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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 ,并发挥其促血小板生成的作用
OBJECTIVE: To study the in vivo transfer of recombinant human thrombopoietin (rhTpo) gene by electrical pulse and investigate its effect on platelet production in normal and experimental thrombocytopenic mice. Methods Recombinant plasmid pcDI Tpo was constructed by using gene recombination technique. 200 microgpcDI Tpo plasmid was injected into the quadriceps femoris of normal and experimental thrombocytopenic mice and then stimulated with 10 0 V, 1 Hz, 40 ms electric stimulation 6 Times. The expression of rhTpo gene in skeletal muscle of normal mice was observed by RT PCR and Western blot. The level of plasma Tpo was measured by ELISA. Peripheral blood platelets were counted with a cell counting plate. Results The eukaryotic expression plasmid pcDI Tpo was successfully constructed. After transfected with pcDI Tpo, the expression of rhTpomRNA and protein in skeletal muscle of mice increased from (250 ± 76) ng L to (1185 ± 264) ng L -1. In normal mice, the platelet count of peripheral blood increased from (259 ± 2 7) × 10 9 L to (6 40 ± 31) × 10 9 L, and the lowest platelet count in mice transfused with pcDI Tpo To (138 ± 2 4) × 10 9 L, which was higher than the minimum value of [(98 ± 19) × 10 9 L] and [(92 ± 16) × 10 9 L] At the same time, the platelet recovery time advanced earlier and returned to (2 38 ± 2 6) × 10 9 L. on the 14th day. Conclusion The electrical pulse-mediated gene transfer method can effectively express rhTpo gene in mouse skeletal muscle and exert its effect of promoting platelet production