电脉冲介导的Tpo基因转移对正常及实验性血小板减少小鼠的促血小板生成作用

目的应用电脉冲介导的重组人血小板生成素(thTpo)基因的体内转移,探讨其对正常及实验性血小板减少小鼠的促血小板生成作用。方法应用基因重组技术,构建真核细胞高表达质粒pcDI/Tpo,将200μgpcDI/Tpo质粒注入正常及实验性血小板减少小鼠的股四头肌内,随后给予100V、1Hz、40ms电刺激6次,用RT-PCR及Westernblot方法观察rhTpo基因在正常小鼠骨骼肌内的表达,ELISA法测定小鼠血浆Tpo水平。用细胞计数板计数小鼠外周血血小板。结果成功构建了真核细胞高表达质粒pcDI/Tpo。将pcDI/Tpo转染小鼠后,在小鼠的骨骼肌内有rhTpomRNA及蛋白的表达,血浆Tpo水平由(250±76)ng/L升高至(1185±264)ng/L。正常小鼠的外周血血小板由(259±27)×10

Thrombopoietic effect of recombinant human thrombopoietin gene transferred to mice mediated by electricpulse on normal and experimental thromboeytopenia mice

OBJECTIVE: To investigate the thrombopoietic effect of recombinant human thrombopoietin (rhTpo) gene transferred by electric pulse to normal and experimental thrombocytopenia mice. METHODS: Eukaryotic high expressing plasmid pcDI/Tpo was constructed by gene recombinant technology. 200 microg of the recombinant plasmid was injected into quadriceps femoris muscle of normal and experimental thrombocytopenia mice. Six times of electric pulse at 100v, 1Hz, 40ms were given immediately after the injection.The expression of rhTpo gene and its protein were assayed by RT-PCR and Western Blotting, respectively. Serum Tpo concentration was assayed by ELISA method. RESULTS: The recombinant plasmid pcDI/Tpo was successfully constructed. The expression of mRNA and protein of rhTpo gene was detected in the skeletal muscle of mice after transfection. Serum Tpo level increased from 328 +/- 89 ng/L to 1185 +/- 264 ng/L, and the platelet level of transfected mice increased from (259 +/- 27) x 10(9)/L to (640 +/- 31) x 10(9)/L. After injection with carboplatin, the platelet level decreased, but the nadir point was higher in pcDI/Tpo group than that in control group, and the recovery time of platelet count in pcDI/Tpo group shortened. CONCLUSION: The rhTpo gene could be effectively transfected to mice by electric pulse and played thrombopoietic role in vivo.