【作者】 吴小萍；

【导师】 吴玉章；

【作者基本信息】 第三军医大学 ， 免疫学， 2010， 硕士

【摘要】 胚胎干细胞(Embryonic Stem Cells)的研究一直受到伦理道德问题的束缚与限制,为此众多研究者尝试寻找其他干细胞来源,以期绕过ES这个伦理束缚。近几年的研究结果发现在羊水中存在少量具有ES细胞特性的干细胞,并将其命名为人源羊水干细胞(human Amniotic Fluid Stem Cells, hAFSCs)。该种细胞表达ES细胞和成体干细胞标志基因,体外诱导可分化为包括三个胚层的细胞并通过功能测试。这就表明hAFSCs具有很好的应用价值。另外hAFSCs具有容易获取,不会损害母体及胚胎的特点。综合以上两点,可以认为hAFSCs是一种细胞和组织工程治疗新的种子细胞来源。间充质干细胞(mesenchymal stem cells, MSCs)是一种具有多向分化潜能的成体干细胞,是当前干细胞研究领域的热点。研究MSCs主要集中在两个方面,分别是MSCs的多分化潜能和免疫抑制作用。前者在组织器官缺损性疾病、退行性疾病、自身免疫性疾病和遗传缺陷等疾病的治疗中有着巨大的临床应用前景;后者则主要为细胞疗法用于副作用小的免疫抑制治疗提供了契机。这两方面目前在基础和临床研究中都已取得令人鼓舞的结果。鉴于以上研究背景,本文的第一部分主要从人孕中期羊水中分离纯化得到间充质干细胞。首先从羊水中培养细胞,而后通过形态选择其中的成纤维样细胞,并将这部分细胞进行单克隆培养及扩增,以达到后续实验要求。将分离得到的细胞克隆通过染色体G染色、RT-PCR和流式细胞仪等技术,分析细胞来源及其分子标记,从而初步鉴定为间充质样细胞(mesenchymanl-like cells, MCs)。而后经由诱导分化实验将MCs在特定分化培养基中进行培养,使其向脂肪细胞和骨细胞进行分化,从而证实分离得到的MCs具有分化能力,确定其为MSCs。在此基础上,检测第5代AF-MSCs的增殖曲线及第40代AF-MSCs的生物学特性,发现其具有旺盛的增殖能力,平均约33h增殖一代,且在不断传代过程中,始终保持其干细胞全能性标记Oct-4的表达及正常的细胞倍型。在文章的第二部分主要研究了AF-MSCs的免疫抑制作用及其机制。研究发现AF-MSCs具有抑制受到植物凝集素(Phytohemagglutinin, PHA)刺激的同种异体外周血单核淋巴细胞(peripheral blood mononuclear cells, PBMC)增殖的能力,且这种抑制作用与AF-MSCs细胞的数量呈线性相关性。研究发现AF-MSCs通过分泌TGF-beta1来发挥其免疫抑制作用。进一步研究发现在共培养体系中加入anti-TGF-beta1后发现AF-MSCs抑制PBMC增殖的能力消失且培养体系中CD4+CD25+Foxp3+ T细胞的频率跟本底对比具有显著性提高,证实分泌产生的TGF-beta1不是通过诱导细胞凋亡产生抑制作用而是通过将CD4+CD25- T细胞诱导为具有抑制能力的CD4+CD25+Foxp3+免疫调节T细胞,而后通过调节性T细胞抑制其它细胞的增殖来发挥其免疫抑制功能。更多还原

【Abstract】 Researchers are trying to find out new resources of stem cells because of the ethical arguments of embryonic stem cells (ESs). Recent studies demonstrateded that a few cells displaying ES’s characteristics exist in amniotic fluid, which was named human amniotic fluid stem cell (hAFS cell). hAFS cells that express embryonic and adult stem cell markers can differentiate into cell types of three embryonic germ layer and display specialized functions. All these confirmed that the hAFS cells have value of application. On the other hand, hAFS cells can be obtained easily and do not damage the mother body and the embryo. So the hAFS cells may offer an alternative approach for the cell therapy and tissue engineering.Mesenchymal stem cells (MSCs) resemble a multipotent adult stem cell population capable of differentiating into different mesodermal cell lineages including osteoblasts, chondroblasts and adipocytes. Research on the MSCs mainly concentrated in two areas: the multi-differentiation potential and the immune-suppression. MSCs have generated a great deal of excitement as a potential source of cells for regenerative medicine and tissue engineering owing to their dramatic potential of proliferation and differentiation, and raise high hopes in clinical application. On the other side, this is an opportunity for the cell therapy providing the low side effects.In this study, in the first part, we isolated and purified the mesenchymal stem cells from human amniotic fluid. First, we isolated and cultured the cells in human amniotic fluid, and choosed the fibroblastoid-like cells to purify and expand. Then, we identified the source and molecular markers of cell clones by Chromosome Giemsa staining, RT-PCR and flow cytometer, and they were identified as the mesenchymal-like cells. Mesenchymal-like cells could be induced to differentiate into adipocytes and osteoblasts in the special medium, and they were confirmed as MSCs because of the differentiation ability. Additionally, we found that AF-MSCs, which can double in 33h on average, have strong proliferation ability and they hold the expression of Oct-4 that is the maker of pluripotency and the normal chromosome ploidy in the different passages.In the second part, we discovered that the AF-MSCs have inhibitory effects on the proliferation of peripheral blood mononuclear cells (PBMC). And this inhibition showed a linear correlation with the AF-MSCs’quantity. We further explored that the AF-MSCs could secrete the TGF-beta1, which mediated the AF-MSCs’suppressive effects on the PBMC. Adding anti-TGF-beta1 in co-culture system found that TGF-beta1 does not stimulate cells’apoptosis but induce the CD4+CD25- T cells into CD4+CD25+Foxp3+ T cells, which have the suppressive function to inhibit other cells’proliferation. So AF-MSCs suppress PBMC’s proliferation via secreting TGF-beta1 to induce the regulatory T cells.更多还原