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Experimental study on regeneration of infarcted myocardium by transplantation of cultured self-myoblasts.

培养自体成肌细胞移植促进梗死心肌再生的实验研究

基本信息

批准号：
10557066
负责人：
MASUDA Yoshiaki
金额：
$ 2.24万
依托单位：
Chiba University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B).
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-10557066/
关键词：
skeletal muscle cardiac muscle culture transplantation regeneration myoprotein gene introduction electroporation 接着

项目摘要

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 … More 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

研究了鸡胚骨骼肌成肌细胞和心肌细胞在共培养体系中的发育和生长情况，并对共培养条件进行了优化。共培养液以骨骼肌和心肌培养基1：1混合为宜。当成肌细胞和心肌细胞在共培养开始时以大于1：3的比例混合时，经常观察到骨骼肌成肌细胞（或肌管）和心肌细胞之间的细胞-细胞接触。黏着斑蛋白免疫染色显示接触区呈阳性反应，表明细胞间存在一定的粘附装置。我们采用电穿孔法将报告基因以鸡胚心脏为靶点导入鸡胚，并对各种基因导入条件进行了考察，以获得较好的基因导入率。作为报告基因，使用携带LacZ基因的pmiwZ和包含绿色荧光蛋白基因的pEGFP-C1。这些基因的表达检测肢体 ...更多信息芽和周围的心脏，但不是在心脏。然而，表达效率根据测试条件而变化。48 h胚的发育效果好于72 h胚。较高的DNA浓度（>10 mg/ml）产生较高的表达效率。最小量的磷酸盐缓冲盐水覆盖胚胎和电极尖端是获得表达所必需的。直线形电极（长5 mm，直径1 mm）优于针尖形或圆盘形电极，金电极优于铂、铜或镍铬合金电极。两电极之间的距离也很重要，对于48 h龄的鸡胚，8 mm是最佳的。当以1秒的间隔施加8次高度为25 V、持续时间为50 msec的脉冲时，获得了良好的结果。施加较高电压、较短持续时间或较少次数的脉冲导致表达效率降低。因此，我们可以优化这些影响基因导入动物效率的条件中的一些。少