肝素促进脐血巨核祖细胞体外扩增

Heparin Enhances the Expansion of Cord Blood Megakaryocyte =Progenitor Cells in vitro

目的 :探索肝素在脐带血CD34+ 细胞定向扩增巨核祖细胞中的作用。方法采用免疫磁珠法 (MACS)分选CD34+ 细胞 ,在TPO ,IL 1 1的扩增体系中加入肝素 ,巨核祖细胞集落分析 (CFU MK)测定巨核祖细胞扩增倍数 ,流式细胞仪检测巨核祖细胞分化过程中的特异性标记 (CD34+ ,CD41a+ ,CD61 + ,CD34+ CD41a+ ,CD41a+ CD61 + ) ,巨核细胞特异性抗体 (CD41a)免疫组化染色和透射电镜观察鉴定巨核细胞形态及超微结构 ,血小板体外活化实验及NOD SCID小鼠异种体内移植实验评价扩增的巨核祖细胞的功能。结果 :TPO( 5 0ng ml)与IL 1 1 ( 5 0ng ml)双因子联合应用 ,7天巨核祖细胞克隆扩增倍数为 83 1 7± 39 41倍 ,1 0天为 2 0 5 0 6± 74 2 6倍 ,流式细胞仪分析显示 7天CD34+ CD41a+ 细胞扩增 1 0 5 1± 4 79倍 ,0天加入肝素后 ,7天巨核祖细胞克隆扩增倍数为 1 0 8 2 5± 32 67倍 ,1 0天为 333 0 6± 2 7 5 4倍 ,7天CD34+ CD41a+ 细胞扩增到 2 9 93± 6 39倍 ,为无肝素组的 2 85倍 ,与双因子组相比有统计学差异 (P <0 0 1 ) ,肝素在第 5天及第 7天加入没有增加巨核祖细胞扩增效果。经全身照射预处理的NOD SCID小鼠静脉输注扩增第 7天的巨核细胞 (TPO、IL 1 1、肝素联合 ) 。

Objective: To explore the effect of heparin in the expansion of cord blood megakaryocyte progenitor cells in vitro. Methods: we added heparin to the TPO、IL-11 system to expand the megakaryocyte progenitor after selected CD34 + cells of cord blood, to calculate the expanded fold by CFU-MK analysis, to examine the specific CDs of megakaryocyte differentiation by FACS (CD41a +,CD61 +,CD41a +CD61 +, CD34 +CD41a +), to mark the specific antibody (CD41a +) by histochemisty, to observe the ultrastructure of MK by Electron microscopy, to examine the megakaryocyte function by the activation in vitro and transplantation in vivo. Results: When TPO combined with IL-11, CFU-MK colony was 83 17±39 41 fold at 7day and 205 06±74 26 fold at 10day, The number of CD34 +CD41a + cells was expanded 10 51±4 79 folds on day 7 by FACS ,when heparin was added to the system at 0 day, the fold of CFU-MK colony was 108 25±32 67 and 333 06±27 54 at 7 or 10 day respectively,the number of CD34 +CD41a + cells was expanded 29 93±6 39 fold, was significantly difference than that in TPO and IL-11 group (p<0 01) and no significantly difference when heparin was added to at 5 day or 7 day; The megakaryocyte which been infused to the radiated NOD/SCID mice by expanding with TPO, rhIL-11, heparin 7 days in vitro would help mice recovery the platelet and white blood cell and raise the survival. Megakaryocyte had the normal function by activation in vitro. Conclusion: The fold was further raised when Heparin was added to the TPO and rhIL-11 expansion system and heparin may optimize the expansion system.