pcDNA3.1(+)TPA构建及其在人脐静脉内皮细胞的表达活性研究

Construction of Expression Vector pcDNA3.1(+)TPA and Study of Its Expression Activity in Human Umbilical Vein Endotheliocytes

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 the expression of exogenous tissue plasminogen activator (TPA) gene in human umbilical vein endotheliocytes to provide both theoretical basis and new method for gene therapy of ischemic heart disease and prevention of postoperative vessel re-stenosis. Methods Expression vector pcDNA3 1(+)TPA was constructed, and transfected into human umbilical vein endotheliocytes cultivated in vitro by lyposome. The exogenous TPA expression was observed. Results The expression vector pcDNA3 1(+)TPA could efficaciously express TPA in human umbilical vein endotheliocytes. The protein quantity of TPA in the transfected cells was 568 6ng/10 6cell/24h, when measured by enzyme-linked immunosorbent assay, while that in the non-transfected cells was 17 8ng/10 6 cell/24h. The exogenous TPA's activity in the transfected cells was 108 8IU/10 6cell/24h, when measured by chromogenic substrate assay, while that in the non-transfected cells was 5 6 IU/10 6cell/24h. Conclusion When pcDNA3 1(+)TPA was transfected into human umbilical vein endotheliocytes, exogenous TPA could be expressed efficaciously, which provides theoretical basis and new method for TPA gene therapy of ischemic heart disease.