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643.5 ng/106个细胞,高于未转pcDNA3.1(+)TPA的皮肤成纤维细胞每24小时19.2 ng/106个细胞;发色底物法测得外源性TPA活性为每24小时122.6 U/106个细胞,亦高于未转pcDNA3.1(+)TPA的皮肤成纤维细胞每24小时5.8 U/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 expressing the tissue plasminogen activator (TPA) in human skin fibroblasts so as to develop the function analysis and gene therapy of TPA in ischemic heart diseases. Methods Eukaryotic expression vector pcDNA3.1(+)TPA was constructed and transferred into human skin fibroblasts. After G418 selection, the exogenous expression and activity of TPA were observed subsequently by reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and chromogenic substrate assay. Results Eukaryotic expression vector pcDNA3.1(+)TPA was expressed effectively in human skin fibroblasts. Quantitative ELISA showed that the expression of TPA protein of the experiment group was much higher than that of the hours). And the chromogenic substrate assay showed that the exogenous TPA activity of the experimental group was also much higher than that of the hours). Conclusion The exogenous TPA gene can be expressed effectively after pcDNA3.1(+)TPA was transferred into human skin fibroblasts, suggesting that the cell model will become an important tool in the further study of TPA function and gene therapy in ischemic heart diseases.
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