人源羊水间充质干细胞的分离培养及其免疫抑制作用机制的研究

发布时间：2018-01-24 16:29

本文关键词： 羊水间充质干细胞免疫抑制外周血单核淋巴细胞调节性T细胞　出处：《第三军医大学》2010年硕士论文　论文类型：学位论文

【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。
[Abstract]:The study of embryonic stem cells (ESC) has been restricted by ethical issues. Therefore, many researchers have tried to find other stem cell sources. In order to circumvent the ethical constraints of es, recent studies have found that a small number of es cell-specific stem cells exist in amniotic fluid. It was named human Amniotic Fluid Stem Cells. These cells express marker genes of es cells and adult stem cells. In vitro, hAFSCs can be induced to differentiate into cells containing three embryo layers and pass functional tests. This indicates that hAFSCs has good application value and hAFSCs is easy to obtain. HAFSCs can be considered as a new source of seed cells for cell and tissue engineering therapy. Mesenchymal stem cells (MSCs) is a kind of adult stem cells with multiple differentiation potential. MSCs is a hot topic in stem cell research. The study of MSCs is mainly focused on two aspects: the multi-differentiation potential of MSCs and the immunosuppressive effect. The former is involved in tissue and organ defect diseases and degenerative diseases. The treatment of autoimmune diseases and genetic defects has a great prospect of clinical application. The latter provides an opportunity for cell therapy to be used in immunosuppressive therapy with little side effects, and encouraging results have been obtained in both basic and clinical studies. In the first part of this paper, mesenchymal stem cells were isolated and purified from human amniotic fluid. First, the cells were cultured from amniotic fluid, and then the fibroblasts were selected by morphology. The cells were cultured and amplified by monoclonal method to meet the requirements of subsequent experiments. The isolated cells were cloned by chromosome G staining RT-PCR and flow cytometry. The mesenchymanl-like cells was identified as mesenchymal like cells by analyzing the origin of cells and their molecular markers. Then MCs was cultured in a specific differentiation medium to differentiate into adipocytes and bone cells, which proved that the isolated MCs had the ability of differentiation. On the basis of this, we detected the proliferation curve of the fifth generation AF-MSCs and the biological characteristics of the 40th generation AF-MSCs, and found that it has the exuberant proliferative ability. During the continuous passage, the expression of Oct-4 and normal cell ploidy were maintained. In the second part of this paper, the immunosuppressive effect of AF-MSCs and its mechanism were studied. Phytohemagglutinin. PHA-stimulated peripheral blood mononuclear lymphocytes (PBMCs) proliferate in peripheral blood cells (PBMCs). The inhibitory effect was linearly correlated with the number of AF-MSCs cells. It was found that AF-MSCs exerts its immunosuppressive effect by secreting TGF-beta1. It was found that the ability of AF-MSCs to inhibit the proliferation of PBMC disappeared after the addition of anti-TGF-beta1 in the co-culture system, and CD4 was found in the culture system. The frequency of CD25 Foxp3 T cells was significantly increased compared with background. It is proved that the secreted TGF-beta1 is produced not by inducing apoptosis, but by inducing CD4 CD25- T cells to be CD4 CD25 with inhibitory effect. Foxp3 immunomodulatory T cells. Then T cells inhibit the proliferation of other cells to play its immunosuppressive function.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329

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