晚期内皮祖细胞大量扩增方法

Method of expansion of late endothelial progenitor cells

目的通过实验验证一个体外大量扩增晚期内皮祖细胞（EPC）的方法。方法先预铺经辐射后的滋养层细胞（晚期EPC或脐带脐静脉内皮细胞），而后植入分离的人外周血单个核细胞，计数并对比培养21d后获得的晚期EPC克隆数目，并检测目的细胞免疫表型，应用基质胶（matrigel）鉴定其体外管状结构形成能力。结果21d时在预铺的两种滋养层细胞培养条件下获得的晚期EPC克隆数目均比无滋养层细胞的培养方法高20倍（40．0±3．9、39．3±3．1比2．0±1．3，均P〈0．05）；且细胞克隆出现的早。目的细胞表达CD31、CD34、内皮型一氧化氮合酶（eNOS）、血管内皮细胞生长因子受体Fit-1、CDl46的2个表型（P1H12、Sendo）、血管内皮钙黏蛋白（VEcadherin）、CDll7，且具有能在体外与matrigel形成管状结构的功能。结论通过提供滋养层细胞的方法可以大量扩增晚期EPC，提示微环境在晚期EPC扩增中起重要作用。

Objective To validate a novel method of expanding late endothelial progenitor cells （EPC） in vitro. Methods We cultured mononuclear cells （ MNC ） from human peripheral blood on the plate with the feeder layer cells, i.e. irradiated late EPC or human umbilical vein endothelial cells. After 21 days, the numbers of late EPC colonies were counted separately. And the surface antigen of late EPC was detected by fluorescence-activated cell sorter （FACS） and their in vitro ability of forming vascular structure examined by matrigel. Results Both colony numbers of late EPC with feeder layer cell culturing were over 20 times than those without （40. 0 ＋3. 9,39. 3 ＋3.1 vs 2. 0 -＋ 1.3 ,both P 〈0. 05）. And the former＇s late EPC colony appeared earlier. The late EPC expressed CD31, CD34, eNOS, Flt-1, P1H12, Sendo, VE eadherin and CDll7. And vascular structures were discerned. Conclusions The method of feeder layer cells may vastly expand the quantity of late EPC. And microenvironment plays an important role expansion.