细胞因子介导的人脐血单个核细胞体外扩增并重建NOD/SCID小鼠造血及免疫功能

背景：细胞因子介导的脐血造血细胞体外扩增有望能解决脐血移植数量不足的问题。目的：实验拟确立在无基质培养条件下体外扩增脐血单个核细胞最合适的细胞因子组合及干细胞因子、FLT3配基协同促聚核细胞生成因子对造血及免疫重建功能的影响。方法：将新鲜脐血标本分离的单个核细胞接种于含有不同细胞因子组合的无血清无基质培养体系中培养7 d，根据不同细胞因子组合分组，将3因子干细胞因子+FLT3配基+促聚核细胞生成因子组合在无血清无基质条件下扩增培养7 d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。在扩增培养0，7 d检测脐血CD34+细胞数及CD34+CXCR4+，CD34+CD62L+，CD34+CD49d+的细胞数。脐血移植6周后通过流式细胞仪，PCR法检测存活小鼠体内的人源性细胞。结果与结论：移植6周后，存活小鼠体内均可检测到人源性CD45+细胞，扩增脐血移植组的NOD/SCID小鼠存活率和人特异性基因捡出率均高于新鲜脐血移植组和高于生理盐水移植组 (P < 0.05)。扩增脐血组存活NOD/SCID小鼠骨髓中可检测到人髓系细胞(CD33+)，T淋巴细胞(CD4+)，B淋巴细胞(CD19+)和人造血干细胞成分(CD34+)细胞的表达。结果提示干细胞因子+FLT3配基+促聚核细胞生成因子3因子组合脐血造血细胞体外扩增是最合适的细胞因子组合，其扩增的脐血单个核细胞能够植入并重建NOD/SCID小鼠的造血及免疫功能。

Ex vivo expanded human umbilical cord blood mononuclear cells and hematopoietic and immune reconstitution with cytokine combination in NOD/SCID mice

BACKGROUND: In vitro amplification of cytokine-mediated cord blood hematopoietic cells can solve the lack of quantity during cord blood transplantation. OBJECTIVE: To explore the effect of the proper combination cytokines without stroma on the expansion of the mononuclear cells from umbilical cord blood in vitro, and to evaluate the effect of stem cell factor + FLT3 ligand + thrombopoietin on the hematopoietic and immune reconstitution.METHODS: Human fresh cord blood mononuclear cells were cultured in serum-free and stroma-free medium containing different combination of cytokines for 7 days. According to the different combination of cytokines, our experiment was divided into groups. Mononuclear cells from cord blood were expanded for 7 days in culture containing stem cell factor + FLT3 ligand + thrombopoietin, and the expanded cells were infused into sublethally irradiated NOD/SCID mice. At days 0 and 7, numbers of cord blood CD34+ cells and CD34+CXCR4+, CD34+CD49d+, CD34+CD62L+ cells were assayed. Six weeks after transplantation, the human cells in mice were assessed by flow cytometry and PCR.RESULTS AND CONCLUSION: Human CD45+ cells could be detected in the recipients 6 weeks after infusion. Detection rate of human specific genes and the survival rate of NOD/SCID mice implanted with expanded cells were higher than mice transplanted with fresh sample and saline (P < 0.05). The human cells of medullary system cells (CD33+), T lymphocytes (CD4+), B lymphocytes (CD19+) and hemopoietic stem cells (CD34+) were detected in bone marrow of NOD/SCID mice. Results indicated that the ex vivo expansion of umbilical cord blood mononuclear cells using the combination of 3 cytokines (stem cell factor + FLT3 ligand + thrombopoietin) is sufficient for clinical application. The expanded cord blood mononuclear cells can insert and reconstruct hematopoietic and immune functions of NOD/SCID mice.