Maruyama H, Sugawa M, Moriguchi Y, Imazeki I, Ishikawa Y, Ataka K, Hasegawa S, Ito Y, Higuchi N, Kazama J J, Gejyo F, Miyazaki J I
Department of Medicine II, Niigata University School of Medicine, Japan.
Hum Gene Ther. 2000 Feb 10;11(3):429-37. doi: 10.1089/10430340050015897.
It has been demonstrated that gene transfer by in vivo electroporation of mouse muscle increases the level of gene expression by more than 100-fold over simple plasmid DNA injection. We tested continuous rat erythropoietin (Epo) delivery by this method in normal rats, using plasmid DNA expressing rat Epo (pCAGGS-Epo) as the vector. A pair of electrodes was inserted into the thigh muscles of rat hind limbs and 100 microg of pCAGGS-Epo was injected between the electrodes. Eight 100-V, 50-msec electric pulses were delivered through the electrodes. Each rat was injected with a total of 400 microg of pCAGGS-Epo, which was delivered to the medial and lateral sides of each thigh. The presence of vector-derived Epo mRNA at the DNA injection site was confirmed by RT-PCR. The serum Epo levels peaked at 122.2 +/- 33.0 mU/ml on day 7 and gradually decreased to 35.9 +/- 18.2 mU/ml on day 32. The hematocrit levels increased continuously, from the preinjection level of 49.5 +/- 1.1 to 67.8 +/- 2.2% on day 32 (p < 0.001). In pCAGGS-Epo treated rats, endogenous Epo secretion was downregulated on day 32. In a control experiment, intramuscular injection of pCAGGS-Epo without subsequent electroporation did not significantly enhance the serum Epo levels. These results demonstrate that muscle-targeted pCAGGS-Epo transfer by in vivo electroporation is a useful procedure for the continuous delivery of Epo.
已证明，通过对小鼠肌肉进行体内电穿孔进行基因转移，与单纯注射质粒DNA相比，基因表达水平提高了100倍以上。我们使用表达大鼠促红细胞生成素（Epo）的质粒DNA（pCAGGS-Epo）作为载体，通过这种方法在正常大鼠中测试了大鼠促红细胞生成素（Epo）的持续递送。将一对电极插入大鼠后肢的大腿肌肉中，并在电极之间注射100μg的pCAGGS-Epo。通过电极施加八个100V、50毫秒的电脉冲。每只大鼠共注射400μg的pCAGGS-Epo，分别注射到每条大腿的内侧和外侧。通过RT-PCR证实了DNA注射部位存在载体衍生的Epo mRNA。血清Epo水平在第7天达到峰值，为122.2±33.0 mU/ml，并在第32天逐渐降至35.9±18.2 mU/ml。血细胞比容水平持续升高，从注射前的49.5±1.1%升至第32天的67.8±2.2%（p<0.001）。在pCAGGS-Epo处理的大鼠中，内源性Epo分泌在第32天被下调。在对照实验中，肌肉注射pCAGGS-Epo而不进行后续电穿孔并没有显著提高血清Epo水平。这些结果表明，通过体内电穿孔进行肌肉靶向pCAGGS-Epo转移是一种持续递送Epo的有用方法。
