2000 Fiscal Year Final Research Report Summary
Experimental study on regeneration of infarcted myocardium by transplantation of cultured self-myoblasts.
| Project/Area Number |
10557066
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| Research Category |
Grant-in-Aid for Scientific Research (B).
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| Allocation Type | Single-year Grants |
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| Section | 展開研究 |
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| Research Field |
Circulatory organs internal medicine
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| Research Institution | Chiba University |
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Principal Investigator |
MASUDA Yoshiaki Chiba University, School of Medicine, Professor, 医学部, 教授 (00009490)
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| Co-Investigator(Kenkyū-buntansha) |
KOMIYAMA Masatoshi Chiba University, School of Medicine, Assistant, 医学部, 助手 (70175339)
TOYOTA Naoji Chiba University, School of Medicine, Lecturer, 医学部, 講師 (00188822)
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| Project Period (FY) |
1998 – 2000
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| Keywords | skeletal muscle / cardiac muscle / culture / transplantation / regeneration / myoprotein / gene introduction / electroporation |
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| Research Abstract |
Development and growth of embryonic chicken skeletal myoblasts and cardiac muscle cells were investigated in co-culture system, and some of co-culture conditions were optimized. Mixture of culture medium for skeletal muscle and that for cardiac muscle at 1 : 1 was optimal as co-culture medium. Cell-cell contact between skeletal myoblasts (or myotube) and cardiac muscle cells was frequently observed when myoblasts and cardiomyocytes were mixed at the ratio of more than 1 : 3 upon start of co-culture. Immunostaining of vinculin revealed positive reaction at the contact area, suggesting the existence of some cell-cell adhesion apparatus.
We introduced reporter genes into chicken embryos aiming at their hearts by the electroporation method, and examined various conditions of gene introduction to obtain a good yield of introduced genes. As reporter genes, pmiwZ carrying LacZ gene and pEGFP-C1 containing green fluorescence protein gene were used. Expression of these genes was detected in limb
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buds and around the heart, but not within the heart. However, expression efficiency varied in accordance with conditions tested. Embryos of 48 h old were better than those of 72 h old. Higher concentration of applied DNA (>10 mg/ml) yielded higher efficiency of expression. Minimal amount of phosphate buffered saline covering both the embryo and the tip of electrodes was necessary to get expression. Linear-shaped electrode (5 mm in length and 1 mm in diameter) was better than pinpoint or disk-shaped one, and gold was better than platinum, copper or Nichrome as its material. Distance between two electrodes was also important and 8 mm was optimal for 48 h old chicken embryos. Good results were obtained when pulses of 25 V in height and 50 msec in duration were applied 8 times with 1 sec intervals. Application of pulses with higher voltage, shorter duration or less number resulted in decrease of expression efficiency. Thus, we could optimize some of these conditions which affect the efficiency of gene introduction into animals. Less
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