Skreb N, Hofman L, Skreb Y, Suchanek E, Serman D
Dev Biol. 1984 Feb;101(2):367-72. doi: 10.1016/0012-1606(84)90150-7.
The modified organ culture of rat embryonic shields provides favorable conditions during 2 weeks for the differentiation of main tissue types. Since the terminal differentiation in explants is inferior to that obtained in the homografts of the same shields under the kidney capsule, we tried to improve the culture medium by adding some known regulatory molecules: db-cAMP, db-cGMP, ATP, AMP, and butyric acid. These agents were added to the liquid medium in the concentration of 1 mM. In the first part of the study the explants were fixed and weighed after 8 or 14 days in vitro culture, and histological sections were examined. When the explants were treated with db-cAMP during the second week of culture, the skeletal muscle appeared more frequently in the treated series than in controls, and the weight of the treated explants was sometimes increased when compared with the control series. The db-cGMP had no effect on differentiation, but stimulated the growth of the explants when applied during the first week of culture. On the contrary, the db-cAMP when added during the first week, severely impeded the growth of explants. Other agents seem to be ineffective. In the second part, the content of cAMP and cGMP was measured in normal explants. The radioimmunoassay showed the same content of cAMP and cGMP during the entire culture period. In the third part of our study the incorporation of tritiated uridine and tritiated thymidine was measured during the second week of culture after the addition of db-cAMP. During the first days of treatment with db-cAMP the uptake of tritiated uridine and thymidine was inhibited, whereas on the seventh day the uptake was similar to that of the control. We can conclude that both cyclic nucleotides have a visible effect on growth whereas only cAMP has a positive impact on the differentiation of myotubes in cultured rat embryonic shields.
大鼠胚胎盾的改良器官培养在两周内为主要组织类型的分化提供了有利条件。由于外植体中的终末分化不如相同盾在肾被膜下的同种移植中获得的分化，我们试图通过添加一些已知的调节分子来改进培养基：二丁酰环磷腺苷（db-cAMP）、二丁酰环磷鸟苷（db-cGMP）、三磷酸腺苷（ATP）、一磷酸腺苷（AMP）和丁酸。这些试剂以1 mM的浓度添加到液体培养基中。在研究的第一部分，外植体在体外培养8天或14天后固定并称重，并检查组织切片。当在培养的第二周用db-cAMP处理外植体时，与对照组相比，处理组中骨骼肌出现的频率更高，并且处理后的外植体重量有时会增加。db-cGMP对分化没有影响，但在培养的第一周应用时会刺激外植体的生长。相反，在第一周添加db-cAMP时，会严重阻碍外植体的生长。其他试剂似乎无效。在第二部分中，测量了正常外植体中cAMP和cGMP的含量。放射免疫测定显示在整个培养期间cAMP和cGMP的含量相同。在我们研究的第三部分中，在添加db-cAMP后的培养第二周测量了氚标记尿苷和氚标记胸腺嘧啶核苷的掺入情况。在用db-cAMP处理的最初几天，氚标记尿苷和胸腺嘧啶核苷的摄取受到抑制，而在第七天，摄取与对照组相似。我们可以得出结论，两种环核苷酸对生长都有明显影响，而只有cAMP对培养的大鼠胚胎盾中肌管的分化有积极影响。
