Myocardial regeneration therapy using nanotechnology enhances self-regeneration in damaged myocardium

1.以27周龄、表现中度心脏重构的BIO -2 （DCM）小仓鼠为受体。从BIO F1B仓鼠中分离的成肌细胞在聚n -异丙基丙烯酰胺包被的培养皿中培养。采用三种不同的治疗方法：(1)片状成肌细胞移植（S组，n=29）；(2)成肌细胞注射（M组，n=28）；假手术组（C组，n=28）。S组在左心室（LV）壁上植入2块片状成肌细胞移植物，M组在右心室（RV）和左室壁上注射成肌细胞。片状成肌细胞移植后，超声心动图显示扩张的左室尺寸明显减小，而其他各组心脏则表现为左室扩张的进展。术后3个月，M组、C组短缩率明显下降，S组短缩率维持在术前水平。组织学检查显示，与其他各组相比，S组左室壁厚度增加，成肌细胞存活，心肌纤维化减少。免疫组化染色显示，S组心肌细胞基底膜上有α -肌聚糖和β -肌聚糖表达，其他各组无。S组患者预期寿命明显延长。在DCM仓鼠中，片状成肌细胞移植可改善心脏功能并延长预期寿命，这与心肌纤维化和细胞骨架蛋白重组的减少有关。大鼠心肌梗死模型成肌细胞片植入左冠状动脉前降支结扎Lewis大鼠心脏（2周），通过成肌细胞注射或植入2个单层构建的成肌细胞片（每张5 x 10(6)个细胞）或通过培养基注射，接受1 × 10(7)个自体骨骼肌细胞移植。超声心动图结果显示，直到细胞移植后8周，成肌细胞片组心脏功能的改善高于其他组。组织学比较显示，仅在成肌细胞片组心脏中，在明显均匀增厚的壁内，细胞更丰富，新毛细血管广泛分布。与骨成肌细胞注射相比，骨成肌细胞片植入可显著减少纤维化。心肌成肌细胞薄片组心肌梗死区造血干细胞（c-kit、干细胞抗原1和CD34）明显增多。逆转录聚合酶链反应结果显示基质衍生因子1、肝细胞生长因子和血管内皮生长因子的表达如下：成肌细胞片>成肌细胞注射>对照。成肌细胞片修复受损的心肌，减少纤维化，并通过释放基质衍生因子1和其他生长因子来阻止与造血干细胞募集相关的重塑。自杀基因系统调控梗死大鼠心脏血管生成的作用我们利用单纯疱疹病毒（TK）胸苷激酶基因和更昔洛韦（GCV）系统制备了人类HGF （hHGF）产生细胞，该细胞调节hHGF的产生。我们测试了这些细胞是否诱导和调节梗死心肌的血管生成作用。用hHGF cDNA表达质粒（NIH/HGF）稳定转染NIH3T3细胞。接着，用TK （NIH/HGF/TK）稳定转染NIH/HGF细胞。结扎重度联合免疫缺陷大鼠心脏左前降支，移植4种材料：1)NIH/HGF (n=10), 2)NIH/HGF/TK，同时口服GCV （n=10), 3)NIH3T3 (n=10), 4）培养基（n=10）。在体外，GCV可抑制NIH/HGF/TK细胞的增殖。在体内，移植后4周，NIH/HGF组和NIH/HGF/TK组心脏功能和血管生成显著增加。NIH/HGF组虽然检测到肿瘤病灶，但在NIH/HGF/TK组肿瘤病灶的生长完全受到控制。利用TK-GCV自杀基因系统进行血管生成基因细胞治疗，在细胞生长的控制下诱导和调节血管生成。少

1.Sheet-shaped myoblast implantation in dilated cardiomyopathic hamstersMale 27-week-old BIO TO-2 (DCM) hamsters that showed moderate cardiac remodeling were used as recipients. Myoblasts isolated from BIO F1B hamsters were cultured on dishes coated with poly(N-isopropylacrylamide). Three different therapies were conducted : (1)sheet-shaped myoblast graft implantation (S group, n=29) ; (2)myoblast injection (M group, n=28) ; and (3)sham operation (C group, n=28). In the S group, two sheet-shaped myoblast grafts were implanted on the left ventricle (LV) wall, and in the M group, myoblasts were injected into the right ventricle (RV) and LV walls. After the sheet-shaped myoblast grafts were implanted, echocardiography demonstrated that the dilated LV dimension was significantly reduced, whereas the hearts in other groups showed a progression of LV dilation. The fractional shortening in the M and C groups decreased significantly while that in the S group was maintained at the preoperative … More level for 3 months after the operation. Histological examination demonstrated that in the S group, the LV wall thickness was increased, with viable myoblasts, and myocardial fibrosis was decreased compared with the other groups. Immunohistochemical staining demonstrated alpha-sarcoglycan and beta-sarcoglycan expression on the basement membrane of the cardiomyocytes in the S group but not in the other groups. The life expectancy was significantly prolonged in the S group. Sheet-shaped myoblast graft implantation improved cardiac performance and prolonged life expectancy, associated with a reduction in myocardial fibrosis and re-organization of the cytoskeletal proteins in DCM hamsters.2.Myoblast sheet implantation in rat myocardial infarction modelLeft anterior descending coronary artery-ligated Lewis rat hearts (2 weeks) received 1 x 10(7) autologous skeletal myoblasts by means of cell transplantation either through myoblast injection or implantation of 2 monolayer-constructed myoblast sheets (5 x 10(6) cells per sheet) or through medium injection. Echocardiographic results indicated higher improvement of cardiac performance in the myoblast sheet group than among the other groups until 8 weeks after cell transplantation. Histologic comparison revealed greater cellularity and abundant widespread neocapillaries within the noticeable uniform thickened wall in myoblast sheet group hearts only. Fibrosis was substantially reduced with skeletal myoblast sheet implantation compared with skeletal myoblast cell injection. Obviously higher numbers of hematopoietic stem cells (c-kit, stem cell antigen 1, and CD34) were observed in the myoblast sheet group infarct heart region. Reverse transcription-polymerase chain reaction results showed expression of stromal-derived factor 1, hepatocyte growth factor, and vascular endothelial growth factor as follows : myoblast sheets > myoblast injection > control. Myoblast sheets repaired the impaired myocardium, reduced fibrosis, and prevented remodeling in association with recruitment of hematopoietic stem cells through the release of stromal-derived factor 1 and other growth factors.3.Suicide gene system regulates the effect of angiogenesis in infarcted rat heartWe developed human HGF (hHGF)-producing cells that regulated hHGF production using the thymidine kinase gene of Herpes Simplex Virus (TK) and the Ganciclovir (GCV) system. We tested whether these cells induced and regulated angiogenic effects in infarcted myocardium. NIH3T3 cells were stably transfected with an hHGF cDNA expression plasmid (NIH/HGF). Next, the NIH/HGF cells were stably transfected with TK (NIH/HGF/TK). The left anterior descending artery was ligated in the heart of severe combined immunodeficiency rats, and four materials were transplanted : 1)NIH/HGF (n=10), 2)NIH/HGF/TK, with orally administered GCV (n=10), 3)NIH3T3 (n=10), and 4)culture medium (n=10). In vitro, the proliferation of NIH/HGF/TK cells was suppressed by GCV. In vivo, significant increases in cardiac performance and angiogenesis were observed in the NIH/HGF and NIH/HGF/TK groups 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. Angiogenic gene cell therapy using the TK-GCV suicide gene system induces and regulates angiogenesis under the control of cell growth. Less