Ang1、Ang2和Tie2在血管內(nèi)皮細胞和體外三維培養(yǎng)血管樣結(jié)構(gòu)中的表達及意義
發(fā)布時間：2019-01-09 10:53
【摘要】： [目的]構(gòu)建體外血管生成的三維培養(yǎng)模型,研究體內(nèi)臍靜脈內(nèi)皮細胞(HVUECs)、體外培養(yǎng)的HVUECs和體外三維培養(yǎng)的血管樣結(jié)構(gòu)中促血管生成素1、2及其受體(Ang1、2及Tie2)的表達水平,旨在探討其在體外血管生成中的作用,為將來進一步在體外進行抑制血管生成的實驗研究打下基礎(chǔ)。 [方法]以酶消化法獲得HVUECs,經(jīng)免疫細胞化學(xué)染色技術(shù)鑒定,采用夾心培養(yǎng)法構(gòu)建體外血管生成的三維培養(yǎng)模型,獲得三維血管樣結(jié)構(gòu)。采用逆轉(zhuǎn)錄-聚合酶鏈式反應(yīng)(RT-PCR)的方法分別檢測10例體內(nèi)HVUECs、體外培養(yǎng)的HVUECs及體外三維培養(yǎng)模型的血管樣結(jié)構(gòu)中Ang1、Ang2及Tie2信使核糖核酸(mRNA)的表達水平;同時采用免疫組化法和免疫細胞化學(xué)染色法檢測10例體內(nèi)HVUECs、體外培養(yǎng)的HVUECs中Ang1、Ang2及Tie2蛋白的表達水平。 [結(jié)果] 1、以酶消化法可獲得以HVUECs為主的混合細胞懸液,經(jīng)反復(fù)貼壁法分離純化內(nèi)皮細胞,采用免疫細胞化學(xué)染色技術(shù)對內(nèi)皮細胞進行鑒定并確定了所純化的細胞為HVUECs。采用夾心培養(yǎng)法成功構(gòu)建了體外血管生成的三維培養(yǎng)模型,獲得三維血管樣結(jié)構(gòu)。 2、體外三維培養(yǎng)模型中Ang2mRNA、Tie2mRNA表達水平均高于體內(nèi)外HVUECs組(P0.05);體內(nèi)外HVUECs組間Ang2mRNA、Tie2mRNA表達水平相比差異無顯著性意義(P0.05)。 3、體內(nèi)外HVUECs及體外三維培養(yǎng)模型中Ang1mRNA表達水平相比差異無顯著性意義(P0.05)。 4、體內(nèi)外HVUECs中Ang1、Ang2及Tie2蛋白的表達均為陽性,兩組的內(nèi)皮細胞光密度值均無明顯差異(P㧐0.05)。 [結(jié)論] 1、夾心培養(yǎng)法是較為成熟的體外血管生成的三維培養(yǎng)方法,具有操作簡便、三維血管樣結(jié)構(gòu)形成數(shù)量多、網(wǎng)絡(luò)結(jié)構(gòu)復(fù)雜等特點,適用于體外血管生成影響因素的實驗研究。 2、Ang/Tie2體系在體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.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R329;R73-36
本文編號：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.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R329;R73-36
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