脐带血干细胞的培养与收获

发布时间：2018-11-10 12:34

【摘要】： 脐带血(Umbilical cord blood,UCB)中同时含有造血干细胞(Hematopoietic stemcells,HSCs)和间充质干细胞(Mesenchymal stem cells,MSCs),造血干细胞(UCB-HSCs)已被用于基因治疗、肿瘤净化和造血重建等方面,另一种干细胞间充质干细胞(UCB-MSCs),不仅可以作为滋养层细胞支持HSCs在体外的大规模扩增,在造血移植过程中还能够降低并发症的发生率以及加速造血重建功能的恢复。因此,在一次原代实验中同时培养并收获这两种细胞具有重要的临床应用价值。但是这两种细胞的生长方式不同,UCB-HSCs为悬浮生长而UCB-MSCs为贴壁生长,传统的培养方法很难在一次原代实验中同时培养并分别收获两种细胞。本文利用微载体为UCB-MSCs提供贴壁表面,寻找适宜的培养基来同时培养脐带血中的两种干细胞。培养结束后采用自由沉降的方法方便地将悬浮的UCB-HSCs和粘附在微载体上的UCB-MSCs两种干细胞分离并收获。本文通过正交实验证实血清是原代UCB-MSCs在微载体表面成功贴附并生长的重要影响因素,而在一般的血清培养中使用的是胎牛血清(FBS)等动物血清,这种体系下培养扩增的细胞在临床应用时会有免疫排斥反应等。本文从同一份脐带血中提取了人自体血清,作为FBS的替代血清加入到培养基中,亦可同时收获UCB-HSCs和UCB-MSCs,原代培养UCB-MSCs的成功率为71.4%。本文同时考察了不同血清用量对UCB-HSCs和UCB-MSCS体外扩增的影响。细胞悬液中的细胞计数及CD34~+细胞流式分析结果表明,随着血清含量的增加,UCB-MSCs的扩增能力略有增加,但是并无显著性差异,而UCB-HSCs的扩增效果在人自体血清含量为5.6%时最好。在96孔板培养体系(100μl)中,经过六天培养可使UCB-HSCs扩增1.88±0.33倍。
[Abstract]:Umbilical cord blood (Umbilical cord blood,UCB) contains both hematopoietic stem cells (Hematopoietic stemcells,HSCs) and mesenchymal stem cells (Mesenchymal stem cells,MSCs). Hematopoietic stem cells (UCB-HSCs) have been used in gene therapy, tumor purification and hematopoietic reconstitution. Another type of stem cell mesenchymal stem cells (UCB-MSCs) can not only be used as trophoblast cells to support the large-scale expansion of HSCs in vitro, but also can reduce the incidence of complications and accelerate the recovery of hematopoietic reconstitution function during hematopoietic transplantation. Therefore, it has important clinical application value to culture and harvest these two kinds of cells simultaneously in a primary experiment. However, the growth patterns of these two kinds of cells are different, UCB-HSCs is suspended growth and UCB-MSCs is adherent growth, the traditional culture method is difficult to culture and harvest two kinds of cells simultaneously in a primary experiment. In this paper, microcarriers were used to provide adherent surface for UCB-MSCs and to find suitable medium for simultaneously culturing two kinds of stem cells in umbilical cord blood. Two kinds of stem cells, suspended UCB-HSCs and UCB-MSCs adhered to microcarriers, were isolated and harvested by free sedimentation after culture. In this paper, it is proved by orthogonal experiment that serum is an important factor influencing the successful adhesion and growth of primary UCB-MSCs on the surface of microcarriers, but in the normal serum culture, the animal serum, such as fetal bovine serum (FBS), is used. In this system, the expanded cells in the clinical application will have immune rejection and so on. Human autologous serum was extracted from the same umbilical cord blood and added to the medium as a substitute for FBS. The success rate of primary culture of UCB-HSCs and UCB-MSCs, was 71.4. The effects of different serum dosage on UCB-HSCs and UCB-MSCS amplification in vitro were also investigated. The results of cell count and CD34~ cell flow analysis showed that with the increase of serum content, the amplification ability of UCB-MSCs increased slightly, but there was no significant difference. The amplification effect of UCB-HSCs was the best when the content of human autologous serum was 5.6. In the 96 well plate culture system (100 渭 l), the UCB-HSCs amplification was 1.88 卤0.33 times after 6 days of culture.
【学位授予单位】：大连理工大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329

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