人羊膜间充质干细胞支持造血的体外研究

发布时间：2018-05-30 00:16

  本文选题：羊膜 + 间充质干细胞　； 参考：《昆明医学院》2010年硕士论文

【摘要】： 目的:探讨人羊膜来源的间充质干细胞(mesenchymal stem cells,MSCs)在体外对造血干细胞(hematopoietic stem cells,HSCs)的扩增是否有支持作用,以促进羊膜间充质干细胞(amniotic mesenchymal stem cells,AMSCs)的临床应用,尤其为临床移植前扩增HSCs数量,以及MSCs和HSCs共移植、提高干细胞移植成功率提供实验依据。 方法:利用成熟的MSCs培养条件,从足月分娩的人羊膜中分离培养AMSCs;用密度梯度离心法分离脐血单个核细胞:用免疫磁珠分选技术分选其中CD34~+细胞:分别用脐血单个核细胞和CD34~+细胞与AMSCs共培养,连续培养四周,每周计悬浮细胞浓度,并把它们分别与骨髓MSCs共培养作为对照。共培养两周后,利用HSCs定向分化的潜能,采用集落形成实验,把扩增的脐血单个核细胞和CD34~+细胞分别接种至甲基纤维素半固体培养基中,14-16天后根据典型形态特征计CFU-GM、BFU-E、CFU-E和CFU-GEMM集落数。结果均以(?)±S表示,各组均数间差异性检验用单因素方差分析。 结果:从人羊膜分离培养出的干细胞在形态和表型上均符合MSCs的特征,细胞呈梭形和多角形。流式分析显示,AMSCs高表达CD29、CD44、CD105,不表达CD31、CD34、CD45、pan-CK等内皮、造血及上皮细胞表型,HLA-DR呈阴性。AMSCs可促进人脐血单个核细胞和CD34~+细胞扩增,扩增后的两者在甲基纤维素半固体培养基中能够形成造血祖细胞集落,其造血支持作用与骨髓MSCs相似,两者对比无明显差异。而无MSCs支持的脐血单个核细胞和CD34~+细胞,其扩增能力和集落形成能力明显减弱。 结论:用组织块培养法可以从人羊膜中成功分离培养出MSCs;AMSCs体外具有与骨髓MSCs相似的造血支持作用,有可能为HSCs体外扩增及临床MSCs与HSCs联合移植、提高HSCs移植成功率提供一种新的、更加理想的MSCs新来源。
[Abstract]:Objective: to investigate whether mesenchymal stem cells (MSCs) derived from human amniotic membrane can support the expansion of hematopoietic stem cells (HSCs) in vitro, so as to promote the clinical application of amniotic mesenchymal stem cells / amniotic cells. Especially, it provides experimental basis for increasing the number of HSCs before transplantation and co-transplantation of MSCs and HSCs to improve the success rate of stem cell transplantation. Methods: using mature MSCs culture conditions, AMSCs were isolated from human amniotic membrane during term delivery, cord blood mononuclear cells were isolated by density gradient centrifugation, CD34 ~ cells were separated by immunomagnetic bead sorting technique: cord blood mononuclear cells and CD34 ~ cells were co-cultured with AMSCs for four weeks, respectively. Suspension cell concentration was measured weekly and co-cultured with bone marrow MSCs as control. After two weeks of co-culture, colony forming experiments were carried out using the potential of HSCs directional differentiation. The expanded cord blood mononuclear cells and CD34~ cells were inoculated into methylcellulose semisolid medium for 14-16 days. The colony numbers of CFU-GMU BFU-EU CFU-E and CFU-GEMM were calculated according to the typical morphological characteristics. The results were all expressed in the form of + S, and the analysis of variance was used to test the difference of the mean of each group by single factor analysis of variance (ANOVA). Results: the stem cells isolated from human amniotic membrane were morphologically and phenotypically consistent with the characteristics of MSCs, and the cells were fusiform and polygonal. Flow cytometry showed that AMSCs overexpressed CD29, CD44, CD105, and did not express CD31, CD34, CD45, pan-CK and other endothelium. HLA-DR negative phenotype of hematopoietic and epithelial cells could promote the expansion of mononuclear cells and CD34~ cells in human umbilical cord blood. The hematopoietic progenitor cell colony was formed in methyl cellulose semisolid medium, and the hematopoietic supporting effect was similar to that of bone marrow MSCs, but there was no significant difference between them. However, the ability of expansion and colony formation of cord blood mononuclear cells and CD34 ~ cells without MSCs support was significantly decreased. Conclusion: MSCs can be successfully isolated from human amniotic membrane by tissue mass culture method and have similar hematopoietic support effect to bone marrow MSCs in vitro, which may be HSCs amplification in vitro and clinical MSCs combined with HSCs transplantation. Increasing the success rate of HSCs transplantation provides a new and more ideal source of MSCs.
【学位授予单位】：昆明医学院
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329

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相关期刊论文 前2条

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