胎盘脐带源间充质干细胞的分离、生物学特性及其内皮分化的研究

发布时间：2018-06-03 19:13

本文选题：胎盘 + 脐带　；参考：《中国协和医科大学》2007年硕士论文

【摘要】： 间充质干细胞(mesenchymal stem cells,MSCs)是来源于中胚层的具有高度自我更新能力和多向分化潜能的成体干细胞,广泛存在于全身结缔组织和器官间质中。由于MSCs具有多向分化的潜能,而且体外容易扩增,很快成为组织工程的理想的种子细胞。胎盘和脐带都属于胎儿附属物,出生后即被遗弃。二者都具有胚外中胚层来源的结构,因此,如果从胎盘和脐带获取间充质干细胞,将为组织工程提供巨大的种子细胞库。本研究体外分离和培养胎盘脐带MSCs,比较两种MSCs与骨髓来源的MSCs在形态、生长特性、表面标志、分化潜能等生物学特征的区别,进而对它们在组织工程中的应用以及向内皮分化时基因的差异表达做了进一步的研究。第一部分研究体外分离和培养胎盘脐带单克隆MSCs,比较两种MSCs与骨髓来源的MSCs在形态、生长特性、表面标志、分化潜能等生物学特征的区别,取上述所得的单克隆来源MSCs分别向脂肪、成骨、内皮定向诱导。结果提示,可以获得性状稳定的单细胞克隆,三种来源的MSCs在形态、免疫表型、分化能力方面并无明显差异。但是骨髓来源MSCs免疫原性较强,倍增时间略长于另外两种MSCs。第二部分研究胎盘MSCs的组织工程应用。第一部分实验中发现胎盘MSCs具有获得容易分离成功率高,细胞增殖能力强等优点,故选用该细胞作为组织工程的种子细胞,将胎盘来源的MSCs和MSC-EC接种于三维支持物上,与培养板表面的MSCs生长情况对比。结果提示,MSCs和MSC-EC可以在三维支持物上生长,增值,但与培养板表面的细胞生长有差距。MSCs和MSC-EC之间并无明显差异。第三部分研究间充质干细胞向血管内皮细胞诱导分化后差异表达基因的微阵列分析,使用芯片技术高通量筛选大量基因,寻找内皮细胞化过程中的基因表达改变。结果提示,在内皮细胞化过程中有多种编码粘附分子和ECM、血管内皮细胞功能相关蛋白和受体基因发生变化。该研究首先完成了胎盘脐带间充质干细胞的单克隆分离、生物学特性检测,为后续工作奠定了基础,而后又探索了胎盘MSCs在组织工程中的初步应用以及向血管内皮细胞诱导分化后基因表达的改变,为今后MSCs的应用及研究打下了基础。
[Abstract]:Mesenchymal stem cells (MSCs) are adult stem cells with high self-renewal and multi-differentiation potential from mesoderm, which are widely found in connective tissues and organs of the whole body. Because MSCs has the potential of multidirectional differentiation and is easy to expand in vitro, it becomes an ideal seed cell for tissue engineering. Placenta and umbilical cord are fetal appendages and are abandoned after birth. Both have the structure of ectodermal origin, so if mesenchymal stem cells were obtained from placenta and umbilical cord, they would provide a huge seed cell bank for tissue engineering. In this study, we isolated and cultured placental umbilical cord MSCs in vitro, and compared the differences between MSCs and bone marrow-derived MSCs in morphology, growth characteristics, surface markers, differentiation potential and other biological characteristics. Their applications in tissue engineering and differential expression of genes in endothelial differentiation were further studied. In the first part, we studied the isolation and culture of placental umbilical cord monoclonal MSCs in vitro, and compared the differences between the two kinds of MSCs and bone marrow-derived MSCs in morphology, growth characteristics, surface markers, differentiation potential and other biological characteristics. The MSCs derived from the above monoclonal source was directed to adipose, osteoblast and endothelium respectively. The results showed that the single cell clones with stable characters could be obtained, and there was no significant difference in morphology, immunophenotype and differentiation ability of MSCs from the three sources. However, the immunogenicity of bone marrow derived MSCs was stronger, and the doubling time was slightly longer than that of the other two. The second part studies the application of placental MSCs in tissue engineering. In the first part of the experiment, it was found that placental MSCs had the advantages of high success rate of easy isolation and strong ability of cell proliferation, so it was selected as seed cells of tissue engineering, and MSCs and MSC-EC from placenta were inoculated on three-dimensional support. Compared with the growth of MSCs on the surface of culture plate. The results suggested that MSCs and MSC-EC could grow on three dimensional support and increase in value, but there was no significant difference between MSCs and MSC-EC in cell growth on the surface of culture plate. In the third part, microarray analysis of differentially expressed genes after differentiation of mesenchymal stem cells into vascular endothelial cells was studied. A large number of genes were screened by high-throughput microarray technique to find the gene expression changes in the process of endothelial cell formation. The results suggest that there are many kinds of coding adhesion molecules and ECM during the process of endothelial cellulization and the changes of vascular endothelial cell function-related proteins and receptor genes. In this study, we first completed the monoclonal isolation of placental umbilical cord mesenchymal stem cells and the detection of biological characteristics, which laid the foundation for further work. Then the preliminary application of placental MSCs in tissue engineering and the changes of gene expression after differentiation into vascular endothelial cells were explored, which laid a foundation for the application and study of MSCs in the future.
【学位授予单位】：中国协和医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R329

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