体外扩增的脐血单个核细胞植入NOD/SCID小鼠的研究

为了探讨在无血清、无基质培养条件下SCF、FL和TPO 3种因子组合体外扩增的脐血单个核细胞(MNC)的最佳移植时机及植入潜能,将SCF,FL和TPO 3种因子组合体外扩增的脐血单个核细胞培养14天,在0、7、10和14天检测有核细胞数(TNC),CD34 细胞数,CD34 CXCR4 细胞数,CD34 CD49d 的细胞数及集落形成单位(CFU)数,并将SCF FL TPO 3种因子组合的无血清无基质条件下扩增培养7天前后的脐血单个核细胞移植给经亚致死量照射的NOD/SCID小鼠,6周后用流式细胞术,PCR法检测存活小鼠体内的人源性细胞.结果表明,经过14天的培养,脐血细胞得到了有效的扩增,TNC数,CD34 细胞数,CD34 CD49d 的细胞数于7天达高峰,其后开始下降,而CFU数,CD34 CXCR4 细胞数于第10天达高峰.在移植6周后,扩增脐血移植组的NOD/SCID小鼠的存活率和人源性CD45 细胞的检出率分别为56.25%和(1.39±0.63)%,高于新鲜脐血移植组31.25%和(0.73±0.16)%,亦高于生理盐水移植组(0和0),差异有显著性(p＜0.05),扩增脐血移植组有6只NOD/SCID小鼠骨髓细胞中可检测到人特异ALU序列的表达.结论体外培养7-10天可能是收获细胞的最佳时机;SCF FL TPO 3种因子组合扩增7天的脐血单个核细胞能够植入NOD/SCID小鼠,其植入水平优于未扩增的脐血;上调脐血造血细胞上CXCR4,CD49d的表达可能会增加脐血造血细胞的植入能力.

Engraftment of Ex Vivo Expanded Mononuclear Cells from Human Umbilical Cord Blood in NOD/SCID Mice

This study was purposed to explore the effect of combination of SCF, FL, TPO without serum and stroma on the ex vivo expansion of the mononuclear cells derived from umbilical cord blood (CB) and to evaluate the proper infused time and implantation of expanded cells . Mononuclear cells derived from CB were cultured with the combination of SCF, FL and TPO for 14 days. At 0, 7, 10 and 14 days, the total mononuclear cells in culture were counted, CD34 cells and CD34 CXCR4 , CD34 CD49d cells were assayed by flow cytometry, and CFU were determined. Mononuclear cells from CB cultured for 7 days ex vivo in serum- and stroma- free medium containing FL SCF TPO were infused into sublethally irradiated NOD/SCID mice. Six weeks after transplantation, the human cells were assessed by flow cytometry and PCR. The results showed that after culturing ex vivo in the presence of SCF FL TPO for 14 days, the total number of nuclear cells significantly increased in the early stage of culture, CD34 ,CD34 CD49d cells reached a peak level at 7 days and then decreased, while the peak level of CFU, CD34 CXCR4 cells was at 10 days, then decreased. Human hematopoietic cells could be detected in the marrow of the recipients at 6 weeks after infusion and the rate of success of NOD/SCID mice implanted with the expanded cells was higher than mice transplanted with fresh sample and control (p<0.05). It is concluded that day 7-10 may be an optimal harvest time for transplantation. The expanded cells with FL SCF TPO can be engrafted into the bone marrow of sublethally irradiated NOD/SCID mice. The engrafting potential of cord blood may be enhanced via up-regulating CXCR4 and CD49d expression on the CD34 cells.