羊膜间充质干细胞克隆的分离和生物学特性的研究

发布时间：2018-01-02 10:46

本文关键词：羊膜间充质干细胞克隆的分离和生物学特性的研究　出处：《华东理工大学》2010年硕士论文　论文类型：学位论文

【摘要】： 羊膜因来源充足、含有丰富的间充质干细胞以及没有伦理争议等优点,而成为再生治疗新的细胞来源。人羊膜间充质干细胞(Human amnion derived mesenchymal stem cells, hAMCs)具多向分化潜能和低免疫原性等特点,日渐成为细胞移植治疗的热点。本文以人类羊膜为研究对象,通过酶解-贴壁方法分离到羊膜间充质干细胞,考察其自我更新能力和多向分化潜能；通过有限稀释法获得形态各异的3支细胞克隆,对比克隆间以及克隆和杂合细胞间形态、自我更新能力、多向分化潜能、表面标记和干细胞基因表达等生物学特性；最后,对比静态和动态培养前后克隆与杂合细胞在数量及质量上的差异,探索规模化扩增克隆细胞的可行方法,为临床和基础研究提供基础。胰酶和胶原酶作用羊膜组织,通过贴壁培养可以获得以细长梭形为主兼有其它形态的单核细胞,该细胞具有形成成纤维样的细胞集落和多向分化的能力,在体外可向骨、脂肪和软骨细胞分化,鉴定其为羊膜间充质干细胞,并命名为杂合细胞。通过有限稀释法获得的3支克隆细胞分别命名为8B、11D和11F,在体外传代过程中保持其各自特有的细胞形态。第6代克隆和杂合细胞表现出不同的体外扩增特性,细长型克隆(8B和11F)具有更强的集落形成能力。克隆和杂合细胞均表达CD29、CD44和CD105,不表达CD14、CD34和CD45,其中CD105在克隆细胞系的表达明显高于杂合细胞。克隆和杂合细胞在干细胞基因Oct-4和Nanog-3表达上有差异。克隆和杂合细胞表现出不同的三向分化能力：8B和11F细胞体外成骨和成脂肪能力明显优于杂合细胞,而11D细胞略低于杂合细胞；除11D外,其它细胞系均能向软骨分化。转瓶动态培养能够实现克隆细胞的扩增。与静态培养系统中收获的细胞相比,在转瓶动态培养中收获的克隆和杂合细胞,能维持较好的集落形成能力和成骨成脂肪分化潜能。研究表明,可以从羊膜中分离获得间充质干细胞,但该细胞群为多种细胞形态构成的杂合细胞。通过克隆分析发现,Nanog-3和CD105高表达的克隆具有更强的三向分化潜能,并且克隆细胞可以通过转瓶动态培养实现规模化扩增。
[Abstract]:Amniotic membrane due to adequate sources, rich in mesenchymal stem cells and the lack of ethical controversy has become a new cell source for regeneration. Human amniotic mesenchymal stem cells (Human amnion derived mesenchymal stem cells, hAMCs) with the characteristics of multilineage differentiation and low immunogenicity, has gradually become a hot spot in cell transplantation. The human amniotic membrane as the research object, through enzymatic hydrolysis and adherent separation method to amniotic mesenchymal stem cells, investigate its self-renewal and multilineage differentiation potential; 3 cell clones of different forms are obtained by the method of limited dilution cloning and comparison between cloned and heterozygous cell morphology, self-renewal capacity, differentiation potential, surface markers and gene expression in stem cells and other biological characteristics; finally, comparison of static and dynamic culture before and after cloning and heterozygous cells in the quality and quantity of the difference, explore The feasible method of scale amplification of cloned cells provides a basis for clinical and basic research.
Trypsin and collagenase effects of amniotic tissue by adherent culture can be mononuclears with other forms with a slender spindle, the formation of cells with fibroblast like cell colony and differentiation ability in vitro into bone, fat and cartilage cell differentiation, identification of the amniotic mesenchymal stem cells, and named heterozygous cells. 3 clones obtained by limited dilution method respectively named 8B, 11D and 11F, it maintains its own unique form in the process of body cell generation. The sixth generation of cloned and heterozygous cells showed different growth characteristics in vitro expansion, slender clones (8B and 11F) has a stronger ability of colony formation. Cloning and heterozygous cells were positive for CD29, CD44 and CD105, the expression of CD14, CD34 and CD45, the expression of CD105 in clonal cell lines was significantly higher than that in heterozygous cells. Cloning and heterozygous cells in stem cell gene Oct-4 and Na Nog-3. Cloning and differential expression of heterozygous cells showed three different differentiation capacity: 8B and 11F cells in vitro osteogenic and adipogenic ability is obviously better than that of heterozygous cells, while 11D cells were slightly lower than heterozygous cells; in addition to 11D, other cells can differentiation into cartilage. Dynamic culture bottle to achieve the amplification of cloned cells. Compared with the static culture system of harvested cells, in turn bottle dynamic culture harvested and heterozygous clone cells can maintain good colony forming ability and osteogenic and adipogenic differentiation potential.
Research shows that can isolate mesenchymal stem cells from amniotic membrane, but the cells were heterozygous cells composed of various cell morphology. By cloning analysis, cloning and high expression of Nanog-3 and CD105 three have stronger differentiation potential, and you can turn the bottle cell dynamic culture to achieve the scale of expansion.

【学位授予单位】：华东理工大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329

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