SAWA Yoshiki Osaka University, Graduate School of Medicine, Professor, 医学系研究科, 教授 (00243220)
MIYAMOTO Yuji Hyogo College of Medicine, Faculty of Medicine, Professor, 医学部, 教授 (80229898)
FUKUSHIMA Norihide Osaka University, Graduate School of Medicine, Lecturer, 医学系研究科, 講師 (30263247)
MATSUMIYA Goro Osaka University, Graduate School of Medicine, Assistant, 医学系研究科, 助手 (20314312)
|Budget Amount *help
¥45,890,000 (Direct Cost: ¥35,300,000、Indirect Cost: ¥10,590,000)
Fiscal Year 2005: ¥13,520,000 (Direct Cost: ¥10,400,000、Indirect Cost: ¥3,120,000)
Fiscal Year 2004: ¥13,520,000 (Direct Cost: ¥10,400,000、Indirect Cost: ¥3,120,000)
Fiscal Year 2003: ¥18,850,000 (Direct Cost: ¥14,500,000、Indirect Cost: ¥4,350,000)
1.Sheet-shaped myoblast implantation in dilated cardiomyopathic hamsters
Male 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
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 model
Left 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 heart
We 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