两步法体外定向诱导小鼠胚胎干细胞发育为CD_(34)^+/Sca-1^+造血干细胞的研究  

Generation of CD_(34)^+/Sca-1^+ cells from mouse embryonic stem cells with two-step differentiation in vitro

作　　者：何志旭[1] 黄绍良[2] 周其峰[3] 李树浓[3] 

机构地区：[1]贵阳医学院干细胞研究中心贵阳医学院附属医院儿科,550001 [2]中山大学附属第二医院儿科 [3]中山大学中山医学院病理生理教研室

出　　处：《中华儿科杂志》2004年第11期830-834,共5页Chinese Journal of Pediatrics

基　　金：中国博士后基金项目 (中博基 2 0 0 0年 3 1号 ) ;贵州省国际科技合作重点项目 (黔科通 2 0 0 3年 71号 )

摘　　要：目的　建立一种体外诱导胚胎干细胞 (embryonicstemcell,ESC)定向发育为CD+ 3 4 /Sca 1+ 造血干细胞 (hematopoieticstemcell,HSC)的实验方法。方法　联合应用血管内皮生长因子(vascularendothelialcellgrowthfactor ,VEGF)、干细胞因子 (stemcellfactor ,SCF)、白细胞介素 3(interleukin 3,IL 3)、白细胞介素 6 (interleukin 6 ,IL 6 )和促红细胞生成素 (erythropoietin ,EPO)分阶段首先诱导ESC形成富含CD+ 3 4 /Sca 1+ 细胞的胚胎体 (embryoidbody ,EB) ,然后分别观察EB细胞在甲基纤维素半固体培养体系和骨髓基质细胞饲养层培养体系中进一步分化为HSC的情况。结果VEGF联合其他细胞因子能有效促进EB中HSC的形成 ,CD+ 3 4 /Sca 1+ 细胞数高达 (13 72± 1 92 ) %。收获此阶段的EB进一步诱导发现 ,甲基纤维素培养体系造血克隆形成率明显低于骨髓基质细胞培养体系 ,在甲基纤维素中CD+ 3 4 /Sca 1+ 细胞数最高只能达到 (2 0 5 2± 2 78) % ,而基质细胞中CD+ 3 4 /Sca 1+ 细胞数可达 (34 6 0± 3 71) % (t=5 2 6 ,P <0 0 0 1)以上 ,且来自骨髓基质细胞培养体系的造血分化细胞在造血集落培养时能形成类型和数量更多的造血克隆。结论　使用VEGF联合SCF、IL 3、IL 6、EPO分阶段诱导ESC形成富含CD+ 3 4Objective Embryonic stem ce ll s (ESCs) are derived from totipotent cells of early embryo and they are potentia l to differentiate to any kind of cells of tissues in the body. Some reports sh owed that ESCs had broad capabilities of differentiating to variety of hematopot ietic cells, such as erythroid, granulocyte/macrophage, megakaryocyte, mast and lymphocyte precursors. However, it is very difficult to control the phase of di fferentiation for ESCs in vitro. There is few report about hematopotietic s tem cells (HSCs) from ESCs. Therefore, this research was designed to establish a culture system for generation of CD+ 34/Sca-1+ HSC from ESC in vit ro. Methods Single mouse E 14.1 cells were suspended in methylcellulose medium, containing 40 ng/ml stem cell factor (SCF) and 20 ng /ml vascular endothelial growth factor (VEGF) and incubated at 37℃ with 5%CO 2 . In order to ensure the viability of the primary differentiation cultures over an extended period of time, the cultures were fed on day 7 with a dilute methyl cellulose medium containing VEGF, SCF, interleukin-3 (IL-3), IL-6 and erythro poietin (EPO), which promoted their primary differentiation into embryoid bodies (EBs) with more CD+ 34/Sca-1+ cells. Then, EBs with peak level of CD + 34/Sca-1+ cells were dispersed into single cells and replanted eithe r in methylcellulose medium or in bone marrow stromal cells differentiation syst em containing 15% fetal bovine serum (FBS), 160 ng/ml SCF, 20 ng/ml VEGF, 30 ng/ ml IL-3, 30 ng/ml IL-6, 3 U/ml EPO and 20% BIT for HSC into second-step diffe rentiation. The HSCs were characterized by flow cytometric analysis, colonogeni c cell assay and Wright-Giemsa stains. Results VEGF had the strongest stimulatory effect on the enhancement of the CD+ 34/Sca-1 + cells population when combined with SCF, IL-3, IL-6 and EPO. It could mar kedly accelerate mouse E14.1 cells to differentiate into EB with more CD+ 34/Sca-1+ cells. Cell cytometric analysis showed CD+ 34/Sca-1+ c ells were up to (1.91±0.

关 键 词：CD34^+ SCA-1^+细胞 骨髓基质细胞 HSC ESC 造血干细胞 VEGF 小鼠胚胎 发育 体外定向诱导 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]