人羊膜间充质干细胞的培养及造血支持的初步研究

发布时间：2018-11-13 10:30

【摘要】： 目的应用成熟的人羊膜间充质干细胞(A-MSC)的分离培养技术,深入研究其生物学特性及支持造血的功能,以促进羊膜间充质干细胞临床的广泛应用,尤其为临床进行MSC与HSC共移植、提高HSC移植成功率提供实验证据。方法1、采用已建立的间充质干细胞(MSC)分离培养体系进行羊膜间充质干细胞的分离、培养、纯化及扩增;2、收集传代培养的第三代细胞10~6个数量级,采用RT-PCR方法在mRNA水平上分析LIF,SCF,M-CSF,G-CSF,MG-CSF,IL-3,IL-6,IL-11等因子在体外培养的人羊膜间充质干细胞(A-MSC)中的表达情况;3、将去贴壁细胞的脐血单个核细胞接种到经X射线照射的第三代人羊膜MSC单层滋养层上,采用长期培养体系(MethoCult~(TM)H5100)培养,每周半量换液,脐血单个核细胞单独培养作为对照,分别收集两组第3d、第7d、第14d、第21d培养液中的悬浮细胞,通过细胞计数,评价人羊膜MSC体外支持造血功能。结果可检测到人羊膜间充质干细胞(A-MSC)表达LIF,SCF,M-CSF,G-CSF,MG-CSF,IL-3,IL-6,IL-11等多种造血调控因子;通过定期计数培养液中的悬浮细胞,表明羊膜MSC具有体外支持造血的功能。结论1、组织块植块培养法是一种比较理想的体外分离、培养、纯化、扩增人羊膜间充质干细胞的方法。2、本实验证实了人羊膜组织与造血之间的相关性,同时初步证实人羊膜间充质干细胞具有造血支持作用,提示人羊膜间充质干细胞与造血干细胞联合移植将可能有效地促进造血恢复和植入。
[Abstract]:Objective to study the biological characteristics and hematopoietic support of mature human amniotic mesenchymal stem cells (A-MSC) in order to promote the clinical application of amniotic mesenchymal stem cells (MMSCs). Especially for clinical MSC and HSC co-transplantation to improve the success rate of HSC transplantation to provide experimental evidence. Methods 1. The amniotic membrane mesenchymal stem cells (MSCs) were isolated, cultured, purified and amplified by the established mesenchymal stem cell (MSC) isolation and culture system. 2. The third generation cells of 10 ~ 6 orders of magnitude were collected and LIF,SCF,M-CSF,G-CSF,MG-CSF,IL-3,IL-6, was analyzed on the mRNA level by RT-PCR method. Expression of IL-11 and other factors in human amniotic mesenchymal stem cells (A-MSC) cultured in vitro; 3. Umbilical cord blood mononuclear cells (UCB) were inoculated into the third generation of human amniotic membrane MSC monolayer trophoblastic layer irradiated by X ray. The mononuclear cells were cultured in a long-term culture system (MethoCult~ (TM) H5100). Cord blood mononuclear cells were cultured alone as control. Suspension cells were collected from the culture medium on the 3rd, 7th, 14th and 21st day, respectively. The hematopoietic function of human amniotic membrane MSC was evaluated by cell count. Results Human amniotic mesenchymal stem cells (A-MSC) expressed LIF,SCF,M-CSF,G-CSF,MG-CSF,IL-3,IL-6,IL-11 and other hematopoietic regulatory factors. The suspension cells in the culture medium were counted regularly, indicating that amniotic membrane MSC has the function of supporting hematopoiesis in vitro. Conclusion 1. Tissue block culture is an ideal method for isolating, culturing, purifying and amplifying human amniotic mesenchymal stem cells in vitro. 2. The correlation between human amniotic membrane tissue and hematopoiesis was confirmed. At the same time, human amniotic mesenchymal stem cells had hematopoietic support, suggesting that combined transplantation of human amniotic mesenchymal stem cells and hematopoietic stem cells might promote hematopoietic recovery and implantation.
【学位授予单位】：昆明医学院
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329

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