人脐带间充质干细胞的分离培养及其多向分化潜能的研究

发布时间：2018-03-12 06:43

本文选题：人脐带间充质干细胞　切入点：细胞分化　出处：《内蒙古农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：角质形成细胞是皮肤表皮组织中最主要的细胞类型,也是对皮肤损伤进行细胞治疗时最重要的细胞类型。间充质干细胞可经诱导分化为多种细胞,具有极低的免疫原性,在细胞治疗领域是一种理想的种子细胞。本研究通过改良的"组织块二次贴壁法",从脐带华通氏胶中高效、大量地获得了人脐带间充质干细胞(human umbilical cord mesenchymal stem cells,hUCMSCs),在特殊的诱导培养基中培养时可分化为多种细胞类型。主要研究结果如下:1.建立了 hUCMSCs细胞系,细胞形态为长梭形、呈漩涡状生长。第三代(P3)细胞表达干细胞转录因子Sox2、间充质干细胞表面标志物基因CD29、CD90和CD105,不表达造血系细胞表面标志物基因CD34、CD45及人白细胞抗原基因HLA-DR。经测定,其生长曲线呈"S"型,细胞倍增时间为24.7 h,生长良好、活性很高,染色体为46,XY正常男性核型,遗传稳定。2.建立了 hUCMSCs多向诱导分化培养体系,诱导其向外胚层(神经细胞、角质形成细胞)和中胚层(成骨细胞)来源的细胞分化,染色鉴定结果均为阳性。3.建立了角质形成细胞诱导分化培养体系,hUCMSCs经诱导培养后分化为角质形成细胞,蛋白质水平染色结果显示其表达细胞角蛋白,基因水平检测到其特异基因CK19的表达量逐日增高。综上所述,本实验创建了 hUCMSCs原代培养的新方法,并成功建立了 hUCMSCs细胞系,获得的hUCMSCs具有多向分化的能力,同时建立了向角质形成细胞诱导分化的培养体系,为间充质干细胞的临床应用尤其是大面积皮肤损伤的细胞治疗提供了实验依据。
[Abstract]:Keratinocytes are the most important cell types in skin epidermis and the most important cell types in the treatment of skin injury. Mesenchymal stem cells can be induced to differentiate into a variety of cells with very low immunogenicity. It is an ideal seed cell in the field of cell therapy. A large number of human umbilical cord mesenchymal stem cells were obtained from human umbilical cord mesenchymal stem cells, which could differentiate into many cell types in a special induction medium. The main results are as follows: 1. The cell line of hUCMSCs was established, and the cell morphology was fusiform. The third generation of P3 cells expressed stem cell transcription factor Sox2, mesenchymal stem cell surface marker gene CD29, CD90 and CD105, but not hematopoietic cell surface marker gene CD34, CD45 and human leukocyte antigen gene HLA-DR. Its growth curve is "S" type, the cell doubling time is 24.7 h, the cell growth is good, the activity is very high, the chromosome is 46 XY normal male karyotype, the heredity is stable .2.It has established the culture system of hUCMSCs multidirectional induction differentiation, induced it to the ectoderm (nerve cell), The differentiation of keratinocytes from keratinocytes) and mesoderm (osteoblasts) were positive. 3. The culture system of keratinocyte induced differentiation was established and then differentiated into keratinocytes. The results of protein level staining showed that it expressed cytokeratin, and the expression of its specific gene CK19 was increased day by day. In conclusion, a new method of primary culture of hUCMSCs was established, and the hUCMSCs cell line was successfully established. The obtained hUCMSCs has the ability of multi-differentiation, and the culture system of inducing differentiation into keratinocytes has been established, which provides the experimental basis for the clinical application of mesenchymal stem cells, especially for the treatment of extensive skin injury.
【学位授予单位】：内蒙古农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R329.2

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