人脐带血间充质干细胞混合培养体系的建立

Establishment of co-culture system of the mixed human umbilical cord blood-derived mesenchymal stem cells in vitro

目的优化脐带血单个核细胞的分离方法,培养、纯化及鉴定脐血间充质干细胞(UCB-MSCs),并观察其生物学特性,探讨单份和混合UCB-MSCs的区别。方法采用四联袋方法和试管方法分离脐带血单个核细胞,比较两种方法分离后单个核细胞的回收率、细胞数及红细胞混入量。鉴定UCB-MSCs向神经样细胞横向分化的能力。酶联免疫吸附试验(ELISA)方法分析单份组和混合组培养液上清中白细胞介素(IL)-2和γ-干扰素(IFN-γ)含量。符合正态分布并方差齐的,根据数据类型应用参数检验中的单因素方差分析或t检验,不符合正态分布的应用非参数检验中秩和检验。结果试管法和四联袋法分离的单个核细胞回收率分别为(60.23±6.34)%、(71.92±11.30)%(P<0.05)。试管法和四联袋法分离后单个核细胞(MNC)数量分别为(6.12±1.21)×10^7、(9.29±3.11)×10^7个(P<0.05)。混合组的培养液上清中IL-2和IFN-γ含量高于单份组。单份组和混合组的IL-2水平分别为(0.52±0.13)、(1.50±0.23)pg/ml(P<0.05);单份组和混合组IFN-γ水平分别为(43.16±4.75)、(56.07±6.67)pg/ml(P<0.05)。巢蛋白(Nestin)、神经元特异性烯醇化酶(NSE)及神经胶质原纤维酸性蛋白(GFAP)染色均为阳性表达。结论采用四联袋方法分离的单个核细胞回收率和细胞数均高于试管方法,四联袋法分离单个核细胞是一种高效、高质量和安全的分离方法,UCB-MSCs可以向神经样细胞分化,混合组UCB-MSCs增殖活跃,分泌更多IL-2和IFN-γ细胞因子。

Objective To optimizate mononuclear isolation methods from human umbilical cord blood,and characterizate human umibilical cord blood-derived mesenchymal stem cells(UCB-MSCs),study its biological characteristics and further investigate the differences between the single and the mixed UCB-MSCs.Methods Four sterile plastic bags and tube method isolated mononuclear cells from umibilical cord blood and compared two methods in recovery rate of mononuclear cells and red blood cells.UCB-MSCs were identifid into nerve-like cells.Enzyme linked immunosorbent assay(ELISA)analyzed the interleukin(IL)-2 and interferon(IFN)-γlevels in supernatant medium of the single and mixed groups.Results The recovery rate of mononuclear cells isolated by tube method and quadruple bag method were(60.23±6.34)%and(71.92±11.30)%,respectively(P<0.05).The number of mononuclear cells(MNC)was(6.12±1.21)×10^7 and(9.29±3.11)×10^7 cells(P<0.05).The content of IL-2 and IFN-γin the supernatant of mixed group was higher than that of single group.The levels of IL-2 in single dose group and mixed group were(0.52±0.13)pg/ml and(1.50±0.23)pg/ml respectively(P<0.05);the levels of IFN-γin single and mixed groups were(43.16±4.75)pg/ml and(56.07±6.67)pg/ml respectively(P<0.05).Nestin,neuron specific enolase(NSE)and glial fibrillary acidic protein(GFAP)of cells was positive through immunohistochemical staining.Conclusion The number of mononuclear cells and recovery rate of four sterile plastic bags were higher than tube method.Separation of mononuclear cells from human umbilical cord blood with four sterile plastic bags was an efficient and safe method.UCB-MSCs could differentiate to osteogenic,fat and nerve-like cells.The mixed UCB-MSCs proliferated actively and secreted more cytokines.