rAAV-HTK重组体对人脐静脉内皮细胞功能的影响
发布时间：2018-08-22 11:51
【摘要】：目的:利用已经构建好的腺相关病毒-人组织激肽释放酶基因重组体 (rAAV-HTK),感染人工培养的人脐静脉内皮细胞(HUVEC),观察HUVEC 细胞合成组织激肽释放酶蛋白量的变化,以及rAAV-HTK 转染对内皮细 胞功能的影响,探讨利用rAAV-HTK 治疗高血压、缺血性心脏病的可行 性。 方法:1.将已经构建好的rAAV-HTK 重组质粒感染人工培养的HUVEC 细胞。2. 应用ELISA 方法测定HUVEC 细胞上清液和细胞内人组织激肽释放酶 (HTK)的含量。3.应用半定量RT-PCR 方法检测感染rAAV-HTK 前后 HUVEC 中内皮型一氧化氮合酶(eNOS)、凋亡蛋白酶(caspase-3)、 内皮素-1(ET-1)、血管内皮生长因子(VEGF)、内皮素B_1受体(ETR-B_1) 以及缓激肽B_1受体、缓激肽B_2受体的mRNA 表达量变化情况。 结果:1.转染有rAAV-HTK 的HUVEC 细胞内HTK 含量比对照组增高3 倍。2.在 人工培养的HUVEC 细胞上清液中没有检测到HTK。3.转染有rAAV-HTK 的HUVEC 细胞与对照组相比,其细胞内eNOS 的mRNA 合成量增加, caspase-3 的mRNA 表达量降低,VEGF、ET-1、ETR-B_1、缓激肽B_1受体、 缓激肽B_2受体的mRNA 表达量没有变化。 结论:rAAV-HTK 重组体感染HUVEC 细胞可以使HUVEC 细胞合成HTK 增多,eNOS 的mRNA 表达量增高,NO 产生增加,caspase-3 的mRNA 表达量减低。 这些作用提示HTK 能够改善内皮细胞功能,在实践上为高血压等血管 内皮功能异常的心血管疾病的基因治疗奠定了基础。
[Abstract]:Aim: to investigate the changes of tissue kallikrein protein in HUVEC cells infected with human umbilical vein endothelial cells (HUVEC),) by using the recombinant adeno-associated virus human tissue kallikrein gene (rAAV-HTK). The effect of rAAV-HTK transfection on endothelial fine cell function and the feasibility of using rAAV-HTK to treat hypertension and ischemic heart disease were discussed. Method 1: 1. The constructed rAAV-HTK recombinant plasmid was infected with cultured HUVEC cells. The content of human tissue kallikrein (HTK) in supernatants and cells of HUVEC cells was determined by ELISA method. Semi-quantitative RT-PCR was used to detect endothelial nitric oxide synthase (eNOS), apoptotic protease (caspase-3), endothelin 1 (ET-1), vascular endothelial growth factor (VEGF), endothelin B1 receptor (ETR-B_1) and bradykinin B1 receptor in HUVEC before and after rAAV-HTK infection. Changes of mRNA expression of bradykinin B 2 receptor. The result is 1: 1. The content of HTK in HUVEC cells transfected with rAAV-HTK was increased by 3 times. 2 times than that in control group. HTK.3 was not detected in the supernatant of cultured HUVEC cells. Compared with the control group, the mRNA synthesis of eNOS was increased in HUVEC cells transfected with rAAV-HTK, the mRNA expression of caspase-3 was decreased, the expression of bradykinin B1 receptor and bradykinin B2 receptor was not changed. Conclusion infection of HUVEC cells with the recombinant fraction of rAAV-HTK can increase the mRNA expression of Enos in HUVEC cells and increase the production of no, increase the expression of caspase-3 in HUVEC cells. These effects suggest that HTK can improve endothelial cell function and lay a foundation for gene therapy of vascular endothelial dysfunctional cardiovascular diseases such as hypertension.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R346
[Abstract]:Aim: to investigate the changes of tissue kallikrein protein in HUVEC cells infected with human umbilical vein endothelial cells (HUVEC),) by using the recombinant adeno-associated virus human tissue kallikrein gene (rAAV-HTK). The effect of rAAV-HTK transfection on endothelial fine cell function and the feasibility of using rAAV-HTK to treat hypertension and ischemic heart disease were discussed. Method 1: 1. The constructed rAAV-HTK recombinant plasmid was infected with cultured HUVEC cells. The content of human tissue kallikrein (HTK) in supernatants and cells of HUVEC cells was determined by ELISA method. Semi-quantitative RT-PCR was used to detect endothelial nitric oxide synthase (eNOS), apoptotic protease (caspase-3), endothelin 1 (ET-1), vascular endothelial growth factor (VEGF), endothelin B1 receptor (ETR-B_1) and bradykinin B1 receptor in HUVEC before and after rAAV-HTK infection. Changes of mRNA expression of bradykinin B 2 receptor. The result is 1: 1. The content of HTK in HUVEC cells transfected with rAAV-HTK was increased by 3 times. 2 times than that in control group. HTK.3 was not detected in the supernatant of cultured HUVEC cells. Compared with the control group, the mRNA synthesis of eNOS was increased in HUVEC cells transfected with rAAV-HTK, the mRNA expression of caspase-3 was decreased, the expression of bradykinin B1 receptor and bradykinin B2 receptor was not changed. Conclusion infection of HUVEC cells with the recombinant fraction of rAAV-HTK can increase the mRNA expression of Enos in HUVEC cells and increase the production of no, increase the expression of caspase-3 in HUVEC cells. These effects suggest that HTK can improve endothelial cell function and lay a foundation for gene therapy of vascular endothelial dysfunctional cardiovascular diseases such as hypertension.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R346
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