内皮细胞靶向的可溶性Notch配体hD1R蛋白对脐血造血干/祖细胞增殖和植入的影响  

Effect of endothelial cell-targeted soluble Notch ligand hD1R protein on expansion and engraftment of cord blood hematopoietic stem/progenitor cells

作　　者：田登梅[1] 梁英民[2] 韩骅[3] 张永清[1] 

机构地区：[1]解放军第309医院血液科,北京100091 [2]第四军医大学唐都医院血液科 [3]第四军医大学遗传与发育教研室

出　　处：《中华血液学杂志》2014年第10期885-890,共6页Chinese Journal of Hematology

基　　金：国家高技术研究发展计划（863计划）（2006AA02A111）

摘　　要：目的 探讨内皮细胞靶向的可溶性Notch配体hD1R蛋白对脐血造血干/祖细胞增殖和植入的影响.方法 诱导、表达及纯化内皮细胞靶向的可溶性hD1R融合蛋白.以人脐静脉内皮细胞（HUVEC）作为支持细胞,联合应用5种人源性生长因子SCF、TPO、FL、IL-6、IL-3及hD1R蛋白,与人脐血CD34＋细胞共培养,分析在PBS组（PBS代替hD1R）、hD1R组、sup组（HUVEC上清代替HUVEC）、fix组（被固定HUVEC代替HUVEC）、Day 0组（未培养的CD34＋细胞）5种不同培养条件下CD34＋细胞增殖、凋亡及细胞周期,并将培养后的细胞经尾静脉移植给亚致死剂量照射的NOD/SCID小鼠,12周后用流式细胞术分析人源性细胞在小鼠体内植入的情况.结果 hD1R组的培养条件对CD34＋细胞有最佳的扩增作用,hD1R组扩增的细胞是Day 0组的87.50倍,是PBS组的7.98倍.hD1R组扩增的细胞约77.0％处于G0/G1期,hD1R明显抑制细胞凋亡,促进细胞增殖.体内移植实验显示hD1R组扩增的细胞在小鼠体内有明显植入优势.结论 成功建立了基于Notch信号的体外扩增体系,hD1R蛋白促进脐血造血干/祖细胞体外增殖及体内植入.Objective To evaluate the effects of endothelial cell-targeted soluble Notch ligand hD1R protein on expansion and engraftment of cord blood hematopoietic stem/progenitor cell （CB HSPCs）. Methods Recombinant hD1Rprotein was first induced and purified. Human cord blood CD34＋ cells were co-cultured on human umbilical vein endothelial cells （HUVECs） supplemented with a cocktail containing 5 types of human cytokines including TPO, SCF, FL, IL-6, IL-3 （5GF） and soluble hD1R. The expansion of CD34＋ cells was tested under different culture conditions including PBS group （PBS replaces HUVEC） , hD1R group, sup group （HUVEC supernatant replaces HUVEC） , fix group （fixed HUVEC replaces HUVEC）, Day 0 group （Control）. Cell cycle and apoptosis of cultured cells were also analyzed. Their progeny expanded in PBS or hD1R group were transplanted into sublethally irradiated NOD/SCID mice. The percentages of human CD45＋ （hCD45＋） cells in the marrow of recipient mice were determined by FACS 12 weeks later. Results hD1R induced more expansion in the total number of CD34~ cells co- cultured with HUVECs plus 5GF, which was 87.50-fold increase compared to the Day 0 group, and 7.98- fold increase than that of PBS group. FACS analysis also showed that the percentage of CD34＋ cells was 77.0% in G0/G1 phase in the hD1R group, which indicated that hD1R enhanced HSPCs expansion and inhibited apoptosis. Moreover, hD1R significantly promoted human HSPC engraftment after BM transplantation in irradiated mice. Conclusion The Notch-mediated ex vivo expansion system has been established and hD1R promoted expansion and engraftment of human CB HSPCs, which provided the evidence for further clinical application.

关 键 词：hD1R蛋白 Notch信号系统 造血干/祖细胞 增殖 植入 

分 类 号：R457.7[医药卫生—治疗学]