Regulation Of Skeletal Growth

纵向骨生长发生在生长板，一薄层软骨，由三个主要区域组成：静止区，增殖区和肥大区。随着年龄的增长，生长板软骨细胞增殖减慢，导致纵向骨生长减慢并最终停止。我们以前的研究表明，增殖减慢，因为干细胞样细胞，位于生长板的休息区，有一个有限的增殖能力，逐渐耗尽。当许多类型的动物细胞被置于原代细胞培养中时，也会发生类似的复制性衰老，这种效应被称为海弗利克现象。然而，我们发现，兔静止区软骨细胞在培养中的群体倍增数并不依赖于从其中收获细胞的动物的年龄，这表明限制生长板软骨细胞在体内的复制能力的机制是不同的，从那些负责限制在体外复制。 复制性衰老可能部分由表观遗传变化介导，包括基因组DNA甲基化的丧失。生长板软骨细胞在体外暴露于去甲基化剂，经历终末分化。此外，SNF2样基因PASG（DNA甲基化的正常表达所需的基因）的破坏导致生长迟缓和过早衰老。为了研究这种可能的机制，我们测量了不同年龄兔生长板软骨中基因组DNA甲基化的总体水平。我们发现生长板衰老与静止区软骨细胞DNA甲基化的丧失有关。在增殖区和肥大区软骨细胞中观察到类似的甲基化随年龄的丢失，增殖区和肥大区软骨细胞被认为是静止区软骨细胞的后代。然而，在每个年龄段内，生长板不同区域之间的DNA甲基化水平没有显著差异。因此，甲基化的丢失似乎特别发生在静止区软骨细胞的复制过程中，而不是在增殖区软骨细胞更快速的增殖过程中。这一发现表明，甲基化的丧失可能与导致软骨细胞复制随年龄增长而减慢的时间限制有关，但与导致软骨细胞增殖随着细胞进一步下降而减慢的空间限制无关。

Longitudinal bone growth occurs at the growth plate, a thin layer of cartilage which consists of three principal zones: the resting zone, the proliferative zone, and the hypertrophic zone. With age, growth plate chondrocyte proliferation slows down, causing longitudinal bone growth to slow and eventually stop. Our previous studies suggest that proliferation slows because stem-like cells, located in the resting zone of the growth plate, have a finite proliferative capacity which is gradually exhausted. Similar replicative senescence occurs when many types of animal cells are placed in primary cell culture, an effect known as the Hayflick phenomenon. However, we found that the number of population doublings of rabbit resting zone chondrocytes in culture did not depend on the age of the animal from which the cells were harvested, suggesting that the mechanisms limiting replicative capacity of growth plate chondrocytes in vivo are distinct from those responsible for limiting replication in vitro. Replicative senescence may be mediated in part by epigenetic changes, including loss of genomic DNA methylation. Growth plate chondrocytes exposed to a demethylating agent in vitro, undergo terminal differentiation. Furthermore, disruption of the SNF2-like gene, PASG, which is required for normal maintainance of DNA methylation, results in growth retardation and premature aging. To investigate this possible mechanism, we measured the overall level of genomic DNA methylation in growth plate cartilage from rabbits of different ages. We found that growth plate senescence is associated with a loss of DNA methylation in resting zone chondrocytes. A similar loss of methylation with age was observed in the proliferative and hypertrophic zone chondrocytes which are thought to be progeny of the resting zone chondrocytes. However, within each age, there was no significant difference in the level of DNA methylation between the different zones of the growth plate. Therefore, loss of methylation appears to occur specifically during replication of resting zone chondrocytes but not during the more rapid proliferation of proliferative zone chondrocytes. This finding suggests that loss of methylation might be involved in the temporal limits that cause chondrocyte replication to slow with age but not the spatial limits that cause chondrocyte proliferation to slow as the cells descend farther down the chondrocyte columns.