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

[利用细胞心肌成形术和基因转染技术开发新的细胞疗法]采用刘易斯大鼠左前降支（LAD）近端结扎模型。LAD结扎后2周，进行三种不同的处理：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），和3）组合（T-H组）。注射部位为心肌梗死瘢痕区。对照组注射培养液（C组）。超声心动图显示T-H组在注射后4周和8周心脏功能明显改善。对比超声心动图也显示T-H组的心肌灌注显著增加，但其他组没有。在T-H组中，组织学上观察到新生血管形成和纤维化显著减少。在免疫组化研究中，β 1-整联蛋白，α-和β-肌营养不良蛋白聚糖的强染色主要在移植后8周的T-H组中的肌细胞的基底膜中发现，虽然在T组中有弱的免疫反应性。[细胞载体和生长因子的控制释放]将人HGF cDNA表达质粒永久转染入NIH 3 T3（NIH/HGF）中。然后将TK永久转染至NIH/HGF（NIH/HGF/TK）中。在SCID大鼠心脏内结扎LAD后，移植4种不同材料：1）NIH/HGF，2）NIH/HGF/TK，口服GCV，3）NIH 3 T3，4）培养液（C组）。体外实验表明，GCV对NIH/HGF/TK细胞的增殖有明显的抑制作用。在体内实验中，NIH/HGF组和NIH/HGF/TK组移植后4周心脏功能和血管生成明显增加。尽管在NIH/HGF组中检测到肿瘤病变，但在NIH/HGF/TK组中它们的生长被完全控制。

[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.