脐血单个核细胞体外扩增的实验研究

Studies on ex vivo expansion of cord blood mononuclear cells for clinical trial

目的探讨含有细胞因子的无血清培养基对脐血单个核细胞（MNC）体外培养后的扩增情况和用于移植的安全性。方法从新鲜脐血中分离出的MNC，在含细胞因子的无血清培养体系中培养。分别将培养前和培养第10天时的造血细胞进行细胞计数、细胞活力分析、集落分析、流式细胞仪检测表面标记、彗星试验分析DNA的损伤情况、无菌性分析及移植至NOD／SCID小鼠体内等项研究。结果经过体外短期培养，脐血中MNC、CD34^＋、CD133^＋、CD34^＋CXCR4^＋及CD34^＋VLA-4^＋细胞扩增倍数均比培养前增高（P〈0．05）；半固体培养基可支持多系集落的生长；培养前和培养第10天时脐血细胞DNA损伤率均低于5％；无菌性分析提示细胞未受污染。将体外扩增后的脐血造血细胞移植入NOD／SCID小鼠体内，与新鲜脐血移植相比，小鼠的存活时间及植入能力的差异均无统计学意义（P〉0.05）。结论脐血造血细胞体外扩增是解决脐血造血细胞数量不足的有效方法。脐血造血细胞经短期培养能为造血干细胞移植提供安全而具植入能力的造血细胞。

Objective To explore the expansion effect and safety of cord blood mononuclear cells （MNCs） after culture ex vivo in serumfree system in combination with cytokines for transplantation. Methods MNCs separated from cord blood were cultured in serum free system in combination with cytokines. Analyses were performed before and after culture for viability, colony, CD34^＋ , CD133^＋ , CD34^＋ CXCR4^＋ and CD34^＋ VLA-4^＋ cells, sterility, comet assay and NOD/SCID mouse transplantation. Results Ex vivo results demonstrated that this culture system induced expansion of MNCs, colony, CD34^＋ , CD133^＋ , CD34^＋ CXCR4^＋ and CD34^＋ VLA-4^＋ cells. Comet assay showed that the DNA damage rate was less than 5 % before and after short-term culture. Sterility assay showed that the culture system was safe. The animal trial demonstrated the cord blood MNCs could maintained their repopulating ability in NOD/SCID mice after culture. Conclusions Ex vivo expansion of umbilical cord blood MNCs is an effective way to overcome the barrier of small number of hematopoietic cells. The expansion procedures could offer safe cord blood MNCs for clinical use.