能的能力。Objective To search for a good method for generation of hematopoietic stem cells（HSC） by embryonic stem cells（ESC）,and to investigate the potential of HSC derived from ESC to reconstruct hematopoiesis in vivo.Methods Using a three-step method to induce a mice ESC line,E14.1-into HSC.And identifying HSC by flow cytometry analysis cell markers with CD34^＋/ Sca-1^＋,then HSC（1×10^9 L^-1）from ESC differentiation were injected into severe combined immunodeficency（SCID） mice for observing teratoma formation.To validate function of HSC by colonogenic cells assay and to reconstitute the hematopoiesis in lethally irradiated mice.Results Combining to use more hematopoietic stimulating factor to availably promote the E14.1 cell into embryoid body（EBs） with abundant hematopoietic progenitors.EBs were induced after 14 day to fast differentiate for HSC with peak percentages of CD34^＋/Sca-1^＋ cells reached to（13.72±2.07）%.To harvested cells from EBs by day 14 for second-step HSC differentiation,percent of CD34^＋/ Sca-1^＋ cells rise to（24.62±2.50）% after day 16 induction.Cloning forming units（CFU） analysis showed that more Erythro-myeloid cloning generation was observed at this period.Cells obtained in the second step are subsequently plated on monolayer of mice bone marrow stromal cells,in the presence of TPO,FLt3 ligand and supernatant of mice fetal liver stromal cells,cultured for additional 15 days,followed fast expansion of CD34^＋/ Sca-1^＋ to maximally（58.64 ± 4.20）% with more CFU-E,CFU-GM and CFU-GEMM population.Wright-Giemsa stain showed that its had the character of hematopoietic progenitors.Cells from the third-step were injected into SCID mice,but no teratomas were recovered in 2 mices after 6 weeks.Positive selection of CD34^＋/Sca-1^＋cells by magnetic sorting from the third-step differentiation were transplanted into 7 lethally irradiated female mice while predominant hematopoietic reconstitution were observed in 10 days after transplantation,with 71.4% succes

关 键 词：胚胎干细胞 造血干细胞 移植 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]