人海绵状血管瘤内皮细胞特性研究和人海绵状血管瘤动物模型的建立

发布时间：2018-01-14 01:11

本文关键词：人海绵状血管瘤内皮细胞特性研究和人海绵状血管瘤动物模型的建立　出处：《中国协和医科大学》2005年博士论文　论文类型：学位论文

【摘要】：血管瘤是最常见的婴幼儿良性血管增生或血管畸形，好发于颜面部，常因引起严重的面部畸形而影响患者身心健康。尽管血管瘤的形成可能与活跃的血管生成有关，但是其发病机制还不明了。而且目前对血管瘤的治疗尚缺乏安全有效的药物，因此，研究血管瘤内皮细胞的表型和功能特性，，并建立一个血管瘤实验动物模型对血管瘤发病机制的研究和临床治疗药物的筛选有非常重要的意义。目的研究人海绵状血管瘤内皮细胞在形态、表型、功能和基因方面的改变，探索海绵状血管瘤的发生机制；建立人海绵状血管瘤的实验动物模型，并筛选对海绵状血管瘤有效的治疗药物。方法从人海绵状血管瘤组织分离、纯化血管瘤内皮细胞。应用免疫荧光染色和流式细胞术鉴定其内皮细胞标志性分子的表达。应用透射电子显微镜观察海绵状血管瘤内皮细胞的超微结构的变化；应用流式细胞术和免疫细胞化学方法分析海绵状血管瘤内皮细胞的表型特征；应用Matrigel比较血管瘤内皮细胞与正常血管内皮细胞形成毛细血管样网络能力的差别：应用RT-PCR研究海绵状血管瘤内皮细胞上VEGF及其受体、Ang-1、Ang-2和Tie2的表达。海绵状血管瘤内皮细胞表达和分泌的基质金属蛋白酶分别通过明胶酶谱法、RT-PCR、Western blot和细胞免疫荧光染色进行分析。应用酶联免疫吸附测定检查海绵状血管瘤内皮细胞释放的U-PA、PAI-1和TF。通过将荧光染料标记的血小板与内皮细胞共培养比较血管瘤内皮细胞和正常内皮细胞与血小板粘附能力的差别。此外，应用RT-PCR方法，比较252-基因在血管瘤内皮细胞，正常血管内皮细胞和肿瘤血管内皮细胞的表达差别，并应用透射电子显微镜观察252-基因转染后对人海绵状血管瘤内皮细胞超微结构异常的影响。
[Abstract]:Hemangioma is the most common benign vascular hyperplasia or vascular malformation in infants. Physical and mental health is often affected by severe facial malformations, although the formation of hemangiomas may be associated with active angiogenesis. However, the pathogenesis of hemangioma is not clear, and the current treatment of hemangioma is still lack of safe and effective drugs, therefore, to study the phenotypic and functional characteristics of endothelial cells of hemangioma. The establishment of an experimental animal model of hemangioma is of great significance for the study of the pathogenesis of hemangioma and the screening of clinical therapeutic drugs. Objective to study the morphological phenotypic functional and genetic changes of human cavernous hemangioma endothelial cells and to explore the pathogenesis of cavernous hemangioma. To establish an experimental animal model of human cavernous hemangioma and to screen effective drugs for treatment of cavernous hemangioma. Methods Human cavernous hemangioma was isolated. The expression of iconic molecules in endothelial cells was identified by immunofluorescence staining and flow cytometry. The ultrastructural changes of endothelial cells in cavernous hemangioma were observed by transmission electron microscope (TEM). The phenotypic characteristics of endothelial cells in cavernous hemangioma were analyzed by flow cytometry and immunocytochemistry. Matrigel was used to compare the ability of angioma endothelial cells and normal vascular endothelial cells to form capillary networks. RT-PCR was used to study VEGF and its receptor on endothelial cells of cavernous hemangioma. Expression of Ang-1 Ang-2 and Tie2. The matrix metalloproteinases secreted by endothelial cells of cavernous hemangioma were detected by reverse transcriptase polymerase chain reaction (RT-PCR). Western blot and cellular immunofluorescence staining were used to detect U-PA released from endothelial cells of cavernous hemangioma by enzyme-linked immunosorbent assay (Elisa). PAI-1 and TF. The difference of adhesion between endothelial cells of hemangioma and that of normal endothelial cells was compared by co-culture of fluorescent dye labeled platelets and endothelial cells. To compare the expression of 252-gene in angioma endothelial cells, normal vascular endothelial cells and tumor vascular endothelial cells by RT-PCR. The effect of 252-gene transfection on the ultrastructure of human cavernous hemangioma endothelial cells was observed by transmission electron microscope.
【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】：2005
【分类号】：R-332

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