C反应蛋白对内皮祖细胞功能的影响及其机制研究

发布时间：2018-05-29 04:36

本文选题：内皮祖细胞 + 细胞培养　；参考：《中国协和医科大学》2009年博士论文

【摘要】：第一部分人外周血内皮祖细胞的分离培养与鉴定目的:探讨人外周血来源的内皮祖细胞(endothelial progenitor cells,EPCs)体外诱导培养和鉴定的方法,以期证实人外周血是基础研究与临床实践的一个理想的内皮祖细胞来源,并且建立一个内皮祖细胞培养与鉴定有效的技术平台。方法:密度梯度离心法获取健康成年男性外周血单个核细胞(peripheral bloodmononuclear cells,PBMNCs),接种在人纤维连接蛋白预先包被的培养板培养,用EndoCult~(TM)内皮祖细胞培养基诱导培养,观察内皮祖细胞集落形成单位,收集贴壁细胞,根据内皮祖细胞的特性,通过倒置荧光显微镜、流式细胞术、RT-PCR 3种方法鉴定内皮祖细胞。结果:从成人外周血分离获得的PBMNCs经体外诱导培养,可以形成内皮祖细胞的早期集落形成单位,表达内皮祖细胞的相对特异性抗原,包括AC133、CD34和VEGFR-2,并显示出具有能内吞乙酰化低密度脂蛋白(acetylate low-densitylipoprotein,acLDL)和结合荆豆凝集素(ulex europaeus agglutinin-1 lectin,UEA-1lectin)的功能,同时可表达内皮源型一氧化氮合酶基因(endothelial nitric oxidesynthase,eNOS),所诱导培养的细胞具有内皮祖细胞的特征,鉴定为内皮祖细胞。结论:通过EndoCult~(TM)内皮祖细胞培养基及其标准操作流程可以在人外周血中诱导培养EPCs,培养出的EPCs可表达内皮祖细胞相对特异性抗原,包括AC133、CD34利VEGFR-2及具有内皮细胞的内吞乙酰化低密度脂蛋白和结合荆豆凝集素,以及eNOS基因表达的功能。eNOS基因表达的检测或可简化EPCs的鉴定。我们的实验为EPCs的分离鉴定方法提供了借鉴。第二部分C反应蛋白对内皮祖细胞白介素8表达的影响及其机制目的:血管内皮祖细胞(endothelial progenitor cells,EPCs)是血管内皮细胞的前体细胞,不仅参与胚胎血管生成,也参与出生后的血管生发过程。C反应蛋白(C-reactive protein,CRP)最初被认为只是一种炎症标志物,并不直接参与炎症反应过程,但新近的证据证实在粥样硬化性心血管疾病的发生和发展中,CRP可能参与了这一慢性炎症损伤过程。白细胞介素8(Interleukin-8,IL-8)是由内皮祖细胞分泌的一个重要的促血管新生因子。本研究旨在探讨C反应蛋白对内皮祖细胞IL-8表达的影响并探讨其可能的机制。方法:以不同浓度(5～25μg/ml))人重组C反应蛋白及不同时间段(3～48 hours)刺激人外周血分离培养的内皮祖细胞,检测其培养液上清IL-8因子的水平和细胞IL-8mRNA的水平。抗CD16、CD32抗体和P38-MAPK信号通路抑制剂预处理内皮祖细胞后,再检测CRP对其IL-8表达水平的影响。结果:不同浓度的CRP加入到EPCs培养基中混合培养内皮祖细胞12 hours后,real-time PCR和ELISA检测IL-8的表达水平,结果显示CRP能抑制EPCs IL-8的表达,并且这一效应与CRP浓度相关,10μg/ml的CRP即可显著抑制IL-8的表达,25μg/ml浓度的CRP作用更强;P38-MAPK信号通路抑制剂SB203580及抗CD32抗体预处理可以部分阻断CRP对内皮祖细胞抑制IL-8分泌的作用;而抗CD16抗体预处理内皮祖细胞则无此作用。结论:C反应蛋白直接抑制内皮祖细胞IL-8的表达,CD32可能是内皮祖细胞表面CRP的受体之一,P38-MAPK信号传导通路可能是CRP作用于EPCs的信号转导通路之一。
[Abstract]:Part 1 isolation, culture and identification of endothelial progenitor cells from human peripheral blood
Objective: To explore the methods of inducing culture and identification of endothelial progenitor cells (EPCs) from human peripheral blood in vitro, in order to prove that human peripheral blood is an ideal source of endothelial progenitor cells in basic and clinical practice, and to establish an effective technical platform for the culture and identification of inner skin progenitor cells.
Methods: density gradient centrifugation was used to obtain the peripheral blood mononuclear cells (peripheral bloodmononuclear cells, PBMNCs) of healthy adult male, inoculated with the culture plate of human fibronectin prepackaged by human fibronectin, and used EndoCult~ (TM) endothelial progenitor cell culture medium to induce culture, observe the colony forming unit of endothelial progenitor cells and collect the adherent cells. The characteristics of endothelial progenitor cells were identified by inverted fluorescence microscopy, flow cytometry and RT-PCR. 3 methods were used to identify endothelial progenitor cells.
Results: the PBMNCs obtained from the adult peripheral blood was induced and cultured in vitro to form an early colony forming unit of endothelial progenitor cells, expressing the relative specific antigens of endothelial progenitor cells, including AC133, CD34 and VEGFR-2, and showing the presence of acetylate Low-densitylipoprotein, acLDL, and binding of endocytic acetylated lipoprotein (acLDL). The function of Ulex europaeus agglutinin-1 lectin (UEA-1lectin) can also express the endothelial nitric oxide synthase gene (endothelial nitric oxidesynthase, eNOS). The induced cells have the characteristics of endothelial progenitor cells and are identified as inner skin progenitor cells.
