组织型纤溶酶原激活因子构建及其在人脐静脉内皮细胞的表达活性

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SmithKline Beecham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pcDNA3.1(+)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568.6 ng/106细胞/24 h,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17.8 ng/106细胞/24 h.②组织型纤溶酶原激活因子活性为108.8 IU/106细胞/24 h,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/24 h.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.

Construction of plasminogen activator and its expressive activity in human umbilical vein endothelial cell

BACKGROUND: Tissue-typed plasminogen activator (t-PA) is considered as the key enzyme to clear thrombus at the early stage in the vascular system.OBJECTIVE: To construct human umbilical vein endothelial cell (HUVEC) transfected by eukaryotic expression vector pcDNA3.1 (+) t-PA and observe the expression of ectogenous t-PADESIGN: A randomly-controlled trial.SETTING: Second Xiangya Hospital, Central South University and National Key Laboratory of Medical GeneticsMATERIALS: This experiment was conducted in the National Key Laboratory of Medical Genetics between September 2002 and June 2003. HUVEC was obtained from the umbilical cord of placenta of healthy parturient in the Second Xiangya Hospital of Central South University, and pcDNA3.1 was from SmithKline Beecham Company.METHODS: Eukaryotic expression vector pcDNA3.1 (+) t-PA was constructed and transferred into HUVEC. The expression of t-PA was detected with quantitative enzyme-linked immunosorbent assay and engenous t-PA activity was detected with chromogenic substrate assay. HUVECs treansfected by t-PA pcDNA3.1 (+)were set as observed subjects and those not transfected by t-PA as controls.MAIN OUTCOME MEASURES: Quantitative measurement of expressing protein of t-PA and the activity of exogenous t-PA.ng/106 cells per 24 hours, and HUVEC of non-transfected pcDNA3.1 (+)tIU/106 cells per 24 hours, and HUVEC of non-transfected pcDNA3.1 (+)tPA was 5.6 IU/106 cells per 24 hours.CONCLUSION: After HUVEC transfected by pcDNA3.1 (+)t-PA, exogenous t-PA gene has obtained effective expression which provide the effective basis for pcDNA3.1 (+)t-PA transfecting into human cells.