Ang1、Ang2和Tie2在血管内皮细胞和体外三维培养血管样结构中的表达及意义
发布时间：2019-01-09 10:53
【摘要】： [目的]构建体外血管生成的三维培养模型,研究体内脐静脉内皮细胞(HVUECs)、体外培养的HVUECs和体外三维培养的血管样结构中促血管生成素1、2及其受体(Ang1、2及Tie2)的表达水平,旨在探讨其在体外血管生成中的作用,为将来进一步在体外进行抑制血管生成的实验研究打下基础。 [方法]以酶消化法获得HVUECs,经免疫细胞化学染色技术鉴定,采用夹心培养法构建体外血管生成的三维培养模型,获得三维血管样结构。采用逆转录-聚合酶链式反应(RT-PCR)的方法分别检测10例体内HVUECs、体外培养的HVUECs及体外三维培养模型的血管样结构中Ang1、Ang2及Tie2信使核糖核酸(mRNA)的表达水平;同时采用免疫组化法和免疫细胞化学染色法检测10例体内HVUECs、体外培养的HVUECs中Ang1、Ang2及Tie2蛋白的表达水平。 [结果] 1、以酶消化法可获得以HVUECs为主的混合细胞悬液,经反复贴壁法分离纯化内皮细胞,采用免疫细胞化学染色技术对内皮细胞进行鉴定并确定了所纯化的细胞为HVUECs。采用夹心培养法成功构建了体外血管生成的三维培养模型,获得三维血管样结构。 2、体外三维培养模型中Ang2mRNA、Tie2mRNA表达水平均高于体内外HVUECs组(P0.05);体内外HVUECs组间Ang2mRNA、Tie2mRNA表达水平相比差异无显著性意义(P0.05)。 3、体内外HVUECs及体外三维培养模型中Ang1mRNA表达水平相比差异无显著性意义(P0.05)。 4、体内外HVUECs中Ang1、Ang2及Tie2蛋白的表达均为阳性,两组的内皮细胞光密度值均无明显差异(P㧐0.05)。 [结论] 1、夹心培养法是较为成熟的体外血管生成的三维培养方法,具有操作简便、三维血管样结构形成数量多、网络结构复杂等特点,适用于体外血管生成影响因素的实验研究。 2、Ang/Tie2体系在体外血管生成中可能起重要的作用。
[Abstract]:[objective] to establish a three-dimensional culture model of angiogenesis in vitro and to study the expression levels of angiopoietin 1 (Ang1,2) and its receptors (Ang1,2 and Tie2) in HVUECs cultured by umbilical vein endothelial cells (HVUECs),) in vitro and in vascular like structure in vitro. The aim of this study was to investigate the role of angiogenesis in vitro and to lay a foundation for further experimental research on the inhibition of angiogenesis in vitro. [methods] HVUECs, obtained by enzyme digestion was identified by immunocytochemical staining and three-dimensional angiogenesis model was constructed by sandwich culture. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the expression levels of Ang1,Ang2 and Tie2 mRNA (mRNA) in 10 cases of HVUECs cultured in vivo and 3 D model in vitro. The expression of Ang1,Ang2 and Tie2 protein in 10 cases of HVUECs cultured in vivo and in vitro were detected by immunohistochemistry and immunocytochemical staining. [results] 1Endothelial cells were isolated and purified by enzyme digestion, and the mixed cell suspensions, which were mainly HVUECs, were isolated and purified by repeated adherent method. The endothelial cells were identified by immunocytochemical staining and the purified cells were identified as HVUECs.. A three-dimensional culture model of angiogenesis in vitro was successfully constructed by sandwich culture, and the three-dimensional vascular-like structure was obtained. 2. The expression level of Ang2mRNA,Tie2mRNA in three-dimensional culture model in vitro was higher than that in HVUECs group in vivo and in vitro (P0.05), but there was no significant difference in Ang2mRNA,Tie2mRNA expression level between in vivo and in vitro HVUECs group (P0.05). 3. There was no significant difference in the expression of Ang1mRNA between in vivo and in vitro HVUECs and in vitro three dimensional culture model (P0.05). 4. The expression of Ang1,Ang2 and Tie2 protein was positive in HVUECs in vivo and in vitro, and there was no significant difference in the optical density of endothelial cells between the two groups (P0. 05). [conclusion] 1. The sandwich culture method is a mature three-dimensional culture method for angiogenesis in vitro, which has the advantages of simple operation, large number of three-dimensional angioid structures, complex network structure, and so on. It is suitable for the experimental study of factors affecting angiogenesis in vitro. 2 Angr / Tie2 system may play an important role in vitro angiogenesis.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329;R73-36
本文编号：2405527
[Abstract]:[objective] to establish a three-dimensional culture model of angiogenesis in vitro and to study the expression levels of angiopoietin 1 (Ang1,2) and its receptors (Ang1,2 and Tie2) in HVUECs cultured by umbilical vein endothelial cells (HVUECs),) in vitro and in vascular like structure in vitro. The aim of this study was to investigate the role of angiogenesis in vitro and to lay a foundation for further experimental research on the inhibition of angiogenesis in vitro. [methods] HVUECs, obtained by enzyme digestion was identified by immunocytochemical staining and three-dimensional angiogenesis model was constructed by sandwich culture. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the expression levels of Ang1,Ang2 and Tie2 mRNA (mRNA) in 10 cases of HVUECs cultured in vivo and 3 D model in vitro. The expression of Ang1,Ang2 and Tie2 protein in 10 cases of HVUECs cultured in vivo and in vitro were detected by immunohistochemistry and immunocytochemical staining. [results] 1Endothelial cells were isolated and purified by enzyme digestion, and the mixed cell suspensions, which were mainly HVUECs, were isolated and purified by repeated adherent method. The endothelial cells were identified by immunocytochemical staining and the purified cells were identified as HVUECs.. A three-dimensional culture model of angiogenesis in vitro was successfully constructed by sandwich culture, and the three-dimensional vascular-like structure was obtained. 2. The expression level of Ang2mRNA,Tie2mRNA in three-dimensional culture model in vitro was higher than that in HVUECs group in vivo and in vitro (P0.05), but there was no significant difference in Ang2mRNA,Tie2mRNA expression level between in vivo and in vitro HVUECs group (P0.05). 3. There was no significant difference in the expression of Ang1mRNA between in vivo and in vitro HVUECs and in vitro three dimensional culture model (P0.05). 4. The expression of Ang1,Ang2 and Tie2 protein was positive in HVUECs in vivo and in vitro, and there was no significant difference in the optical density of endothelial cells between the two groups (P0. 05). [conclusion] 1. The sandwich culture method is a mature three-dimensional culture method for angiogenesis in vitro, which has the advantages of simple operation, large number of three-dimensional angioid structures, complex network structure, and so on. It is suitable for the experimental study of factors affecting angiogenesis in vitro. 2 Angr / Tie2 system may play an important role in vitro angiogenesis.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329;R73-36
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