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背景:组织型纤溶酶原激活因子被认为是血管系统内初期血栓清除的关键酶。目的:构建pcDNA3.1(+)组织型纤溶酶原激活因子真核表达载体转染人脐静脉内皮细胞,观察外源性组织型纤溶酶原激活因子的表达情况。设计:随机对照实验。单位:中南大学湘雅二医院及中国医学遗传学国家重点实验室。材料:实验于2002-09/2003-06在中国医学遗传学国家重点实验室完成。脐静脉内皮细胞取自中南大学湘雅二医院健康产妇分娩后胎盘脐带,pcDNA3.1为SmithKlineBeecham公司赠送。方法:构建真核表达载体pcDNA3.1(+)组织型纤溶酶原激活因子,并将pcDNA3.1(+)组织型纤溶酶原激活因子转入人脐静脉内皮细胞,用酶联免疫吸附法定量检测转基因后组织型纤溶酶原激活因子蛋白表达情况、发色底物法检测外源性组织型纤溶酶原激活因子活性。以转pcD-NA3.1(+)组织型纤溶酶原激活因子基因人脐静脉内皮细胞为观察对象,未转pcDNA3.1(+)组织型纤溶酶原激活因子基因人脐静脉内皮细胞作为对照。主要观察指标:组织型纤溶酶原激活因子蛋白定量及活性测定结果。结果:①组织型纤溶酶原激活因子蛋白定量表达结果为568.6ng/106细胞/24h,未转pcDNA3.1(+)组织型纤溶酶原激活因子的人脐静脉内皮细胞测得为17.8ng/106细胞/24h。②组织型纤溶酶原激活因子活性为108.8IU/106细胞/24h,未转pcDNA3.1(+)组织型纤溶酶原激活因子的人脐静脉内皮细胞测得为5.6IU/106细胞/24h。结论:pcDNA3.1(+)组织型纤溶酶原激活因子转染人脐静脉内皮细胞后,外源性组织型纤溶酶原激活因子基因获有效表达,为pcDNA3.1(+)组织型纤溶酶原激活因子转入人体细胞确立了有效依据。
BACKGROUND: Tissue-type plasminogen activator is considered as a key enzyme in initial thrombosis in the vascular system. Objective: To construct pcDNA3.1 (+) tissue plasminogen activator eukaryotic expression vector for transfection of human umbilical vein endothelial cells to observe the expression of exogenous tissue plasminogen activator. Design: Randomized controlled experiment. Organization: Second Xiangya Hospital of Central South University and Chinese State Key Laboratory of Medical Genetics. MATERIALS: Experiments were performed at the State Key Laboratory of Medical Genetics in China from September 2002 to June 2003. Umbilical vein endothelial cells taken from Xiangya Second Hospital of Central South University healthy maternal postpartum placenta umbilical cord, pcDNA3.1 for the gift of SmithKlineBeecham company. Methods: The eukaryotic expression vector pcDNA3.1 (+) tissue plasminogen activator was constructed and the pcDNA3.1 (+) tissue plasminogen activator was transfected into human umbilical vein endothelial cells Adsorption method was used to detect the expression of tissue-type plasminogen activator protein after transgene and the chromogenic substrate method was used to detect the activity of exogenous tissue-type plasminogen activator. To take pcD-NA3.1 (+) tissue-type plasminogen activator gene human umbilical vein endothelial cells as the observation object, did not transfer pcDNA3.1 (+) tissue-type plasminogen activator gene human umbilical vein endothelial cells as comparison. MAIN OUTCOME MEASURES: Quantification and activity determination of tissue-type plasminogen activator protein. Results: (1) The quantitative expression of tissue plasminogen activator protein was 568.6ng / 106cells / 24h, and the untransfected human umbilical vein endothelial cells of tissue plasminogen activator was 17.8 ng / 106 cells / 24h. ② The activity of tissue-type plasminogen activator was 108.8 IU / 106 cells / 24h, the untreated pcDNA3.1 (+) tissue-type plasminogen activator of human umbilical vein endothelial cells measured 5.6IU / 106 cells / 24h. Conclusion: The pcDNA3.1 (+) tissue plasminogen activator is transfected into human umbilical vein endothelial cells and the expression of exogenous tissue plasminogen activator gene is efficiently expressed. Plasminogen activator into human cells established an effective basis.