【摘要】 目的建立并稳定和优化诱导胚胎干细胞（ES）向造血干细胞定向分化的方法。方法应用体外胚胎干细胞分化系统，观察了多种因素对原始细胞成集落细胞（BLast Colony-Forming Cell，BL-CFC）形成集落的影响。结果植入不同数量的３．５d胚体衍生的细胞与BL-CFC集落生成数量之间呈高度相关，大约每植入１００个３．５d胚体衍生的细胞，能生成１．０８～１．２个BL-CFC集落；２０％与３０％浓度的D４T条件培养液促BL-CFC集落生成作用的效率最高；单用D４T条件培养液、EPO或KL对BL-CFC集落的生长无明显促进作用（P＞０．０５）；但单用VEGF对BL-CFC集落的生成有极显著的促进作用（P＜０．００１）。但当这四种因素两两组合使用时，均比单用VEGF具有明显的促BL-CFC集落生成作用。在VEGF＋KL＋D４T合用的基础上加用EPO，则促BL-CFC集落生成作用大大增强，与其它各组相比较，BL-CFC集落数极显著地增多（P＜０．００１）。大约每植入１００个３．５d胚体衍生的细胞，能生成１．５～１．６个BL-CFC集落。结论影响BL-CFC生长的主要因素可能为VEGF；D４T条件培养液具有强大的协同作用；EPO和KL为诱导BL-CFC向造血细胞系分化的主要因素；３．５d胚体衍生的细胞比３．２５d胚体衍生的细胞对各种刺激因子反应更敏感，生成B更多还原

【Abstract】 Objective To explore a stable technology for inducing ES cell to committed hematopoietic diff-eren tiation．Methods The effects of various inductive factors on BLast Colony-Forming Cell（BL-CFC）were investigated.In vitro differentiative system for ES cells was employed in this study.Results A high linear correlation between the number of D3.5EB-derived cells plated and the number of blast cell colonies was developed,r=0.9931.With high frequency of blast colonies observed(1.08～1.2colonies per100cells).20%～30%D4T conditioned medium (D4T CM)showed the most significant growth potentials of blast colonies.D4T CM,EPO or KL alone had no blast colony growth prom-oting effect (P>0.05).But VEGF alone had high significant blast colony growth promoting effect(P<0.001=.However,any two factors combination from above four factors exerted better growth promoting effect than VEGF alone(EPO+D4T CM,P<0.05;KL+D4T CM,P<0.01;VEGF+D4T CM,P<0.001=.There were no significant difference among VEGF+KL and EPO+D4T CM or KL+D4T CM,and KL+D4T CM (P>0.05).While the combination of VEGF+D4T CM was better than KL+D4T CM,VEGF+KL or EPO+D4T CM(P<0.001=.Moreover,the combination of VEGF+KL+D4T CM+EPO,had the highest significant blast colony growth promoting effect(P<0.001=.And the highest frequency of blast colonies was observed(1.5～1.2colonies per100cells).Conclusion VEGF may be the main factor which stimulates the growth of significant numbers of blast cell colonies.D4T CM maybe contains strong cofactors.EPO and KL are the main factors for the induction of BL-CFC to committed hematopoietic differentiation.D3.5EB-derived cells are more sensitive to various stimu -lators and have strong blast colony growth promoting effect than that of D3.25EB-derived cells.更多还原