微环境在体外大量扩增晚期内皮祖细胞中的作用

Role of Microenvironment in the Process of Expansion of Late Endothelial Progenitor Cell in vitro

目的探讨做为微环境的滋养层细胞在体外大量扩增晚期内皮祖细胞(EPC)中的作用。方法预铺辐射后的滋养层细胞,而后植入分离的外周血单个核细胞,计数培养21 d后获得的晚期EPC克隆数目,并检测目的细胞免疫表型,用Matrigel鉴定其体外血管形成能力。采用动态显微镜追踪单个干细胞在滋养层细胞上的分化过程。结果培养21 d时,在预铺晚期EPC和人脐静脉内皮细胞(HUVEC)为滋养层的培养环境下,每100ml外周血单个核细胞分别获得(40.0±3.9)和(39.3±3.1)个晚期EPC克隆,均明显高于无滋养层条件下的(2.0±1.3)个(P均<0.05)。目的细胞可表达CD31、CD34、eNOS、FLt-1、P1H12、Sendo、VEcadherin和CD117,并在体外与Matrigel形成管状结构。动态追踪示单个干细胞的扩增是在有滋养层细胞参与下完成不对称分裂的。结论通过提供滋养层细胞方法可以大量扩增晚期EPC,滋养层细胞可能参与了早阶段内皮干细胞的不对称分裂扩增。

Objective To study the role of the feeder layer cells as niche in the process of expansion of late endothelial progenitor cell in vitro. Methods We cultured mononuclear cells （MNC） from human pe- ripheral blood （PB） on the plate with the feeder layer cells which were irradiated late endothelial progenitor cells （EPC） or human umbilical vein endothelial cells （HUVEC） by EGM-2. After 21 days, the numbers of obtained late EPC colonies were counted separately, and their surface antigen of the late EPC was verified by fluorescence-activated cell sorter （FACS） analysis, and their ability of forming vessel structure with Matrigel in vitro. The differentiation of single stem cell on the feeder layer cell was traced by video-microscopy. Results After 21 days of culture, （40. 0±3.9） and （39.3±3.1 ） late EPC colonies that MNC of a handred milliliterPB were cultured, respectively, on the feeder layer cells of EPC and HUVEC were much more than （2.0±1.3 ） colonies cultured on without the feeder layer cells （ all P 〈 O. 05 ） . These cells also expressed CD31, CD34, eNOS, FLt-1, P1H12, Sendo, VEcadherin, and CDll7, as shown by FACS analysis. Further- more, they formed vessel structure with Matrigel in vitro. The video-microscopy showed the asymmetric cell di- vision was participated by the feeder layer cell during the expansion of single stem cell. Conclusion The mas- sive expansion of late EPC can be achieved by the provision of the feeder layer cells, which may be involved in the stem cell asymmetric cell division.