Establishment of Regenerative Therapy for Patients with Left Ventricular Assist Device 〜Aiming to 'Bridge to Recovery〜

1.扩张的心肌病仓鼠中的片状成肌细胞植入显示中度心脏重塑的27周龄雄性BIO TO-2（DCM）仓鼠用作受体。将从BIO F1 B仓鼠分离的成肌细胞在用聚（N-异丙基丙烯酰胺）包被的培养皿上培养。实验分为三组：（1）成肌细胞片状移植组（S组，n=29）;（2）成肌细胞注射组（M组，n=28）;（3）假手术组（C组，n=28）。在S组中，将两个片状成肌细胞移植物植入左心室（LV）壁，在M组中，将成肌细胞注入右心室（RV）和LV壁。植入片状成肌细胞移植物后，超声心动图显示扩张的LV尺寸显著减小，而其他组的心脏显示LV扩张进展。术前M组和C组的短轴缩短率显著降低，而S组的短轴缩短率维持在术前水平。 ...更多信息 术后3个月维持水平。组织学检查显示，S组左室壁厚度增加，成肌细胞存活，心肌纤维化程度减轻。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，而其他组无表达。S组平均寿命明显延长。片状成肌细胞移植物植入改善了心脏性能，延长了预期寿命，并减少了心肌纤维化和细胞骨架蛋白的重组. 12只犬以230次/min连续心室起搏4周后，将成肌细胞片（n=5）移植于左心室壁或假手术（n=7）。将细胞数量调整为1.5 × 2.5 × 10（6）个细胞/移植物，每只犬接受约20个移植物。细胞片很容易移植到大面积的左心室表面，没有严重的后遗症。移植物植入后4周，超声心动图显示左心室射血分数和缩短分数显著改善，左心室扩张减少，室壁厚度增加。组织学检查显示移植的成肌细胞存活，纤维化和凋亡明显减少，增殖明显增加.自杀基因系统调控大鼠心肌梗死后血管生成的效应我们利用单纯疱疹病毒（HerpesSimplex Virus，TK）的胸苷激酶基因和更昔洛韦（Ganciclovir，GCV）系统构建了人肝细胞生长因子（humanHGF，hHGF）产生细胞，并对其产生进行调控。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NIH 3 T3细胞。接着，用TK稳定转染NIH/HGF细胞（NIH/HGF/TK）。结扎重症联合免疫缺陷大鼠心脏左前降支，移植NIH/HGF（n=10）、NIH/HGF/TK（n=10）+GCV（n=10）、NIH 3 T3（n=10）和4）培养基（n=10）。GCV可抑制NIH/HGF/TK细胞的增殖。在体内，移植后4周，NIH/HGF和NIH/HGF/TK组中观察到心脏性能和血管生成显著增加。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。使用TK-GCV自杀基因系统的血管生成基因细胞疗法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 cardioyopathic 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 preoperati … More ve 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. Myobalst sheet implantation in pacing -induced canine modelTwelve dogs were given continuous ventricular pacing at 230 beats/min for 4weeks ; then the myoblast sheets (n=5) were grafted onto the left ventricular wall or a sham operation was performed (n=7). The number of cells was adjusted to 1.5〜2.5 x 10(6) cells per graft, and each dog received approximately 20 grafts. The cell sheets were easily grafted onto a large area of the left ventricular surface, and there were no serious sequelae. Four weeks after graft implantation, echocardiography demonstrated that the left ventricular ejection fraction and fractional shortening were significantly ameliorated with reduced left ventricular dilatation and increased wall thickness. Histologic evidence indicated the grafted myoblasts had survived, accompanied by a significant reduction in fibrosis and apoptosis, and a significant increase in proliferation.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