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-I和胰岛素样生长因子-II信使RNA的表达类型。在软骨组织中，除1类外，其余各类IGF-II基因均有表达。另一方面，包括第一类在内的所有类都在肝脏中表达。采用逆转录-聚合酶链式反应(RT-PCR)定量检测20~40个循环的软骨组织中IGF-I和IGF-II的表达。此外，IGF-I在软骨中的表达明显低于IGF-II，提示IGF-II在生长软骨中是一种重要的旁分泌因子。细胞在小室中增殖，直到10d，然后细胞形态发生变化，并刺激尿酸的积累，这是一个软骨活动的指标。植入60d后，这些分化的软骨细胞大部分被占据在腔内。然后，分别饲喂高蛋白饲料或低蛋白饲料，添加或不添加旁路蛋氨酸。然后，将放置分离的软骨细胞的房间植入这些羔羊的皮下。在绵羊饲喂高蛋白日粮时，添加蛋氨酸可促进细胞增殖。60d后各组尿酸含量均有升高，但组间无明显差异。当羔羊饲喂不含蛋氨酸的低蛋白日粮时，IGF-I的mRNA含量增加。其余各组IGF-I mRNA表达均降低。体内孵育过程中IGF-II的mRNA含量没有变化，也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.