Novel Therapy for Heart Failure of cardiomyopathic Hamster with Gene Transfection of Hepatocyte Growth Factor

1)我们将大鼠肝细胞生长因子CDNA （rHGF）整合到具有β-actin启动子和肌细胞亲和力的PCAGGS质粒载体（PCAGGS-rHGF）中。同样，我们制作了PCAGGS-human HGR，我们可以单独确认rHGF的表达，并将其用于实验。2)单纯超声对rHGF转染无促进作用。微泡超声处理使大鼠心肌培养细胞向培养基中分泌HGF。3)我们尝试将基因直接注入心肌病仓鼠的心脏。报告基因LacZ在心肌中被转染。HGF蛋白未排泄到外周血中。4)在仓鼠骨骼肌内电穿孔LacZ和白细胞介素5成功转染了这些基因。我们采用该方法，6个脉冲100V 50 ms，作为标准的体内转染方法。5)在心肌病仓鼠to -2胫骨前肌内电灌注rHGF，剂量分别为200、400、800 μg。剂量为800 μg时HGF血药浓度最高。该剂量用标准体内转染法证明HGF浓度持续升高超过5 ng/mL。与正常对照组和安慰剂对照组相比，hgf治疗仓鼠的心脏功能通过回声多普勒测量得到改善。与给予安慰剂的仓鼠相比，hgf处理的仓鼠心肌纤维化受到抑制，毛细血管密度增加。MMP-1活性的增加可能归因于HGF治疗心肌病仓鼠心力衰竭的有益作用的可能机制之一（提交）。

1) We integrated rat Hepatocyte growth factor CDNA (rHGF) into PCAGGS plasmid vector, which has β-actin promoter and affinity to muscle cells (PCAGGS-rHGF). Likewise, we made PCAGGS-human HGR We could confirm expression of rHGF alone, and used this forthe experiment.2) Ultrasound alone did not enhance transfection of rHGF. Sonoporation wrth microbubble made rat myocardial culture cells excrete HGF into medium.3) We tried direct injection of genes into the heart of cardiomyopathic hamster. Reporter gene LacZ was found to be transfected in the myocaraium. There was no excretion of HGF protein into peripheral blood.4) In vivo electroporation of LacZ and interleukin 5 into skeletal musde of hamster successfully transfected these genes. We employed this method, 6 pulses of 100V 50 ms, as a standard in vivo transfection method.5) We tried in vivo electroponation of rHGF into tibialis anterior musde of cardiomyopathic hamsterTO-2 using the dose of 200, 400 and 800 μg. Dose of 800 μg. resulted into maximum blood concentration of HGF. This dose with standard in vivo transfection method turned out to be a sustained elevation of HGF concentration over 5 ng/mL. Cardiac function estimated by echo-Doppler improved in HGF-treated hamsters compared with normal control and placebo given hamsters. Myocardial fibrosis was suppressed and capillary density was increased in HGF-treated hamsters compared with placebo-given hamsters. Increase in MMP-1 activity might be attributable to one of possible mechanisms of beneficial effects of HGF treatment for heart failure in cardiomyopathic hamsters (submitted).