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目的 建立TPA基因在人脐静脉内皮细胞外源性表达的方法 ,为缺血性心脏疾病的基因治疗及防止血管再狭窄提供理论依据和新方法。方法 构建真核表达载体pcDNA3 1( +)TPA ,并采用脂质体法将其转入体外培养的人脐静脉内皮细胞 ,并观察外源性TPA表达情况。结果 真核表达载体pcDNA3 1( +)TPA在人脐静脉内皮细胞中获有效表达 ,酶联免疫吸附法TPA蛋白表达定量检测结果为 5 68 6ng/10 6细胞 /2 4h ,未转pcDNA3 1( +)TPA的人脐静脉内皮细胞测得为 17 8ng/10 6细胞 /2 4h ;发色底物法测得外源性TPA活性为 10 8 8IU/10 6细胞 /2 4h ,未转pcDNA3 1( +)TPA的人脐静脉内皮细胞测得为 5 6IU/10 6细胞 /2 4h。结论 pcD NA3 1( +)TPA转入人脐静脉内皮细胞后 ,外源性TPA基因获有效表达 ,为缺血性心脏病的TPA基因治疗提供了理论依据和新方法。
OBJECTIVE: To establish a method for the exogenous expression of TPA gene in human umbilical vein endothelial cells for the gene therapy of ischemic heart disease and the prevention of restenosis. Methods The eukaryotic expression vector pcDNA3 1 (+) TPA was constructed and transfected into human umbilical vein endothelial cells by liposome method. The expression of exogenous TPA was observed. Results The eukaryotic expression vector pcDNA3 1 (+) TPA was effectively expressed in human umbilical vein endothelial cells. The quantitative analysis of TPA protein expression by enzyme-linked immunosorbent assay was 5686ng / 106cells / 24h, without transfecting pcDNA3l ( +) TPA of human umbilical vein endothelial cells measured 17 8ng / 106 cells / 2 4h; chromogenic substrate method measured exogenous TPA activity of 10 8 8IU / 106 cells / 2 4h, did not turn pcDNA3 1 (+) TPA human umbilical vein endothelial cells measured 5 6IU / 106 cells / 2 4h. Conclusions The exogenous TPA gene is efficiently expressed after transfection of pcD NA3 1 (+) TPA into human umbilical vein endothelial cells, which provides a theoretical basis and a new method for TPA gene therapy of ischemic heart disease.