Development of novel myocardial regeneration therapy for severe heart failure using cellular cardiomyoplasty and gene transfection

[应用细胞性心肌成形术和基因转染法开发新的细胞治疗方法]我们采用Lewis大鼠冠状动脉左前降支(LAD)近端结扎模型。LAD结扎2周后，进行3种不同的处理：1)新生大鼠心肌细胞组(T组)，2)携带hHGF基因的HVJ脂质体组(H组)，3)联合组(T-H组)。注射部位为心肌梗死疤痕区域。对照组注射培养液(C组)。超声心动图显示，注射后4周和8周，T-H组的心功能明显改善。超声心动图也显示T-H组的心肌灌注显著增加，而其他组则无明显增加。在T-H组，组织学观察到新生血管和明显的纤维化减轻。免疫组织化学检测显示，移植后8周，T-H组大鼠心肌细胞基底膜β1-整合素、α-和β-抗肌营养不良蛋白主要呈强阳性表达，而T组则呈弱阳性表达。将TK基因永久性地转入NIH/HGF(NIH/HGF/TK)中。将LAD结扎于SCID大鼠心脏，移植4种不同材料：1)NIH/HGF，2)NIH/HGF/TK，口服GCV，3)NIH3T3，4)培养液(C组)。体外实验表明，GCV对NIH/HGF/TK细胞的增殖有较好的抑制作用。体内实验显示，移植后4周，NIH/HGF组和NIH/HGF/TK组的心功能和血管生成均显著增加。尽管NIH/HGF组有肿瘤病变，但NIH/HGF/TK组肿瘤生长完全受控。

[Exploitation of new cellular therapy using cellular cardiomyoplasty and gene transfection]We used a ligation model of proximal left anterior descending coronary artery (LAD) of Lewis rats. Two weeks after LAD ligation, three different treatments were conducted; 1) neonatal rat cardiomyocytes group (T group), 2) HVJ-liposomes bearing the hHGF gene group (H group), and 3) combined (T-H group). The injection site was the scar area of myocardial infarction. For control, culture medium was injected (C group). Echocardiography demonstrated that cardiac performance was significantly ameliorated in the T-H group four and 8 weeks after injection. Contrast echocardiography also showed a marked increase in myocardial perfusion in the T-H group but not in the other groups. In the T-H group, neovascularization and a marked reduction of fibrosis were observed histologically. In an immunohistochemical study, strong staining for beta1-integrin, alpha- and beta-dystroglycan were found principally in the basement membrane of myocytes in the T-H group 8 weeks after transplantation, although there was weak immunoreactivity in the T group.[Cellular vector and control release of growth factors]Human HGF cDNA expression plasmid was permanently transfected into NIH3T3 (NIH/HGF). Then, TK was permanently transfected into NIH/HGF (NIH/HGF/TK). After LAD was ligated in the heart of SCID rat, four different materials were transplanted; 1) NIH/HGF , 2) NIH/HGF/TK, oral administration of GCV, 3) NIH3T3 , 4) culture medium (C group). In vitro study, proliferation of NIH/HGF/TK cells was well suppressed under GCV. In vivo study, significant increase of cardiac performance and angiogenesis were observed in the NIH/HGF and the NIH/HGF/TK group 4 weeks after transplantation. Although tumorous lesions were detected in the NIH/HGF group, their growth was completely controlled in the NIH/HGF/TK group.