大鼠骨髓和外周血早晚期内皮祖细胞的分离培养和鉴定

发布时间：2018-04-10 16:26

本文选题：内皮祖细胞 + 血管新生　；参考：《吉林大学》2011年硕士论文

【摘要】：当机体发生缺血性脑卒中时,血管损伤是重要的病理基础。目前治疗均不能完全恢复血管的解剖结构,恢复脑卒中后缺血半暗带及坏死区的血液供应,促进缺血区血管生长的意义更为重大。血管新生和血管发生是血管生长的两种主要形式。血管新生即在已有的血管处芽生出新的毛细血管。血管发生是在无血管区形成新的血管,即EPCs选择性募集到受损或缺血组织区形成血管。机体发生缺血缺氧时,两种成血管方式相互配合促进新生血管形成。EPCs在缺血性疾病领域的研究已经越来越多,本实验旨在从大鼠骨髓及外周血中分离出EPCs并对其进行鉴定,发现其特点及区别。实验使用密度梯度离心及贴壁筛选法从大鼠骨髓和外周血中分离获得单个核细胞(MNC),进行诱导培养,观察并记录贴壁细胞的生物学特征；选取EPCs特异性表面标志CD133、CD34、KDR对原代细胞进行免疫荧光检测,利用流式细胞学技术检测KDR、CD34,并通过吞噬功能实验进一步鉴定培养细胞。实验结果： (1)大鼠骨髓和外周血能够分离获得内皮祖细胞； (2)骨髓及外周血EPCs的生长共性：1.贴壁延迟性2.多种细胞形态：圆形细胞、长梭形细胞,有中央聚集成簇的圆形细胞和周边放射状细胞组成的“血岛”样细胞集落、条索样、管状、鹅卵石样； (3)骨髓及外周血EPCs的生长异同：1、骨髓细胞集落数量多于外周血细胞集落数量；2、细胞培养前3d骨髓中单个核细胞贴壁明显较外周血中多,但杂细胞较多；3、骨髓中EPCs细胞活性较强,增殖能力强,可见大量空腔样或管腔样,桥接样细胞生长,可以大量传代、冻存及复苏。外周血中EPCs活性略差,一般生长4周后逐渐出现大量细胞变圆凋亡。 (4)贴壁细胞免疫荧光检测CD34、CD133、KDR表达阳性； (5)流式细胞学检测CD34、KDR表达阳性,阳性率分别为骨髓69.0%、84.8%,外周血5.4%、32.7%。 (6)细胞能够吞噬ac-LDL,结合UEA-1,说明所获细胞为功能完整的EPCs。 EPCs是一种能直接分化为血管内皮细胞的前体细胞。它不仅参与了胚胎早期血管的形成,更是成年个体中与血管新生和血管形成关系最为密切的干细胞,在血管修复及侧支血管网的建立中具有重要作用,具有促进新生血管形成、参与缺血组织血管再生、促进神经再生、预测脑缺血发展及预后等功能。近年来, EPCs移植应用于血管相关性疾病的治疗,利用EPCs作为种子细胞对血管新生的作用更加显著,联合基因治疗效果更佳。我们以临床应用为目的,在动物实验的基础上,选取来源大鼠骨髓及外周血提取并培养EPCs,探寻出了稳定、实用的EPCs体外分离、培养和鉴定的方法,探寻EPCs的最佳扩增体系以提供足量的有活力的可用于临床的细胞源。
[Abstract]:Vascular injury is an important pathological basis when ischemic stroke occurs.Angiogenesis and angiogenesis are two main forms of angiogenesis.Angiogenesis is the formation of new capillaries in the bud of an existing vessel.Angiogenesis is the formation of new blood vessels in the absence of blood vessels, that is, the selective recruitment of EPCs to the damaged or ischemic tissue regions to form blood vessels.In the process of ischemia and hypoxia, there have been more and more researches on how to promote neovascularization. EPCs are isolated and identified from bone marrow and peripheral blood of rats.Find its characteristics and differences.The mononuclear cells were isolated from rat bone marrow and peripheral blood by density gradient centrifugation and adherent screening. The mononuclear cells were induced and cultured. The biological characteristics of adherent cells were observed and recorded.The EPCs specific surface marker CD133G CD34KDR was selected to detect the primary cells by immunofluorescence, and the KDRN CD34 was detected by flow cytometry, and the cultured cells were further identified by phagocytosis assay.Experimental results:1) Endothelial progenitor cells were isolated from rat bone marrow and peripheral blood.The growth commonness of EPCs in bone marrow and peripheral blood was 1: 1.Adhesion delay 2.A variety of cell morphology: round cells, long fusiform cells, there are central clusters of round cells and peripheral radial cells composed of "blood island" like cell colony, like, tubular, cobblestone;(3) the growth similarities and differences of EPCs in bone marrow and peripheral blood were observed. The number of bone marrow cells was more than that of peripheral blood cells. The number of mononuclear cells in bone marrow was significantly more than that in peripheral blood 3 days before cell culture, but the number of miscellaneous cells was more than that in peripheral blood, and the activity of EPCs cells in bone marrow was stronger.The proliferation ability is strong, a large number of cavity or tube cavity, bridging like cell growth, can be a large number of passage, cryopreservation and recovery.The activity of EPCs in peripheral blood was slightly poor, and a large number of cells became round and apoptotic gradually after 4 weeks of growth.The positive expression of KDR was detected by immunofluorescence in adherent cells.The positive rate of CD34 KDR expression was 69.0% in bone marrow and 84.8% in bone marrow, and 5.44% and 32.7% in peripheral blood respectively.(6) the cells could phagocytosis ac-LDL and combined with UEA-1, which indicated that the cells obtained were EPCs with complete function.EPCs is a kind of progenitor cells that can directly differentiate into vascular endothelial cells.Participate in vascular regeneration of ischemic tissue, promote nerve regeneration, predict the development and prognosis of cerebral ischemia.For the purpose of clinical application, on the basis of animal experiments, EPCs were extracted and cultured from rat bone marrow and peripheral blood, and a stable and practical method for isolation, culture and identification of EPCs in vitro was found.To explore the best amplification system of EPCs to provide a sufficient number of viable cell sources for clinical use.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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