人骨髓间充质干细胞体外向血管内皮细胞诱导分化的研究

发布时间：2018-12-30 12:22

【摘要】： 目的:国内外多项研究初步表明,人骨髓间充质干细胞(hMSCs)具有多向分化潜能,已有实验证明在体内、体外不同细胞因子作用下,可以诱导分化为骨、软骨和脂肪细胞,参与组织更新、损伤的修复和器官的重建。本课题将通过体外培养hMSCs ,并通过体外诱导使其向血管内皮细胞方向分化,使目的细胞具有内皮细胞样特性,旨在为临床上骨髓间充质干细胞移植治疗缺血性心脑血管疾病以及为血管组织工程提供新的种子细胞来源。方法:通过密度梯度离心法分离出成人骨髓单个核细胞,接着经贴壁法获取hMSCs ,体外扩增培养并通过流式细胞仪对其进行鉴定。在hMSCs生长培养基(DMEM/F12)中加入血管内皮细胞生长因子(VEGF)、碱性成纤维细胞生长因子(bFGF),定向诱导hMSCs向血管内皮细胞分化,观察细胞形态学变化,扩增培养后经流式细胞仪进行细胞表型鉴定,荧光显微镜下观察诱导后hMSCs的DiI-ac-LDL摄取能力。结果:人骨髓间充质干细胞经过24小时的培养,贴壁粘附在培养瓶壁上,换液同时弃去未贴壁细胞,继续培养,细胞呈克隆样生长,12天后细胞生长接近融合。消化传代,传代后细胞生长速度加快,约5-7天后细胞生长即融合。传至第四代后,细胞生长趋于稳定,此时开始进行诱导分化。诱导分化24天后,所得细胞具有摄取Dil-ac-LDL功能。诱导分化同时,分别于第0天、14天和24天用流式细胞仪检测细胞表面的表型CD34、CD45、CD106、HLA-DR ,其表达结果如下表: 结论:本实验探索了体外诱导人骨髓间充质干细胞分化为血管内皮细胞的可行性。在诱导后24天,经流式细胞仪检测细胞的表型,包括CD34, CD45, CD106, HLA-DR,其表达量明显增加。与此同时部分细胞可摄取Dil-ac-LDL ,提示实验所得细胞具有内皮细胞特征。这种诱导分化成的具有内皮细胞表型特征的细胞在形态学上变化不大,仍呈梭形或多角形,提示通过实验培养并诱导分化后所得到的内皮样细胞与真正的内皮细胞存在一定差异。
[Abstract]:Objective: many studies at home and abroad have shown that human bone marrow mesenchymal stem cell (hMSCs) has the potential to differentiate into bone, cartilage and adipocytes in vivo and in vitro, and has been proved to be able to differentiate into bone, cartilage and adipocytes by different cytokines in vivo and in vitro. Participate in tissue renewal, repair of injury and reconstruction of organs. In this study, hMSCs was cultured in vitro and induced to differentiate into vascular endothelial cells (VEC) in order to make the target cells have the characteristics of endothelial cells. The aim is to provide a new source of seed cells for clinical transplantation of bone marrow mesenchymal stem cells for the treatment of ischemic cardio-cerebrovascular diseases and vascular tissue engineering. Methods: adult bone marrow mononuclear cells were isolated by density gradient centrifugation and hMSCs was obtained by adherent method. Vascular endothelial growth factor (VEGF),) basic fibroblast growth factor (bFGF),) was added to hMSCs growth medium (DMEM/F12) to induce hMSCs to differentiate into vascular endothelial cells (VEC). The phenotype of hMSCs was identified by flow cytometry, and the ability of DiI-ac-LDL uptake of induced hMSCs was observed under fluorescence microscope. Results: after 24 hours of culture, human bone marrow mesenchymal stem cells adhered to the wall of culture bottle, then abandoned the unadherent cells and cultured on the same time. After 12 days, the cells grew close to fusion. After digestion and passage, the cell growth rate was accelerated, and the cell growth was fused after about 5-7 days. After the fourth passage, the cell growth tended to stabilize, and then began to induce differentiation. After induced differentiation for 24 days, the cells had the function of Dil-ac-LDL uptake. At the same time, the phenotypic CD34,CD45,CD106,HLA-DR on cell surface was detected by flow cytometry on day 0, day 14 and day 24, respectively. Conclusion: this study explored the feasibility of inducing human bone marrow mesenchymal stem cells to differentiate into vascular endothelial cells in vitro. At 24 days after induction, the phenotypes, including the expression of CD34, CD45, CD106, HLA-DR, were detected by flow cytometry. At the same time, some cells could absorb Dil-ac-LDL, which suggested that the cells had the characteristics of endothelial cells. The cells with phenotypic characteristics of endothelial cells, which were induced to differentiate into cells, had little morphological changes, but still appeared fusiform or polygonal. These results suggest that there is a certain difference between the endothelial like cells and the real endothelial cells after cultured and induced differentiation.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329

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