重组人血小板生成素基因在COS-7细胞及在小鼠体内的转移与表达

Transfer and Expression of Recombinant Human Thrombopoietin Gene in COS-7 Cells and Mice In Vivo

为了观察重组血小板生成素 (thrombopoietin ,TPO)基因在COS 7细胞及在小鼠体内的表达 ,应用DNA重组技术构建了含TPO基因的真核表达质粒 pcd2 TPO ,用脂质体法将其转染COS 7细胞 ,用裸质粒DNA直接注射加电脉冲的方法将 pcd2 TPO质粒转移到小鼠骨骼肌中。用RT PCR及ELISA法检测到TPO基因在COS 7细胞的瞬时表达 ,MTT法显示其有刺激TPO依赖细胞系增殖的活性。RT PCR及免疫组织化学染色可检测到TPO基因在转基因小鼠骨骼肌的表达 ,ELISA法定量检测转基因组小鼠血清TPO浓度为 (1185± 2 64)ng L ,明显高于正常小鼠的TPO浓度 (2 5 0± 76)ng L。本实验实现了TPO基因在小鼠体内和COS 7细胞的转移 。

In order to investigate the expression of recombinant TPO gene in COS-7 cells and in vivo of the mouse model, eukaryotic expressing plasmid pcd2/TPO with human TPO cDNA was constructed with DNA recombinant techniques. The plasmid pcd2/TPO was transiently transfected into the COS-7 cells by means of lipofection, the naked pcd2/TPO plasmid was injected into the skeletal muscle of mice with electric pulses. RT-PCR and ELISA methods were used to detect the TPO expression of the transfected COS-7 cells, both showed high level expression. The MTT test showed the expressed TPO had proliferative activity to TPO-dependent cell line. High efficiency of gene transfer in transgenic mice was also observed by RT-PCR and immunohistochemical methods. The serum TPO level〔(1 185±264) ng/L〕 in transgenic mice was quite different compared with the normal mice〔(250±76) ng/L〕. All these results provided solid foundations for the research of TPO gene therapy in the future.