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目的:建立在人皮肤成纤维细胞中稳定表达的细胞株,为组织型纤溶酶原激活因子(TPA)功能分析和对缺血性心脏疾病基因治疗提供理论依据。方法:构建真核表达载体pcDNA3.1(+)TPA,然后将其转入人皮肤成纤维细胞,经G418筛选后,用RT-PCR,ELISA和发色底物法观察外源性TPA表达情况。结果:真核表达载体pcDNA3.1(+)TPA在人皮肤成纤维细胞中获有效表达,酶联免疫吸附法TPA蛋白表达定量检测结果为每24小时643.5ng/106个细胞,高于未转pcDNA3.1(+)TPA的皮肤成纤维细胞每24小时19.2ng/106个细胞;发色底物法测得外源性TPA活性为每24小时122.6U/106个细胞,亦高于未转pcDNA3.1(+)TPA的皮肤成纤维细胞每24小时5.8U/106个细胞。结论:pcDNA3.1(+)TPA转入人皮肤成纤维细胞后,外源性TPA基因获有效表达,该细胞模型将成为TPA功能研究和缺血性心脏病基因治疗的重要方法。
OBJECTIVE: To establish a cell line stably expressed in human dermal fibroblasts and to provide a theoretical basis for the functional analysis of tissue-type plasminogen activator (TPA) and gene therapy for ischemic heart disease. Methods: The eukaryotic expression vector pcDNA3.1 (+) TPA was constructed and transfected into human dermal fibroblasts. After screening by G418, the expression of exogenous TPA was observed by RT-PCR, ELISA and chromogenic substrate method . Results: The eukaryotic expression vector pcDNA3.1 (+) TPA was efficiently expressed in human dermal fibroblasts. The quantitative analysis of TPA protein expression by ELISA showed that the TPA protein expression was 643.5 ng / 106 cells per 24 hours, The skin fibroblasts of pcDNA3.1 (+) TPA had 19.2 ng / 106 cells per 24 hours; the exogenous TPA activity measured by the chromogenic substrate method was 122.6 U / 106 cells per 24 hours, Skin fibroblasts of pcDNA3.1 (+) TPA 5.8 U / 106 cells per 24 hours. Conclusion: The transfection of pcDNA3.1 (+) TPA into human dermal fibroblasts effectively expresses the exogenous TPA gene. This cell model will become an important method for the study of TPA function and gene therapy of ischemic heart disease.