Galectin-3调控Tip血管内皮细胞塑管效应的机制研究

发布时间：2018-01-12 03:36

本文关键词：Galectin-3调控Tip血管内皮细胞塑管效应的机制研究　出处：《昆明医科大学》2012年硕士论文　论文类型：学位论文

【摘要】：第一部分人外周血内皮祖细胞及Tip血管内皮细胞的分离培养与鉴定目的：探讨人外周血内皮祖细胞的分离与培养方法,研究人外周血内皮祖细胞的体外诱导培养条件及其生物学活性,拟证实人外周血是内皮祖细胞除了骨髓以外的另一个理想来源。在一定的体外培养条件下,外周血内皮祖细胞还可定向诱导分化为Tip血管内皮细胞,不仅为下一步实验研究提供所需的细胞来源,还为干细胞源性血管新生机制提供可靠的实验室依据。方法：采用Ficoll密度梯度离心法分离人外周血单个核细胞,将其接种在预先包被有人纤维连接蛋白的培养瓶中培养。每日于倒置相差显微镜下观察细胞的生长形态及内皮祖细胞集落形成形态,每隔3-4天换一次液,培养7天后收集贴壁细胞,根据内皮祖细胞的特性,用流式细胞仪检测内皮祖细胞特异性抗原CD34、CD133及VEGFR等表面标志物,以鉴定培养后的细胞主要为内皮祖细胞。培养至21天时倒置相差显微镜下观察和鉴定Tip血管内皮细胞形态,并收集该贴壁细胞进行RT-PCR实验检测Tip血管内皮细胞中vWF、eNOS等血管内皮细胞系特异性基因的表达情况。结果：分离获得的单个核细胞培养4天后多数呈贴壁生长,7天后形成梭状的内皮样细胞,部分细胞积聚成团形成克隆集落,流式细胞仪鉴定该细胞表达的内皮祖细胞特异性抗原CD34、CD133及VEGFR分别为(68.9±2.34)%、(81.7±1.50)%、(97.1±2.01)%。继续培养21天后的细胞呈鹅卵石样改变,并伸出伪足相互连接形成类似血管腔样结构,形态学表现为Tip血管内皮细胞的形态特点,RT-PCR实验检测该细胞有血管内皮细胞系特异性基因vWF、eNOS的表达。结论：可以成功从人外周血中分离具有增殖分化潜能的内皮祖细胞,该细胞可形成内皮祖细胞特有的集落形成单位,并表达内皮祖细胞的相对特异性抗原CD34、CD133及VEGFR。同时经过特定的体外诱导培养体系可将其扩增并定向诱导分化为Tip血管内皮细胞。第二部分Galectin-3对外周血内皮祖细胞源性Tip血管内皮细胞增殖能力的影响目的：内皮祖细胞是血管内皮细胞的前体细胞,是一群具有游走特性,在生理或病理因素的刺激下,可从骨髓动员到外周血,进一步增殖分化并参与受损血管的修复的幼稚内皮细胞。半乳糖凝集素-3(Galectin-3)是一种半乳糖结合蛋白,该蛋白的糖结构识别域在血管生成的糖依赖过程中起着关键的作用。本实验观察Galectin-3对外周血内皮祖细胞源性Tip血管内皮细胞增殖能力的影响,为临床干细胞治疗慢性下肢动脉缺血性疾病提供新的实验室依据。方法：收集第一部分实验中培养21天后的贴壁Tip血管内皮细胞,随机将Galectin-3分为终浓度各浓度组(共6组)：在外周血内皮祖细胞源性Tip血管内皮细胞的培养液中分别加入终浓度为0ug/ml,0.1ug/ml,1.0ug/ml,2.5ug/ml,5.0ug/m1和10ug/ml的Galectin-3培养24小时。MTT比色法观察不同浓度Galectin-3对外周血内皮祖细胞源性Tip血管内皮细胞增殖能力的影响。结果：六组不同终浓度的Galectin-3对外周血内皮祖细胞源性Tip血管内皮细胞增殖能力的影响有差别(F值=37.329,P0.01)。加入Galectin-3的各浓度组外周血内皮祖细胞源性Tip血管内皮细胞的增殖能力均高于Oug/ml组,其中0.10ug/ml组,1.00ug/ml组及2.50ug/ml组与0ug/ml组比较无统计学意义(P0.05),但5.0ug/ml组及10.0ug/ml组明显高于0ug/ml组,0.10ug/ml组,1.00ug/ml组及2.50ug/ml组,差异具有统计学意义(P0.05);10.0ug/ml浓度组也明显高于5.0ug/ml浓度组,差异具有统计学意义(P0.05)。外周血内皮祖细胞源性Tip血管内皮细胞增殖能力与Galectin-3浓度间存在正相关关系(r=0.966),两变量有高度相关关系。结论：Galectin-3可促进体外培养外周血内皮祖细胞源性Tip血管内皮细胞的增殖能力,是Tip血管内皮细胞塑管效应过程中重要的因子之一。
[Abstract]:Isolation , culture and identification of endothelial progenitor cells and vascular endothelial cells from human peripheral blood in the first part Objective : To study the isolation and culture of endothelial progenitor cells from human peripheral blood . It is suggested that human peripheral blood endothelial progenitor cells can induce differentiation into the endothelial progenitor cells in vitro . Methods : Human peripheral blood mononuclear cells were isolated by Ficoll density gradient centrifugation . The growth morphology of endothelial progenitor cells and the formation of endothelial progenitor cells were observed under reversed phase contrast microscope . The surface markers of endothelial progenitor cells specific antigen CD34 , CD133 and VEGFR were detected by flow cytometry after 7 days . Results : After 4 days after the single core cell culture was isolated , most of the cells were adherent . After 7 days , spindle - shaped endothelium - like cells were formed . The specific antigens CD34 , CD133 and VEGFR were ( 68.9 卤 2.34 ) % , ( 81.7 卤 1.50 ) % and ( 97.1 卤 0.01 ) % respectively . Conclusion : Endothelial progenitor cells with proliferative differentiation potential can be isolated from human peripheral blood successfully , which can form a specific colony forming unit of endothelial progenitor cells and express the relative specific antigens CD34 , CD133 and VEGFR of endothelial progenitor cells . Effect of the second part galectin - 3 on the proliferation of endothelial progenitor cells in peripheral vascular endothelial cells Objective : To investigate the effect of Galectin - 3 on the proliferation of vascular endothelial cells derived from vascular endothelial progenitor cells and to provide a new experimental basis for the treatment of chronic lower extremity arterial ischemic diseases by Galectin - 3 . Galectin - 3 is a kind of galactose binding protein . Methods : Galectin - 3 was randomly divided into final concentration groups ( group 6 ) . Galectin - 3 was cultured for 24 hours in the culture solution of peripheral vascular endothelial progenitor cells . The effects of Galectin - 3 on the proliferation of endothelial progenitor cells in peripheral blood were observed by MTT colorimetric method . Results : The effects of Galectin - 3 at different final concentrations on proliferation of endothelial progenitor cells in peripheral blood were different ( F = 37.329 , P0.01 ) . The proliferative capacity of endothelial progenitor cells in peripheral vascular endothelial cells was higher than that in Oug / ml group ( P0.05 ) . Conclusion : Galectin - 3 can promote the proliferation of endothelial progenitor cells derived from endothelial progenitor cells in vitro , which is one of the most important factors in the plastic tube effect of endothelial cells .

【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

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