人骨髓间充质干细胞向血管内皮细胞诱导分化的实验研究及差异表达基因的微阵列分析

发布时间：2018-04-17 13:27

本文选题：骨髓间充质干细胞 + 内皮细胞　；参考：《天津医科大学》2006年博士论文

【摘要】：人骨髓间充质干细胞向血管内皮细胞诱导分化的实验研究及差异表达基因的微阵列分析骨髓间充质干细胞是多能干细胞,已有实验证明在体内和体外细胞因子作用下,可分化为骨、软骨和脂肪细胞,在体内参与组织更新、损伤的修复和重建。本研究探讨人骨髓间充质干细胞诱导分化为内皮细胞的可能性。 1.材料和方法 1.1 人骨髓间充质干细胞的分离培养人骨髓标本来源于39岁男性健康志愿者。按骨髓穿刺的常规方法,抽取骨髓液5ml。肝素抗凝。DMEM+F12培养液稀释后,用人淋巴细胞分离液Ficoll(比重为1.077)密度梯度离心法,分离出单个核细胞。用DMEM+F12洗涤后,加入DMEM+F12与MCBD的混合培养液(3:2,V/V),并加入10%胎牛血清(FCS)、2ng/ml碱性成纤维细胞生长因子(bFGF)、10ng/ml表皮生长因子(EGF)、10ng/ml胰岛素样生长因子(IGF)和双抗。24小时后,吸取培养液,连同未贴壁细胞弃去。贴壁细胞继续培养,每四天更换半量的培养液,12天后细胞融合时,用0.25%胰蛋白酶/0.02%EDTA消化液消化后,细胞传代,每天观察生长情况。 1.2.诱导分化细胞传至第四代后,在培养液中加量bFGF至10ng/ml,加10ng/mlVEGF培养,每四天半量换液,定期观察细胞生长形态,于第0、14、24天分析细胞的表型和功能。 1.3 流式细胞分析细胞收集后,用多聚甲醛固定后用流式细胞分析仪,分析CD34、CD45、CD54、CD106、HLA-DR、vWF及KDR的表达情况。
[Abstract]:Experimental study on differentiation of human bone marrow mesenchymal stem cells into vascular endothelial cells and microarray analysis of differentially expressed genesBone marrow mesenchymal stem cells (BMSCs) are pluripotent stem cells. It has been proved that bone cartilage and adipocytes can be differentiated into bone cartilage and adipocytes under the action of cytokines in vivo and in vitro. Bone marrow mesenchymal stem cells participate in tissue renewal repair and reconstruction of injury in vivo.The aim of this study was to investigate the possibility of differentiation of human bone marrow mesenchymal stem cells into endothelial cells.1.Materials and methods1.1 isolation and Culture of Human Bone Marrow Mesenchymal Stem cellsHuman bone marrow specimens were derived from 39-year-old male healthy volunteers.According to the routine method of bone marrow puncture, 5 ml of bone marrow fluid was extracted.After dilution of heparin anticoagulant, DMEM F12 culture medium, human lymphocytes were separated by density gradient centrifugation with Ficoll1 (specific gravity 1.077), and mononuclear cells were isolated.After washing with DMEM F12, adding the mixture of DMEM F12 and MCBD (3: 2 V / V), and adding 10% fetal bovine serum to 2ng / ml of basic fibroblast growth factor (bFGF-1) and 10ng / ml EGF (10ng / ml) and double antibody (.24h),With unattached cells discarded.The adherent cells were cultured continuously. After 12 days of cell fusion, the cells were digested with 0.25% trypsin / 0.02TA digestible solution, and then the cells were subcultured, and the growth was observed every day.1.2.Induced differentiationAfter the cells were transferred to the fourth passage, bFGF was added to the culture medium to 10 ng / ml, and 10ng/mlVEGF was added to the culture medium. The cell growth morphology was observed regularly every four and a half days. The phenotypes and functions of the cells were analyzed on day 1424.1.3 flow cytometry analysisAfter cell collection, the expression of HLA-DRV WF and KDR was analyzed by flow cytometry.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R329.2

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