体外联合诱导脐血单个核细胞向内皮样细胞分化的实验研究

发布时间：2018-01-06 23:06

本文关键词：体外联合诱导脐血单个核细胞向内皮样细胞分化的实验研究　出处：《福建医科大学》2008年硕士论文　论文类型：学位论文

【摘要】： 目的:探讨细胞外基质、植物血球凝集素(phytohemagglutinin ,PHA-M)、血管内皮生长因子(vascular endothelial growth factor,VEGF)和碱性成纤维生长因子(basic fibroblast growth factor,bFGF),联合诱导人脐血单个核细胞(mononuclear cells,MNC)向内皮样细胞分化的可行性,及此体系中不同细胞外基质(extracellular matrix ,ECM)对内皮样细胞诱导效率的影响。方法:密度梯度离心法分离脐血单个核细胞,在含有PHA、VEGF、bFGF及ECM等具有诱导分化作用的因子的培养基中进行体外培养。倒置显微镜下观察细胞形态及三维体系中血管的形成;应用免疫细胞化学,半定量RT-PCR、流式细胞学等方法检测内皮细胞标记Ⅷ因子、假性血友病因子(von Willebrand factor, vWF)和一氧化氮合酶(endothelial nitric oxide synthase,eNOS)及CD31的表达,并描绘细胞生长曲线,以鉴定诱导的细胞生物学特性及比较不同细胞外基质对诱导效率的影响。结果:①倒置显微镜下观察结果:最初贴壁时细胞为小圆形,PHA刺激24小时后细胞聚集成团,诱导第5天集落周边出现少量梭形细胞且数量逐渐增多;7天可见线状排列,大量典型梭形贴壁细胞生成,在三维培养体系中形成血管样结构。②培养7天,贴壁细胞表达内皮细胞标记FⅧ因子,eNOS、vWF和CD31。③FN组培养7天后eNOS(0.476±0.072)、vWF(0.729±0.114)表达均高于其它两组(P0.05);其CD31的表达率由第7天(48.04±5.34%)到第14天(72.90±8.54%),均高于同一时间胶原组与对照组(P0.05)。结论:①脐血中存在内皮前体细胞,分离得到的单个核细胞能在体外定向诱导分化为内皮样细胞。②证实在不降低诱导效率的前提下,PHA与VEGF、bFGF联合诱导可缩短脐血单个核细胞分化为内皮样细胞的时间,具有协同效应。③纤维连接蛋白较胶原可促进内皮样细胞的诱导。④脐血有望成为治疗缺血性疾病的一种有效的干细胞来源。
[Abstract]:Objective: To investigate the effects of extracellular matrix, phytohemagglutinin (phytohemagglutinin, PHA-M), vascular endothelial growth factor (vascular endothelial, growth factor, VEGF) and basic fibroblast growth factor (basic fibroblast, growth factor, bFGF), induced by human umbilical blood mononuclear cells (mononuclear, cells, MNC) to the feasibility of endothelial like cells and this system, different extracellular matrix (extracellular matrix ECM) effect on endothelial cells induced by efficiency.
Methods: isolated umbilical cord blood mononuclear cells by density gradient centrifugation in VEGF medium containing PHA, bFGF, and ECM factor can induce the differentiation of cultured in vitro. Observation of cell morphology and angiogenesis in 3D system under the inverted microscope; using immunocytochemical, semi quantitative RT-PCR, flow cytometry etc. detection of endothelial cell marker von Willebrand factor, von Willebrand factor (von Willebrand, factor, vWF) and nitric oxide synthase (endothelial nitric oxide synthase, eNOS) and the expression of CD31, and describe the cell growth curve, and the comparison of different extracellular matrix cell characterization induced induction efficiency.
Results: the results were observed by inverted microscope: first adherent cells were round, 24 hours after the stimulation of PHA cells together, after fifth days the colony appeared around a small number of spindle cells and increased gradually; 7 days can be seen in the linear array, a large number of typical spindle shaped adherent cells to form vascular like structure in the three-dimensional culture system. The adherent cells were cultured for 7 days, the expression of endothelial cell markers F factor VIII, eNOS, vWF and CD31. in FN group after 7 days of culture eNOS (0.476 + 0.072), vWF (0.729 + 0.114) expression were higher than that of the other two groups (P0.05); the expression rate of CD31 by seventh days (48.04 + 5.34%) to fourteenth days (72.90 + 8.54%), were higher than the same time collagen group and the control group (P0.05).
Conclusion: endothelial progenitor cells of human umbilical cord blood, mononuclear cells isolated can differentiate into endothelial like cells in vitro. The confirmed in the premise of not reducing the efficiency of induction, PHA and VEGF, can shorten the time of umbilical cord blood mononuclear cells differentiation into endothelial like cells induced by the time bFGF, synergistic effect 3. Compared with collagen induced fibronectin can promote endothelial cells. The umbilical cord is expected to become an effective source of stem cells for the treatment of ischemic diseases.

【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329

【相似文献】