体外诱导成人外周血单个核细胞向血管内皮细胞分化

发布时间：2018-09-02 10:06

【摘要】： 目的: 应用血管内皮细胞生长因子和碱性成纤维细胞生长因子在体外联合诱导人外周血单个核细胞(PBMNCs)定向分化为血管内皮细胞,为外周血干细胞在组织工程血管化及肢体缺血性疾病的细胞移植治疗中的应用提供理论依据。方法: (1)无菌条件下,取重组人粒细胞集落刺激因子刺激后采集的PBMNCs 2ml,分别设立实验组1和对照组;健康人外周血20mL,作为实验组2。肝素抗凝。 (2)Hanks液双倍稀释,按1:2置于淋巴细胞分离液上层,离心后提取分液层与上层交界部位呈混浊的灰白色层,即为单个核细胞层。 (3)取离心后的细胞,向DMEM-F12培养基中分别加入含体积分数为0.2的胎牛血清、10μg/L血管内皮细胞生长因子、10μg/L碱性成纤维细胞生长因子。对照组中不加血管内皮细胞生长因子和碱性成纤维细胞生长因子等诱导剂。吹打均匀并计数,按1×1010 L-1接种于25cm2培养瓶中,于37℃、体积分数为0.05的CO2饱和湿度培养箱中培养,第3天更换培养基,去除未贴壁的细胞,以后每2d换液一次。 (4)倒置相差显微镜下观察细胞单层排列是否呈“铺路石”样结构。运用流式细胞术检测诱导后的细胞表达CD31和vWF情况。透射电镜观察细胞的超微结构。结果: (1)诱导分化后细胞形态学变化:经血管内皮细胞生长因子和碱性成纤维细胞生长因子诱导后的细胞形态上呈典型的“铺路石”样外观,经历从小圆→梭形→扁平细胞的过程,符合内皮前体细胞演变为成熟内皮细胞的过程。 (2)诱导分化后细胞的表面标志鉴定:诱导分化20d,实验组1中CD31+占86.9%、vWF+占82.5%、CD31+/vWF+占70.9%,实验组2中CD31+占62.5%、vWF+占58.2%、CD31+/vWF+占54.3%。 (3)培养细胞的超微结构观察:透射电镜下细胞胞浆内可见特征性W-P小体。结论: 外周血单个核细胞在血管内皮细胞生长因子和碱性成纤维细胞生长因子联合诱导下,可分化为血管内皮细胞。
[Abstract]:Objective:
Vascular endothelial growth factor and basic fibroblast growth factor (bfgf) were combined to induce human peripheral blood mononuclear cells (PBMNCs) to differentiate into vascular endothelial cells (VECs) in vitro.
Method:
(1) PBMNCs 2ml was harvested after stimulation with recombinant human granulocyte colony-stimulating factor (rhGMCSF) under aseptic condition, and the experimental group 1 and the control group were set up respectively.
(2) Hanks solution was diluted twice and placed on the upper layer of lymphocyte separating solution at 1:2. After centrifugation, the grey-white layer, which was turbid at the junction of the lymphocyte separating layer and the upper layer, was extracted.
(3) Fetal bovine serum containing 0.2 volume fraction, 10 ug/L vascular endothelial growth factor and 10 ug/L basic fibroblast growth factor were added to the DMEM-F12 medium after centrifugation. X1010L-1 was inoculated in a 25 cm 2 culture flask and cultured in a saturated humidity incubator with a volume fraction of 0.05 and a temperature of 37 C. On the third day, the medium was replaced to remove the unadhered cells, and then the liquid was changed every 2 days.
(4) Inverted phase contrast microscope was used to observe whether the monolayer arrangement of cells was "paving stone" like structure. Flow cytometry was used to detect the expression of CD31 and vWF in the induced cells.
Result:
(1) Morphological changes after differentiation: the cells induced by vascular endothelial growth factor and basic fibroblast growth factor showed a typical "paving stone" appearance, and experienced a process from small round to spindle to flat cells, which accorded with the evolution of endothelial progenitor cells into mature endothelial cells.
(2) Identification of cell surface markers after induction of differentiation: CD31 + accounted for 86.9%, vWF + accounted for 82.5%, CD31 + / vWF + accounted for 70.9%, CD31 + accounted for 62.5%, vWF + accounted for 58.2%, CD31 + / vWF + accounted for 54.3%.
(3) ultrastructure observation of cultured cells: the characteristic W-P bodies in cytoplasm were observed under transmission electron microscope.
Peripheral blood mononuclear cells can differentiate into vascular endothelial cells induced by vascular endothelial growth factor and basic fibroblast growth factor.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R329

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