大鼠骨髓血管内皮祖细胞分离、培养、诱导分化及鉴定

发布时间：2018-02-16 15:48

本文关键词： 内皮祖细胞骨髓鉴定　出处：《苏州大学》2007年硕士论文　论文类型：学位论文

【摘要】： 目的探索大鼠骨髓内皮祖细胞(EPCs)的分离、培养、诱导分化及鉴定方法，为实验研究和临床应用搭建平台。方法冲洗大鼠骨髓腔，梯度密度离心法获得单个核细胞，内皮系细胞专用培养液EGM-2MV培养，通过观察细胞形态学、增殖活性、内皮细胞系列标志CD34、VEGFR-2、vWF免疫组化，祖细胞标志CD133免疫荧光技术动态鉴定，激光共聚焦显微镜观察其摄取DIL-AC-LDL，结合FITC-Lectin-UEA-1实验，，透射电镜观察超微结构和内皮细胞特征性细胞器以及染色体核型分析进行安全性评价。结果新分离的骨髓单个核细胞(BMMNCs)呈圆形，大小不一，48h后部分细胞开始贴壁，形态有梭形、三角形、纺锤形或不规则形，4～8d出现细胞集落和细胞线状排列，第9～11天接近融合，呈典型鹅卵石样外观。细胞生长曲线呈“S”形，8天和10天的增殖活性强于5天和14天。贴壁细胞CD34、VEGFR-2、vWF、CD133均表达阳性并呈动态变化，能够摄取DIL-AC-LDL和结合FITC-Lectin-UEA-1，透射电镜可见特征性的Weible-Palade(W-P)小体存在，能稳定保持二倍体核型。结论本实验初步建立了一套大鼠骨髓血管内皮祖细胞分离、培养、诱导分化及鉴定方法体系。
[Abstract]:Objective to explore the methods of isolation, culture, differentiation and identification of rat bone marrow endothelial progenitor cells (EPCs), and to establish a platform for experimental research and clinical application. Methods Mononuclear cells were obtained from rat medullary cavity by gradient density centrifugation. EGM-2MV was used as a special culture medium for endothelial cells. The morphology, proliferative activity and endothelial cell marker CD34 VEGFR-2VWF were observed by immunohistochemistry. CD133 immunofluorescence technique was used to dynamically identify progenitor cells. The uptake of DIL-AC-LDLwas observed by confocal laser microscope. The safety of DIL-AC-LDLwas evaluated by FITC-Lectin-UEA-1 assay, ultrastructural observation, characteristic organelles of endothelial cells and chromosome karyotype analysis under transmission electron microscope (TEM). Results the newly isolated bone marrow mononuclear cells (BMMNCs) were round. After 48 hours of different sizes, some of the cells began to adhere to the wall, with fusiform, triangular shape, spindle shape or irregular shape, cell colony and cell line arrangement appeared on the 8th day, and nearly fused on the 911th day. The cell growth curve was 8 and 10 days and the proliferation activity was stronger than that of 5 and 14 days. CD34 + VEGFR-2vWFFtc CD133 was positive and showed dynamic changes. When DIL-AC-LDL and FITC-Lectin-UEA-1 were ingested, the characteristic Weible-Palade W-PEA bodies were observed by transmission electron microscopy, and the diploid karyotype could be maintained stably. Conclusion A set of methods for isolation, culture, induction of differentiation and identification of endothelial progenitor cells from rat bone marrow were established.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R329.2

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