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

研究了鸡胚成骨骼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。骨骼肌培养基与心肌培养基以1:1的比例混合为最佳共培养培养基。当成肌细胞和心肌细胞在共培养开始时以超过1:3的比例混合时，经常观察到骨骼肌细胞（或肌管）与心肌细胞之间的细胞接触。免疫染色显示接触区有阳性反应，提示存在某种细胞-细胞黏附装置。采用电穿孔法将报告基因导入鸡胚心脏，并考察了各种基因导入条件，获得了较好的导入基因产量。报告基因采用携带LacZ基因的pmiwZ和含有绿色荧光蛋白基因的pEGFP-C1。在肢体和心脏周围检测到这些基因的表达，但在心脏内部没有。然而，表达效率随测试条件的不同而变化。48 h的胚胎比72 h的胚胎更好。应用浓度越高（bbb10 mg/ml），表达效率越高。少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。线形电极（长5mm，直径1mm）优于针状或圆盘状电极，金电极优于铂、铜、镍铬合金电极。两个电极之间的距离也很重要，对于48 h龄的鸡胚胎，8 mm为最佳。高25v、持续时间50msec的脉冲以1 sec的间隔施加8次，获得了良好的效果。施加电压较高、持续时间较短或脉冲数量较少的脉冲会导致表达效率下降。因此，我们可以优化一些影响基因导入动物效率的条件。少

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