人外周血内皮前体细胞的体外诱导与培养

发布时间：2018-01-03 04:42

本文关键词：人外周血内皮前体细胞的体外诱导与培养　出处：《山东大学》2005年硕士论文　论文类型：学位论文

【摘要】：胚胎发生时期,内皮前体细胞(endothelial progenitor cells EPCs)参与了原始血管形成的最初过程(血管发生)。已有的证据显示,发育为内皮细胞(endothelial cells ECs)的前体也存在于成人中,正常情况下,EPCs停留在成人的骨髓,但是,可以通过细胞因子或血管生成因子信号被动员到循环血,迁移到生理或病理条件下的新血管形成位点,并原位分化成内皮细胞,有助于血管新生(成人的血管发生)。目前,出生后血管发生的研究是临床研究的热点问题,自源的EPCs原位动员或移植是治疗性血管再生的一个潜在、有效的方法,而且,内皮前体也代表了阻止肿瘤生长的一个潜在的靶位。因此,要达到真正的临床应用,首选的问题之一就是探索EPCs的分离、培养与鉴定。本论文旨在探讨人外周血EPCs的分离、体外诱导与分化,以及培养与鉴定,并研究其体外的生物学活性,为血管生成的细胞治疗提供参考依据。 Ficoll液作为单个核细胞的提取液,用新鲜、抗凝的全血和抗凝血静置后取富集单个核细胞层这两种方法提取单个核细胞,分离的细胞浓度调整为1.0×10~6个/ml,接种在24孔培养板上,培养液分别用含有促血管生长因子VEGF、bFGF、IGF、EGF等的内皮生长基质EGM-2和不含血管生长因子的内皮基础培养液EBM,第4d移走非黏附细胞,继续培养黏附细胞,黏附细胞与Dil-AC-LDL和FITC标记的UEA-1一起孵育,荧光显微镜和激光扫描共聚焦显微镜下观察。第7d,收集黏附细胞,流式细胞仪检测其表面标志CD34、CD31、CD45、KDR的表达。结果显示,用抗凝血静置后取富集单个核细胞层的方法提取单个核细胞,既缩短时间又排除了过多红细胞的干扰。Dil-AC-LDL和FITC标记的UEA-1双阳性的细胞在荧光显微镜和共聚焦显微镜下都能看到。细胞培养7d后,FACS分析,CD34+、CD31+、KDR+的表达分别为2.79%、6.27%、31.24%,而CD45表达呈阴性。由此得出结论:外周血中确实存在EPCs,并且在血管生长因子VEGF、bFGF、IGF、EGF等的刺激下能分化为成熟的Ecs。
[Abstract]:During embryogenesis, endothelial progenitor cells (endothelial progenitor cells EPCs) participate in the initial process of primitive blood vessel formation (angiogenesis). The evidence shows that the growth of endothelial cells (endothelial cells ECs) precursors also exist in the adult, normal condition, EPCs stay in the adult bone marrow, but through cytokine or angiogenic growth factor signals were mobilized into circulation, migrate to the new vascular physiological or pathological conditions and in situ formation sites, differentiate into endothelial cells contribute to angiogenesis (adult blood Guan Fasheng). At present, research on angiogenesis after birth is a hot issue in clinical research, from the source EPCs in situ mobilization or transplantation is a potential therapeutic angiogenesis, and effective method, endothelial precursors also represents a potential target to prevent tumor growth. Therefore, to achieve the real clinical One of the first problems in application is to explore the isolation, culture and identification of EPCs.
The purpose of this study is to explore the isolation, induction and differentiation, culture and identification of human peripheral blood EPCs, and to study its biological activity in vitro, so as to provide reference for angiogenesis therapy.
Ficoll solution as extraction liquid, mononuclear cells with fresh blood, anticoagulation and anti coagulation after static enrichment of mononuclear cell layer of the two extraction methods of mononuclear cells, isolated cell concentration was adjusted to 1 * 10~6 /ml, were cultured on 24 well plates respectively, with vascular growth factor VEGF, bFGF, IGF medium, the growth of endothelial basal endothelial EGF matrix do not contain EGM-2 and vascular endothelial growth factor EBM of the medium, the 4D removal of non adherent cells, cultured cell adhesion, cell adhesion and Dil-AC-LDL and FITC labeled UEA-1 incubated together, fluorescence microscopy and laser scanning confocal microscope - 7d, collect the adherent cells, to detect the surface markers of CD34, CD31, CD45, flow cytometry, and the expression of KDR.
The results showed that using the method of static anti coagulation after enrichment of mononuclear cells from mononuclear cells, not only shorten the time and eliminate excessive interference of red cell.Dil-AC-LDL and FITC labeled UEA-1 double positive cells under fluorescence microscope and confocal microscope. Cell culture can be seen after 7d, FACS analysis CD34+, CD31+, and KDR+ expression were 2.79%, 6.27%, 31.24%, and CD45 negative expression.
It is concluded that EPCs does exist in peripheral blood and can be differentiated into mature Ecs. under the stimulation of vascular growth factor VEGF, bFGF, IGF, EGF and so on.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R329

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