摘要
目的 应用电脉冲介导的重组人血小板生成素 (rhTpo)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 2 0 0 μgpcDI 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 10 0V、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 (2 5 0± 76 )ng L升高至 (1185± 2 6 4)ng L。正常小鼠的外周血血小板由 (2 5 9± 2 7)× 10 9 L最高升高至 (6 40± 31)× 10 9 L ;注射卡铂后 ,转pcDI Tpo组小鼠的血小板最低下降至 (138± 2 4)× 10 9 L ,比转pcDI空载体组 [(98± 19)× 10 9 L]和单纯注射卡铂组 [(92± 16 )× 10 9 L]最低值高 ,同时血小板恢复时间提前 ,第 14天已恢复至 (2 38± 2 6 )× 10 9 L。结论 应用电脉冲介导的基因转移方法 ,可使rhTpo基因在小鼠骨骼肌内有效地表达 。
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 μg 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±89ng/L to 1185±264 ng/L,and the platelet level of transfected mice increased from(259±27)×10 9/L to (640±31)×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.
出处
《中华血液学杂志》
CAS
CSCD
北大核心
2001年第3期128-131,共4页
Chinese Journal of Hematology
基金
"九五"科技攻关基金资助项目!(96 90 6 0 1 19)
