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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 ,为今后TPO基因治疗的研究奠定了实验基础
In order to observe the expression of recombinant thrombopoietin (TPO) gene in COS-7 cells and in mice, a recombinant eukaryotic expression plasmid pcd2 TPO containing TPO gene was constructed by DNA recombination technique and transfected into PC12 cells by lipofectamine COS 7 cells, pcd2 TPO plasmid was transferred to mouse skeletal muscle by direct injection of naked plasmid DNA with electric pulse. The transient expression of TPO gene in COS 7 cells was detected by RT PCR and ELISA. MTT assay showed that TPO gene could stimulate the proliferation of TPO-dependent cell lines. The expression of TPO gene in skeletal muscle of transgenic mice was detected by RT-PCR and immunohistochemistry. The level of TPO in transgenic mice was (1185 ± 2 64) ng L, which was significantly higher than that of normal mice Concentration (250 ± 76) ng L. In this experiment, the transfer of TPO gene in COS7 cells in mice was established, which laid the experimental foundation for the study of TPO gene therapy in the future