人类胚胎干细胞向造血细胞和内皮细胞诱导分化的初步研究  

Differentiation from Human Embryonic Stem Cells to Hematopoietic Cells and Endothelial Cells

作　　者：王建[1] 赵惠萍[1] 谢常青[1] 林戈[1] 杨胜[1] 聂东宋[1] 王绮如[1] 卢光琇[1] 

机构地区：[1]中南大学生殖与干细胞工程研究所,长沙410078

出　　处：《中国实验血液学杂志》2005年第2期222-228,共7页Journal of Experimental Hematology

基　　金：国家863组织工程专项基金(2002AA205181)国家自然科学基金重点项目(3030070)国家自然科学基金项目(30300119)资助

摘　　要：胚胎干(ES)细胞是一种能够自我更新、长期增殖并且具有多向分化潜能的干细胞。近年来的研究表明, 小鼠胚胎干细胞体外分化能够模拟体内造血发育的过程。为探讨诱导人类胚胎干细胞向造血细胞和内皮细胞的 分化的方法,将人类胚胎干细胞去饲养层,形成拟胚体(EB)培养3天后消化;采用基质细胞条件培养基联合细胞 因子的诱导体系,诱导8天后,种植到半固体培养基中培养7-14天。应用RT-PCR检测诱导细胞flk-1、BMI-1、scl、 Zeta-globin造血相关基因的表达;流式细胞术检测KDR、CD34等造血细胞表面抗原的表达;免疫组织化学检测集落 细胞的表面标记。结果表明:①半固体培养基中出现20-30个细胞组成的集落,集落细胞再种植仍然能够形成大 小相似的细胞集落;另外一些较大的细胞集落中CD45细胞呈阳性。②部分细胞贴壁生长形成内皮细胞样集落,Ⅷ 因子、KDR、UEA检测均为阳性。③在ES细胞内有少量flk-1、BMI-1表达,而scl、Zeta-globin基因不表达;自发分化 3天的EB可见flk-1、BMI-1表达上调;消化形成单细胞并诱导4天,检测到flk-1、BMI-1、scl、Zeta-globin基因的表 达,第8天仍然检测到flk-1、BMI-1、scl基因的表达,Zeta-globin基因不表达。④诱导8天的细胞中表达flk-1阳性细 胞的率为9.8％,CD34阳性细胞占总细胞量的16.8％。Embryonic stem cells are pluripotent and their differentiation in vitro can serve as an experimental model to explore the molecular mechanisms of early embryonic development. To investigate the effect of stromal cell conditioned medium combined with cytokines (sccm +cys) on the differentiation from human embryonic stem cells to hematopoietic cells and endothelial cells, the mouse fibroblast feeder cells to make human embryonic stem cells grown into embryonic bodies (EBs) were initially deleted. After culture for 3 days, EB cells were trypsinized into single cells and induced for 8 days by sccm + cys. Then, the differentiated cells were cultured in the semisolid medium containing 0.9% methylcellulose and cytokines to study the colony forming and self-renewal ability of cells. Immunocytochemical staining was used to check the surface markers of the colony cells. During the induction, mRNA expression of flk-1, BMI-1, scl, and Zeta-globin genes was tested by RT-PCR. Surface markers, such as flk-1, CD34 were tested by the flow cytometry. The results demonstrated that: (1) cell clusters containing 20-30 cells were formed after culture for 8-14 days in the semisolid medium, replanting these cells resulted in similar cell cluster forming. In addition, CD45 positive in big cell colonies were also found in the semisolid medium; (2) attached cell colonies appeared after culture for 8 days in the semisolid medium and VIII factor, UEA and KDR could be detected as nigative by immunocytochemical staining; (3) on the 4th day of induction, mRNAs of flk-1, BMI-1 ,scl and Zeta-globin were all expressed. On the 8th day of induction, all of the above genes except Zeta-globin were expressed, while ES cell and EB cells which served as controls did not express scl and Zeta- globin genes; (4) on the 8th day of induction, the proportions of flk-1 + cells and CD34 + cells among all the inducing population were 9. 8% and 16. 8% , respectively, while the corresponding positive populations were 0. 36% and 1. 16% in spontaneously differentiate

关 键 词：胚胎干细胞 诱导分化 造血细胞 内皮细胞 

分 类 号：R331.2[医药卫生—人体生理学] R329.28[医药卫生—基础医学]