Conclusion: EndoCult~ (TM) endothelial progenitor cell culture medium and its standard operation process can be induced to induce EPCs in human peripheral blood. The cultured EPCs can express the relative specific antigen of endothelial progenitor cells, including AC133, CD34 Li VEGFR-2, endothelium endocyacetylation low density lipoprotein and binding bean agglutinin with endothelial cells, and eNOS base. The detection of.ENOS gene expression can simplify the identification of EPCs because of the expression function. Our experiments provide a reference for the isolation and identification of EPCs.
The second part is the effect of C reactive protein on the expression of interleukin 8 in endothelial progenitor cells and its mechanism.
Objective: endothelial progenitor cells (EPCs) is a precursor cell of vascular endothelial cells, which not only participates in the angiogenesis of embryo, but also participates in the.C reactive protein (C-reactive protein, CRP) in the process of postnatal angiogenesis (C-reactive protein, CRP), which was originally considered as an inflammatory marker, not directly involved in the process of inflammation, but recently The evidence suggests that CRP may be involved in the process of chronic inflammatory damage in the occurrence and development of atherosclerotic cardiovascular disease. Interleukin 8 (Interleukin-8, IL-8) is an important neovascularization factor secreted by inner skin progenitor cells. The aim of this study is to explore the effect of C anti stress protein on the expression of IL-8 in endothelial progenitor cells. To discuss the possible mechanism.
Methods: the endothelial progenitor cells isolated from human peripheral blood were stimulated by human recombinant C reaction protein and different time periods (3~48 hours) in different concentrations (5~25 g/ml). The level of IL-8 factor and the level of cell IL-8mRNA in the culture liquid supernatant were detected. The anti CD16, CD32 antibody and P38-MAPK signal pathway inhibitors pretreated the endothelial progenitor cells and then detected CR The effect of P on the level of IL-8 expression.
Results: after adding different concentrations of CRP to EPCs medium, the expression level of IL-8 was detected by real-time PCR and ELISA. The results showed that CRP could inhibit the expression of EPCs IL-8, and this effect was related to CRP concentration. The expression of 10 micron g/ml could inhibit the expression of IL-8 significantly, and the effect of 25 micron concentration was stronger. The preconditioning of MAPK signaling pathway inhibitor SB203580 and anti CD32 antibody can partially block the inhibitory effect of CRP on the inhibition of IL-8 secretion by endothelial progenitor cells, while the anti CD16 antibody pretreated endothelial progenitor cells have no effect.
Conclusion: C reactive protein directly inhibits the expression of IL-8 in endothelial progenitor cells, and CD32 may be one of the CRP receptors on the surface of endothelial progenitor cells. The P38-MAPK signaling pathway may be one of the signal transduction pathways of CRP in EPCs.
【学位授予单位】：中国协和医科大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R329

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