成体组织中“类胚胎干细胞”可塑性的初步研究

发布时间：2018-03-07 21:23

  本文选题：生物信息学　切入点：骨髓源干细胞　出处：《第三军医大学》2009年博士论文　论文类型：学位论文

【摘要】： 干细胞自发现以来一直是研究的焦点,它为许多疾病的治疗提供了新的思路和方法,具有重要意义。由于干细胞表面缺乏明确的标志,很难分离纯化单一克隆的细胞种群,这就导致我们对干细胞的认识存在非常大的局限性。正是由于上述这些原因,干细胞研究既是当前生物医学领域研究的热点之一,更是研究的难点之一。 从干细胞本身发育的特点来看,具有高度的自我复制能力是其重要的特征之一,因此有学者推测:在成体组织中可能残留有胚胎发育过程中不同分化阶段的干细胞群,其中可能就有具有胚胎时期干细胞特性的干细胞亚群。 在目前研究条件下,从成体组织中分离获得的干细胞通常是多种干细胞的混合物,具有多种表面分化抗原。在传统的观念中,细胞表面分化抗原通常标志着细胞具有不同的分化成熟特性。干细胞表面存在多种不同分化抗原是否代表着成体组织中残留有多种不同发育阶段的干细胞亚群?目前还不是很清楚,需要进行深入的研究。 近年来在成体组织中发现一类具有向多胚层发育潜能的多能干细胞。这些研究结果支持了成体组织中可能残留有胚胎发育过程中的、具有不同分化阶段的干细胞群。这些从成体组织中获得的干细胞中,是否存在具有胚胎早期干细胞特征的细胞亚群,目前还缺乏足够的证据,但有个别的研究表明这个情况是可能存在的,Ratajczak等将这类细胞命名为“类胚胎干细胞”。 要证实成体干细胞中残留有胚胎发育早期的干细胞群,还必需找到足够多的实验证据来支持上述观点。基于上述认识和分析,我们通过以下几个部分的研究来证实可能在成体组织中残留有“类胚胎干细胞”。 1.“类胚胎干细胞”标志物的筛选: 采用生物信息学技术,对美国国立图书馆(NCBI)的数据库进行DDD (Digital Differential Display)分析,结果显示,成体组织细胞与胚胎干细胞基因表达谱差异分子是颗粒酶B(Granzyme B,GRB);然后进一步复习文献,发现阶段性胚胎抗原-1(Stage specific embronic antigen-1,SSEA-1)、胚胎干细胞关键蛋白-4(Octamer-4,Oct-4)这两种蛋白在胚胎干细胞发育中具有重要作用。最后我们得出结论:这三种蛋白质可以作为标志物鉴定成体干细胞中是否存在具有胚胎发育早期特性的干细胞亚群。 2.SSEA-1、Oct-4、GRB在骨髓、脂肪、皮肤来源的干细胞中的表达: 为了证实成体组织中可能残留有“类胚胎干细胞”,我们采用不同的分离方法,分别从骨髓、脂肪、皮肤提取成体干细胞,然后采用免疫荧光化学的方法,观察上述选定的三种标记物在这三种不同组织来源的干细胞中表达的情况。 结果显示,骨髓、脂肪、皮肤来源的成体干细胞均能不同程度地表达SSEA-1、Oct-4、GRB。皮肤来源的成体干细胞(Dermal-derived adult stem cells,DSCs)三种标记物表达最弱,而其它两种组织来源的干细胞中表达Oct-4、GRB蛋白的情况接近,骨髓来源的成体干细胞(Bone marrow-derived stem cells,BMSCs)表达SSEA-1最强。 3.SSEA-1表达阳性骨髓成体干细胞的免疫磁珠分选及其生物学特性: 应用SSEA-1抗体,通过免疫磁珠分选法,收获了SSEA-1呈阳性表达的骨髓来源的干细胞亚群(SSEA-1+BMSCs),SSEA-1的表达细胞接近80%;可以进行成脂、成神经和成胰岛诱导分化,表明这类干细胞具有多向分化潜能,其中可能存在“类胚胎干细胞”。骨髓中“类胚胎干细胞”的存在可能是其可塑性分化的另一条途径。 4.骨髓来源的成体干细胞在创伤修复中的动员及募集作用: 将Brdu标记的第三代骨髓来源的成体干细胞输入到5Gy放射性损伤的C57小鼠体内,建立骨髓移植模型;利用骨髓移植模型动物分别建立皮肤缺损和Ccl4肝脏中毒损伤两种实验动物模型,研究骨髓成体干细胞在组织损伤中的作用。 结果显示,嵌合于骨髓中的Brdu标记的干细胞可以通过动员及募集作用参与创伤的修复。
[Abstract]:Stem cells have been the focus of research since the discovery, provides new ideas and methods for the treatment of many diseases, which is of great significance. Because of the lack of clear stem cell surface markers, it is difficult to purify single clone cell population, which leads to our understanding of stem cells exist great limitations. It is precisely because of these reasons, the stem is one of the hot topics in the field of biomedical cell research, is one of the most difficult research.
From the characteristic of stem cells and development of itself, having the ability of self replication is one of its important features, so some scholars speculate that in adult tissue may be residual stem cells at different stages of differentiation during embryonic development, which may have characteristics of embryonic stem cells with stem cell subsets.
In the present study conditions, isolated stem cells are usually a mixture of different types of stem cells from adult tissues, with a variety of surface antigen. In the traditional concept, cell surface differentiation antigen usually marked with different differentiation cells. Stem cells have different surface differentiation antigens represents a there are a variety of tissue residues in different developmental stages of stem cell subsets? Is not clear, the need for in-depth research.
In recent years, found a developmental potential to embryonic pluripotent stem cells in adult tissues. These findings support adult tissues may remain in the process of embryonic development, with different stages of differentiation. These stem cells obtained from adult tissue stem cells, whether there is a cell the characteristics of early embryonic stem cell subsets, there is not enough evidence, but there are a few studies show that this situation is likely to exist, such as the Ratajczak of these cells named "embryonic stem cells".
To prove that adult stem cells are embryonic stem cells remaining in the early group, also must find experimental evidence enough to support this view. The understanding and analysis based on our study of the following several parts to identified in adult tissue residue "embryonic stem cells".
1. screening of the markers of "embryonic stem cell like cells":
閲囩敤鐢熺墿淇℃伅瀛︽妧鏈，

本文编号：1581007