体外定向诱导小鼠胚胎干细胞发育为造血干/祖细胞方法的初步研究  

Primary study on inductive methods of hematopoietic stem cellfrom embryonic stem cells in vitro

作　　者：何志旭[1] 黄绍良[1] 周其峰[2] 李树浓[3] 

机构地区：[1]中山大学附属二院儿科,广东广州510120 [2]中山大学中山医学院免疫学教研室,广东广州510080 [3]中山大学中山医学院病理生理学教研室,广东广州510080

出　　处：《中国病理生理杂志》2003年第4期443-447,T002,共6页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目 ( 30 0 70 72 6 );中国博士后基金资助项目 (中博基 2 0 0 0年第 31号 )

摘　　要：目的 :探讨体外定向诱导胚胎干细胞 (ESC)发育为造血干细胞 (HSC)的方法。方法 :将小鼠E14胚胎干细胞在含干细胞生长因子 (SCF)和血管内皮生长因子 (VEGF)的甲基纤维素培养基中首先诱导发育为胚胎体(EB) ,再将EB置于均含SCF、VEGF、IL - 3、IL - 6及促红细胞生成素 (EPO)的 3种不同培养体系中定向分化为HSC ,并观察HSC表面标志性抗原、造血集落形成及瑞氏 -姬姆萨染色的结果。结果 :经两阶段诱导ESC分化为HSC ,发现在甲基纤维素半固体培养体系中HSC发育缓慢 ,分化 14d后CD34+/Sca - 1+细胞数最高为 ( 31 5± 4 7) %;而在骨髓基质细胞饲养层上HSC发育较快 ,细胞数量较多 ,分化第 10dCD34+/Sca - 1+细胞数即达到峰值 ,为 ( 47 8±6 3) %;骨髓基质细胞饲养层 +胎肝基质细胞上清培养体系中HSC发育同样迅速 ,所产生的CD34+/Sca - 1+细胞数量在 3个体系中最高 ,为 ( 5 3 6± 7 2 ) %。经瑞氏 -姬姆萨染色证实上述细胞为早期造血细胞 ,均有形成各系造血细胞集落的能力。结论 :使用骨髓基质细胞饲养层 +胎肝基质细胞上清培养体系及SCF、VEGF、IL - 3、IL - 6及EPO等细胞因子 ,通过两阶段诱导分化 ,可从小鼠ESC获得较高比例的HSC。AIM: To explore inductive methods of hematopoietic stem cell (HSC) from embryonic stem cells (ESC) in vitro. METHODS: Using mice E14 line, the first step was the primary differentiation in which the ESC form embryoid bodies (EB) in methylcellulose-based cultures with SCF and VEGF. The second step involved the plating of cells originating from the EB into three different system of cultures containing SCF, VEGF,IL-3, IL-6 and EPO for HSC. And identifying HSC by flow cytometry analysis, colonogenic cells assay and Wright-Giemsa stain were also used. RESULTS: By two-step differentiating, it showed that HSC differentiated slowly in methylcellulose medium, percent age of CD34+/Sca-1+ cells slight increased about(31.5±4.7)% after day 14 induction. However, EBs were induced after 10 days to fast differentiate for HSC with more cells population by coculture on bone marrow stromal cells feeder. Flow cytometry analysis showed that percentages of CD34+/Sca-1+ cells might reached to (47.8±6.3)%. The more optimistic system of differentiation was bone marrow stromal cells feeder (BMSCF) in combination with supernatants of stromal cells from mice fetal liver(SSCFL), it significant supported differentiation of ESC into HSC with higher percent (53.6±7.2)%. Colonogenic cell assay and Wright-Giemsa stain confirmed that it possessed character of hematopoietic progenitors. CONCLUSION: Using methods of two-step differentiation, mice ESC were induced to differentiate into HSC by coculturing with BMSCF and SSCFL in combination of SCF,VEGF,IL-3,IL-6 and EPO.

关 键 词：胚胎干细胞 造血干细胞 骨髓细胞 分化 小鼠 体外定向 

分 类 号：R329.28[医药卫生—人体解剖和组织胚胎学]