脐血间充质干细胞联合细胞因子支持人脐血单个核细胞体外扩增的探讨

发布时间：2018-04-17 18:40

本文选题：脐血 + 细胞因子　；参考：《广州医学院》2009年硕士论文

【摘要】： 脐血作为一种造血干细胞资源越来越受到广泛重视,但由于单份脐血中造血干细胞数量不足,不能满足大多数成人和高体重儿童造血干细胞移植重建造血及免疫功能的需要,故限制了其广泛应用。体外扩增虽然可以增加造血细胞数量,但可能导致造血干细胞分化并降低其归巢和长期造血重建能力,因此提高脐血造血干细胞体外扩增质量仍是脐血移植中亟待解决的问题。间充质干细胞作为造血微环境主要细胞成分基质细胞的前体细胞,可以分泌与造血干细胞生长发育相关的黏附分子、细胞外基质和多种细胞因子,为造血干细胞体外扩增提供适宜的微环境。随着培养技术的提高,脐血间充质干细胞有着与其他来源的间充质干细胞所无法比拟的优势。本实验研究脐血间充质干细胞对人脐血单个核细胞体外扩增的支持作用。目的探讨脐血来源间充质干细胞(UCB-MSCs)体外分离、培养和初步鉴定的方法,以及UCB-MSCs联合外源性细胞因子对人脐血单个核细胞(UCB-MNCs)体外扩增的支持作用和最佳收获时间,为配合造血干细胞移植的临床应用提供实验方法。方法用羟乙基淀粉(HES)和人淋巴细胞分离液(Ficoll-Hypaque)以密度梯度离心法分离脐血单个核细胞,采用贴壁筛选法以MesencultTM专用培养基培养出脐血间充质干细胞,并通过流式细胞仪检测其细胞表面抗原。将新鲜脐血标本分离出的脐血单个核细胞接种于无血清培养体系(Stem spanTM)中培养18天,实验分三组,A组:为空白对照组(培养体系中无外源性细胞因子和UCB-MSCs滋养层);B组:为因子组(培养体系中有外源性细胞因子但无UCB-MSCs滋养层);C组:为实验组(培养体系中既有外源性细胞因子又有UCB-MSCs滋养层)。在第0、7、10、14及18天检测有核细胞总数(MNCs)、CD34+细胞数、CD133+细胞数、集落形成单位数(CFU)和细胞在周期(G2+M+S期)含量的变化。结果①从脐血中分离、培养的出间充质干细胞,稳定表达CD29、CD105和CD44,不表达CD34和CD133。②在体外培养过程中,外源性细胞因子及UCB-MSCs均对脐血单个核细胞的扩增起支持作用,但以UCB-MSCs联合外源性细胞因子组效果最好,该组各项指标在同一时间点较其它两组高,有统计学意义(P㩳0.05),且维持造血至少达18天。③以UCB-MSCs联合外源性细胞因子对脐血单个核细胞扩增,第10天上述各项指标达到最高峰,有统计学意义(P㩳0.05)。结论①采用密度梯度离心联合贴壁筛选法并通过流式细胞检测技术,可以成功地实现从脐血中分离、培养出间充质干细胞,并完成其细胞表型的初步鉴定。②UCB-MSCs联合外源性细胞因子可有效扩增脐血单个核细胞。③UCB-MSCs联合外源性细胞因子体外扩增脐血单个核细胞细胞收获的最佳时间为第10-14天。
[Abstract]:As a kind of hematopoietic stem cell resource, umbilical cord blood has been paid more and more attention. However, due to the insufficient number of hematopoietic stem cells in a single cord blood, it can not meet the needs of most adult and high-weight children with hematopoietic stem cell transplantation and reconstitution of hematopoietic and immune function.Therefore, its wide application is limited.Although in vitro amplification can increase the number of hematopoietic cells, it may lead to differentiation of hematopoietic stem cells and decrease their homing and long-term hematopoietic reconstitution ability. Therefore, improving the expansion quality of umbilical cord blood hematopoietic stem cells in vitro is still an urgent problem to be solved in umbilical cord blood transplantation.Mesenchymal stem cells, as precursors of stromal cells, which are the main cell components of hematopoietic microenvironment, can secrete adhesion molecules, extracellular matrix and many cytokines related to the growth and development of hematopoietic stem cells.To provide a suitable microenvironment for the expansion of hematopoietic stem cells in vitro.With the improvement of culture technology, umbilical cord blood mesenchymal stem cells have unparalleled advantages over other mesenchymal stem cells.The purpose of this study was to investigate the supporting effect of umbilical cord blood mesenchymal stem cells on the expansion of human umbilical cord blood mononuclear cells in vitro.Objective to investigate the methods of isolation, culture and identification of UCB-MSCs derived from umbilical cord blood in vitro, and the supporting effect of UCB-MSCs combined with exogenous cytokines on the expansion of human umbilical cord blood mononuclear cells (UCB-MNCs) in vitro and the optimal harvest time.To provide experimental methods for clinical application of hematopoietic stem cell transplantation.Methods umbilical cord blood mononuclear cells were isolated with hydroxyethyl starch (HES) and human lymphocyte isolate (Ficoll-Hypaque) by density gradient centrifugation. Mesenchymal stem cells from umbilical cord blood were cultured in MesencultTM medium by adherent screening method.The cell surface antigen was detected by flow cytometry.Cord blood mononuclear cells isolated from fresh cord blood samples were cultured in serum-free culture system Stem span TM1 for 18 days.The experiment was divided into three groups: blank control group (no exogenous cytokines in culture system and UCB-MSCs trophoblastic trophoblast group B): factor group (exogenous cytokines in culture system but no trophoblastic layer in UCB-MSCs group C): experimental group (culture group)Both exogenous cytokines and UCB-MSCs trophoblast were found in the system.The number of CD133 cells, the number of colony forming units (CFU) and the changes of cell contents in G 2 M S phase were detected on the 14th and 18th day of the 7th day. The total number of nucleated cells and the number of CD34 cells and the number of colony forming units (CFU) were measured.Results 1 Mesenchymal stem cells isolated from umbilical cord blood expressed CD29, CD105 and CD44 stably, but not CD34 and CD133.2. Exogenous cytokines and UCB-MSCs played a supporting role in the expansion of cord blood mononuclear cells in vitro.But the effect of UCB-MSCs combined with exogenous cytokines was the best, and the indexes of the group were higher than those of the other two groups at the same time point.At least 18 days after maintenance, UCB-MSCs combined with exogenous cytokines was used to amplify umbilical cord blood mononuclear cells. On the 10th day, the above indexes reached the highest peak, with statistical significance.Conclusion 1 using density gradient centrifugation combined with adherent screening and flow cytometry, mesenchymal stem cells can be successfully isolated and cultured from umbilical cord blood.The results showed that UCB-MSCs combined with exogenous cytokines could effectively amplify cord blood mononuclear cells. 3UCB-MSCs combined with exogenous cytokines could obtain the best harvest time of UCB-MSCs combined with exogenous cytokines in vitro. The best time for the harvest of UCB-MSCs combined with exogenous cytokines was 10-14 days.
【学位授予单位】：广州医学院
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329

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