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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开发新型心肌再生疗法治疗严重心力衰竭

基本信息

批准号：
13470274
负责人：
SAWA Yoshiki
金额：
$ 10.62万
依托单位：
OSAKA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

项目摘要

[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.

[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。将TK基因永久转染NIH/HGF（NIH/HGF/TK）细胞。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。