CELL-BIOLOGICAL STUDIES ON AMINO ACID NUTRITION AND BONE GROWTH IN SHEEP

用RT-PCR方法检测了10日龄羔羊生长软骨和肝脏中胰岛素样生长因子（IGF）-Ⅰ和-Ⅱ mRNA的表达类型。在软骨中，所有类的IGF-II mRNA的类1以外的表达。另一方面，包括类别1在内的所有类别都在肝脏中表达。采用20 ~ 40个循环的RT-PCR方法定量检测软骨中IGF-Ⅰ和IGF-Ⅱ mRNA的表达。另外，在软骨中IGF-Ⅰ mRNA的表达量明显低于IGF-Ⅱ mRNA的表达量，提示IGF-Ⅱ是软骨生长过程中重要的旁分泌因子。细胞在腔室中增殖，直到植入10天，然后细胞发生形态学变化，并刺激糖醛酸的积累，糖醛酸是软骨活性的指标。植入后60天，这些分化的软骨细胞大量占据小室内部，然后饲喂高蛋白饲料或低蛋白饲料（添加或不添加蛋氨酸）。然后将分离的软骨细胞植入这些羔羊的皮下。在高蛋白日粮中添加蛋氨酸可促进细胞增殖。糖醛酸含量在60天时均有所增加，但各组间差异不明显。当饲喂不含蛋氨酸的低蛋白日粮时，羔羊IGF-I mRNA含量增加。而其他各组IGF-I mRNA表达均降低。IGF-II mRNA的含量在体内孵育过程中没有变化，不受蛋白质营养平面差异的影响。

The expressed classes of messenger RNA of Insulin-like growth factor (IGF) -I and -II were determined in the growth cartilage and the liver of 10 days old lambs using RT-PCR.Three classes of IGF-I mRNA were found in either tissues. In the cartilage, all the classes of IGF-II mRNA other than class 1 were expressed. On the other hand, all the classes including class 1 were expressed in the liver. IGF-I and -II mRNAs were quantitatively determined by RT-PCR in the cartilage when PCR of 20 to 40 cycle was adopted. In addition, it was clear that the expression of IGF-I mRNA was less than that of IGF-II in the cartilage, which suggested that IGF-II is an important paracrine factor in the growth cartilage.Isolated chondrocytes from the growth plate of lambs were placed into the diffusion chambers then the chambers were subcutaneously implanted into lambs. The cells were proliferated in the chamber till 10 days of implantation then the cells were morphologically changed and accumulation of uronic acid, which is an index of cartilage activity, was stimulated. On 60 days of implantation, these differentiated chondrocytes were largely occupied the inside of chamber.Then lambs were fed a high protein diet or low protein diet with or without by-passed methionine. Then the chambers placed isolated chondrocytes were subcutaneously implanted into these lambs. The cellular proliferation was stimulated by addition of the methionine when sheep were fed the high protein diet. The contents of uronic acid was increased in all of the group by 60 days but there was not clear difference among the groups. When lambs were fed the low protein diet without the methionine, IGF-I mRNA content was increased. However, IGF-I mRNA was decreased in the other groups. IGF-II mRNA content was not changed during the in vivo incubation and was not affected by the difference of nutritional plane of protein.