电脉冲介导红细胞生成素基因肌肉转移治疗大鼠肾性贫血

The effects of electroporation mediated erythropoietin (EPO) gene transfer into skeleton muscle on renal anemia

目的 研究电脉冲介导的质粒红细胞生成素 (EPO)基因肌肉转移效率以及对肾性贫血的治疗作用。方法 用腺嘌呤诱导大鼠慢性肾性贫血模型 ,以电脉冲介导质粒EPO基因肌肉转移对6 0只大鼠实施基因治疗 (电脉冲实验组 ) ,治疗期间监测血红蛋白 (Hb)、红细胞压积 (HCT)和血清肌酐(Cr)、血清尿素氮 (BUN) ,并用EPO酶联免疫 (ELISA)试剂盒测定血清EPO水平 ,逆转录聚合酶链反应(RT/PCR)检测EPO基因局部表达情况。结果 电脉冲介导的EPO基因肌肉转移可在局部有效表达和分泌 ,并在 3周内使Hb和HCT分别提高 46 .5 %和 43% (Hb :84g/L± 12g/L ,12 2g/L± 2 8g/L ;HCT :2 6 %± 5 % ,38%± 9% )。 5周Hb和HCT分别上升至正常水平的 91.2 %和 86 .5 % (Hb :12 6g/L± 32g/L ,正常水平 :139g/L± 10g/L ;HCT :40 %± 11% ,正常水平 :46 %± 2 .2 % )并维持高水平至少 9周。动物 9周存活率 (77.8% )是对照组 (16 .7% )的 4倍 ,同时电脉冲实验组其Hb、HCT、EPO均显著高于非电脉冲实验组 ,并呈现一定的剂量效应。结论 电脉冲介导的质粒EPO基因肌肉转移能使EPO基因局部显著表达和分泌 ,对肾性贫血有较好的治疗作用 ,该方法具有很好的临床应用前景。

Objective To investigate the effects of erythropoietin (EPO) gene transfer into skeleton muscle mediated by electroporation on renal anemia. Methods Renal failure models were created by adenine excessive diet (150 mg per day). Plasmid vectors encoding EPO were transferred by electroporation after 80 days when mean blood urea nitrogen level (BUN) had increased from 3.4 mmol/L±1.3 mmol/L to 18.1 mmol/L±4.1 mmol/L and the hematocrit had decreased from 45.6%±2.1% to 25.4%± 3.7%. During the process of treatment, adenine excessive diet was given. Hb, HCT, BUN and Cre in blood were tested by automatic analyzer; EPO level in the serum was tested by EPO ELISA kit, EPO gene expression was proved by RT/PCR.The survival rate was caculated. Results Hematocrit increased to 34.4%±7.5% only 7 days after the treatment and reached 91.4% of normal level (46%±2%) after 5 weeks. The survival rate of test models after 9 weeks was 77.8%, which was remarkably higher than that of controls (16.7%). mRNA level of EPO gene expression was indicated by RT/PCR. Conclusion Electroporation can increase the efficiency of EPO gene transfer and thus greatly improve hematocrit in mice and prolong the life span of chronic renal anemia models. This method can provide a new way for treatment of EPO responsive anemias.