本文關鍵詞： 羊水 間充質(zhì)干細胞 免疫抑制 外周血單核淋巴細胞 調(diào)節(jié)性T細胞　出處：《第三軍醫(yī)大學》2010年碩士論文　論文類型：學位論文

【摘要】： 胚胎干細胞(Embryonic Stem Cells)的研究一直受到倫理道德問題的束縛與限制,為此眾多研究者嘗試尋找其他干細胞來源,以期繞過ES這個倫理束縛。近幾年的研究結果發(fā)現(xiàn)在羊水中存在少量具有ES細胞特性的干細胞,并將其命名為人源羊水干細胞(human Amniotic Fluid Stem Cells, hAFSCs)。該種細胞表達ES細胞和成體干細胞標志基因,體外誘導可分化為包括三個胚層的細胞并通過功能測試。這就表明hAFSCs具有很好的應用價值。另外hAFSCs具有容易獲取,不會損害母體及胚胎的特點。綜合以上兩點,可以認為hAFSCs是一種細胞和組織工程治療新的種子細胞來源。 間充質(zhì)干細胞(mesenchymal stem cells, MSCs)是一種具有多向分化潛能的成體干細胞,是當前干細胞研究領域的熱點。研究MSCs主要集中在兩個方面,分別是MSCs的多分化潛能和免疫抑制作用。前者在組織器官缺損性疾病、退行性疾病、自身免疫性疾病和遺傳缺陷等疾病的治療中有著巨大的臨床應用前景;后者則主要為細胞療法用于副作用小的免疫抑制治療提供了契機。這兩方面目前在基礎和臨床研究中都已取得令人鼓舞的結果。 鑒于以上研究背景,本文的第一部分主要從人孕中期羊水中分離純化得到間充質(zhì)干細胞。首先從羊水中培養(yǎng)細胞,而后通過形態(tài)選擇其中的成纖維樣細胞,并將這部分細胞進行單克隆培養(yǎng)及擴增,以達到后續(xù)實驗要求。將分離得到的細胞克隆通過染色體G染色、RT-PCR和流式細胞儀等技術,分析細胞來源及其分子標記,從而初步鑒定為間充質(zhì)樣細胞(mesenchymanl-like cells, MCs)。而后經(jīng)由誘導分化實驗將MCs在特定分化培養(yǎng)基中進行培養(yǎng),使其向脂肪細胞和骨細胞進行分化,從而證實分離得到的MCs具有分化能力,確定其為MSCs。在此基礎上,檢測第5代AF-MSCs的增殖曲線及第40代AF-MSCs的生物學特性,發(fā)現(xiàn)其具有旺盛的增殖能力,平均約33h增殖一代,且在不斷傳代過程中,始終保持其干細胞全能性標記Oct-4的表達及正常的細胞倍型。 在文章的第二部分主要研究了AF-MSCs的免疫抑制作用及其機制。研究發(fā)現(xiàn)AF-MSCs具有抑制受到植物凝集素(Phytohemagglutinin, PHA)刺激的同種異體外周血單核淋巴細胞(peripheral blood mononuclear cells, PBMC)增殖的能力,且這種抑制作用與AF-MSCs細胞的數(shù)量呈線性相關性。研究發(fā)現(xiàn)AF-MSCs通過分泌TGF-beta1來發(fā)揮其免疫抑制作用。進一步研究發(fā)現(xiàn)在共培養(yǎng)體系中加入anti-TGF-beta1后發(fā)現(xiàn)AF-MSCs抑制PBMC增殖的能力消失且培養(yǎng)體系中CD4+CD25+Foxp3+ T細胞的頻率跟本底對比具有顯著性提高,證實分泌產(chǎn)生的TGF-beta1不是通過誘導細胞凋亡產(chǎn)生抑制作用而是通過將CD4+CD25- T細胞誘導為具有抑制能力的CD4+CD25+Foxp3+免疫調(diào)節(jié)T細胞,而后通過調(diào)節(jié)性T細胞抑制其它細胞的增殖來發(fā)揮其免疫抑制功能。
[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.
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R329

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本文編號：1460480