人脐血间充质干细胞体外分离培养与扩增的实验研究

发布时间：2018-01-28 22:38

本文关键词： 人脐血间充质干细胞体外培养分离扩增　出处：《广州医学院》2010年硕士论文　论文类型：学位论文

【摘要】：【目的】 1.探讨人脐血来源的间充质干细胞(Mesenchymal stem cells,MSCs)在体外分离培养与扩增的可行性。 2.探讨不同培养基浓度下人脐血间充质细胞(HMSCs)培养的差异。【方法】在无菌条件下收集正常足月胎儿的脐带血,经复合枸橼酸钠抗凝,用相对密度为1.077g/L的Ficoll淋巴细胞分离液分离脐血的单个核细胞(Mononuclear,MNC),分两组,一组以20%胎牛血清浓度的培养基进行培养和扩增,一组以30%胎牛血清浓度的培养基进行培养和扩增,用流式细胞仪检测人脐血MSCs的表面标志。【结果】 1.来源于人脐血的单个核细胞接种于20%胎牛血清浓度的培养基中后,可产生贴壁细胞,但数量稀少不能传代,接种于30%胎牛血清浓度的培养基中后,可产生较多的贴壁细胞,主要表现为破骨样细胞和间充质样细胞,经传3代后,可得纯化扩增的人脐血MSCs。流式细胞仪检测结果显示,人脐血MSCs不表达造血干细胞表面标志的相关抗原CD14、淋巴细胞表面标志的相关抗原CD19,强表达间充质干细胞表面标志的相关抗原CD105、CD44。 2.用高浓度胎牛血清培养基培养的单个核细胞形成的克隆数与低浓度胎牛血清培养的单个核细胞形成的克隆数相比差异有显著意义(P0.05)。【结论】 1.来源于人脐血的MSCs在体外可以分离培养、扩增,为人脐血MSCs的进一步研究奠定基础。 2. 30%胎牛血清浓度培养基的应用明显提高人脐血MSCs培养的成功率。
[Abstract]:[purpose] 1. To investigate the feasibility of isolation, culture and expansion of mesenchymal stem cells derived from human umbilical cord blood in vitro. 2. To investigate the difference of human umbilical cord blood mesenchymal cells (HMSCs) culture in different culture medium. [methods] Umbilical cord blood of normal full-term fetus was collected under aseptic condition and anticoagulant with sodium citrate. Mononuclear mononuclear cells (MNCs) were isolated from umbilical cord blood with a relative density of 1.077g / L of Ficoll lymphocytes. The mononuclear cells were divided into two groups. One group was cultured and amplified on the medium of 20% fetal bovine serum concentration, another group was cultured and amplified by 30% fetal bovine serum concentration. The surface markers of human umbilical cord blood MSCs were detected by flow cytometry. [results] 1. Mononuclear cells derived from human umbilical cord blood could produce adherent cells after inoculating in the medium of 20% fetal bovine serum concentration, but the number of adherent cells could not be subcultured. After inoculating in the medium of 30% fetal bovine serum concentration, more adherent cells were produced, mainly osteoclast cells and mesenchymal cells. The purified human umbilical cord blood MSCs.FCM analysis showed that human umbilical cord blood MSCs did not express CD14 associated with hematopoietic stem cell surface markers. CD19, the antigen associated with lymphocyte surface markers, strongly expressed CD105 and CD44, which were associated with the surface markers of mesenchymal stem cells. 2. The clone number of mononuclear cells cultured in high concentration fetal bovine serum medium was significantly different from that in low concentration fetal bovine serum culture medium. [conclusion] 1. MSCs derived from human umbilical cord blood can be isolated, cultured and amplified in vitro, and the further study of MSCs in human umbilical cord blood lay a foundation. 2. The application of 30% fetal bovine serum concentration medium significantly improved the success rate of human umbilical cord blood MSCs culture.
【学位授予单位】：广州医学院
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329

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