Yang E V, Gardiner D M, Carlson M R, Nugas C A, Bryant S V
Developmental Biology Center and Department of Developmental and Cell Biology, University of California, Irvine, California 92697-2275, USA.
Dev Dyn. 1999 Sep;216(1):2-9. doi: 10.1002/(SICI)1097-0177(199909)216:1<2::AID-DVDY2>3.0.CO;2-P.
One of the earliest events in limb regeneration is the extensive remodeling of the extracellular matrix (ECM). Matrix metalloproteinases (MMPs) are a family of matrix degrading enzymes that have been identified in both normal and disease states. Using RT-PCR and cDNA library screening, we have isolated sequences homologous to four different Mmp genes. The spatial and temporal expression of one of these, Mmp-9, has been analyzed during axolotl limb regeneration. Northern blot analysis identifies a 3.8 kb transcript that is abundantly expressed during regeneration, and whole-mount in situ hybridization has uncovered an unusual bi-phasic expression pattern. The first phase begins at 2 hours after amputation, and expression is confined to the healed wound epithelium. This phase continues for 2 days, showing peak expression at 14 hours after amputation. This early phase may be needed to retard reformation of the basal lamina of the epidermis, and thereby facilitate the epidermal-mesenchymal interactions required for successful regeneration. The second phase begins a few days later when a small blastema has formed. During this phase, expression is in the mesenchyme, localized to cells around the tips of the cut skeletal elements. This expression is maintained through several stages until redifferentiation begins. The timing and position of the second phase of expression is consistent with a role for Mmp-9 in the removal of damaged cartilage matrix. We have also discovered that the time of onset of Mmp-9 expression is sensitive to denervation, which causes a delay of several hours. Finally, retinoids, known for their dramatic effects on the pattern of regenerating limbs, can cause a down regulation of Mmp-9 expression. Dev Dyn 1999;216:2-9.
肢体再生过程中最早发生的事件之一是细胞外基质（ECM）的广泛重塑。基质金属蛋白酶（MMPs）是一类在正常和疾病状态下均已被鉴定出的基质降解酶。通过逆转录聚合酶链反应（RT-PCR）和cDNA文库筛选，我们分离出了与四个不同Mmp基因同源的序列。其中之一Mmp-9在蝾螈肢体再生过程中的时空表达已得到分析。Northern印迹分析鉴定出一个3.8 kb的转录本，其在再生过程中大量表达，而整装原位杂交揭示了一种不同寻常的双相表达模式。第一阶段在截肢后2小时开始，表达局限于愈合的伤口上皮。该阶段持续2天，在截肢后14小时表达达到峰值。这个早期阶段可能是为了延缓表皮基底层的重新形成，从而促进成功再生所需的表皮-间充质相互作用。第二阶段在几天后一个小芽基形成时开始。在此阶段，表达位于间充质中，定位于切断的骨骼元件尖端周围的细胞。这种表达在几个阶段中持续存在，直到重新分化开始。第二阶段表达的时间和位置与Mmp-9在去除受损软骨基质中的作用一致。我们还发现Mmp-9表达开始的时间对去神经支配敏感，这会导致数小时的延迟。最后，以其对再生肢体模式有显著影响而闻名的视黄酸，可导致Mmp-9表达下调。《发育动力学》1999年；216:2 - 9。
