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.

abstractsObjective To evaluate the effects of endothelial cell-targeted soluble Notch ligand hD1R protein on expansion and engraftment of cord blood hematopoietic stern/progenitor cell (CB HSPCs).Methods Recombinant hD1R protein 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),hD 1R 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 cocultured 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 hDIR 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.