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目的探讨脐血间充质干细胞(MSC)的生物学特征及其对造血干/祖细胞体外扩增的支持作用。方法用液体培养法分离脐血贴壁细胞,采用ELISA方法检测贴壁细胞条件培养液中细胞因子的表达;用流式细胞术分析其免疫表型特征;在成软骨细胞诱导培养条件下诱导细胞分化,并用RTPCR方法检测分化后细胞原胶原Ⅱ型基因的表达。采用分阶段共培养方法观察脐血贴壁细胞对CD34+细胞体外扩增的支持作用。结果脐血单个核细胞纤维样细胞集落形成率为(3.5±0.7)/106。脐血MSC体外至少可以扩增15代。没有分化的脐血MSC表型为CD13、CD29、CD90、CD105、CD166、SH2、SH3和SH4阳性,CD45、CD34和CD14阴性;脐血MSC培养上清中干细胞因子、IL6和肿瘤坏死因子α检测阳性。在成软骨细胞诱导培养基培养条件下,脐血MSC原胶原Ⅱ型基因mRNA表达阳性。脐血MSC与CD34+细胞共掊养14d,CD34+细胞扩增率高于未共培养组4倍。结论脐血MSC具有类似于成体骨髓MSC的特征,对造血干细胞增殖有明显的支持作用。
Objective To investigate the biological characteristics of umbilical cord blood mesenchymal stem cells (MSCs) and their role in the in vitro expansion of hematopoietic stem / progenitor cells. Methods The cord blood adherent cells were isolated by liquid culture and the expression of cytokines in adherent cells conditioned medium was detected by ELISA. The immunophenotypic characteristics were analyzed by flow cytometry. The cells were induced under chondrogenic induction Differentiation, and RTPCR method was used to detect the expression of procollagen type Ⅱ gene after differentiation. The stably co-culture method was used to observe the supporting effect of cord blood adherent cells on the expansion of CD34 + cells in vitro. Results The rate of fibroblast colony formation in umbilical cord blood mononuclear cells was (3.5 ± 0.7) / 106. Cord blood MSC in vitro can be expanded at least 15 generations. The phenotype of undifferentiated cord blood MSC was positive for CD13, CD29, CD90, CD105, CD166, SH2, SH3 and SH4, but negative for CD45, CD34 and CD14; the levels of stem cell factor, IL6 and tumor necrosis factor alpha Positive. In chondrocyte-induced culture medium, cord blood MSC procollagen type II gene mRNA expression was positive. The cord blood MSC co-cultured with CD34 + cells for 14 days, the rate of CD34 + cells expansion was 4 times higher than that of the non-co-culture group. Conclusion The umbilical cord blood MSCs have characteristics similar to those of adult bone marrow MSCs, and have obvious supportive effect on the proliferation of hematopoietic stem cells.