Fitch J, Fini M E, Beebe D C, Linsenmayer T F
Department of Anatomy and Cellular Biology, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
Dev Dyn. 1998 May;212(1):27-37. doi: 10.1002/(SICI)1097-0177(199805)212:1<27::AID-AJA3>3.0.CO;2-4.
A critical event in avian corneal development occurs when the acellular primary stroma swells and becomes populated by mesenchymal cells that migrate from the periphery. These cells then deposit the mature stromal matrix that exhibits the unique features necessary for corneal function. Our previous work correlated the disappearance of collagen type IX immunoreactivity at stage 27 (5 1/2-6 days) with matrix swelling and invasion. To investigate further the mechanism of this disappearance, we employed immunohistochemistry after tissue fixation with Histochoice, a non-crosslinking fixative, immunoblot analysis of protein extracts, and gel substrate chromatography (zymography) to detect endogenous proteolytic activity. We found that corneas fixed in Histochoice retain immunoreactivity for type IX collagen for 1-2 days after corneal swelling. This immunoreactivity, however, becomes extractable from tissue sections of unfixed corneas at the time of initiation of stromal swelling and mesenchymal cell invasion. Immunoblot analysis confirmed that, following swelling, immunoreactivity for collagen IX decreased substantially in corneas, but not in the vitreous body, which served as a comparison. Analysis of ammonium sulfate (AS) fractions of such extracts indicated that, at the time of swelling, much of the immunoreactivity for type IX collagen in cornea shifted from the AS precipitate (containing high molecular weight molecules) to the AS supernatant (containing smaller fragments). In contrast, collagen IX immunoreactivity from the vitreous was precipitated by ammonium sulfate throughout the period of study. Collagen type II, a major fibrillar collagen in both the corneal stroma and vitreous, remained in the high molecular weight fraction at all times examined. Zymography detected the presence of the latent (proenzyme) form of gelatinase A (MMP-2) before corneal swelling and invasion (4 days), and both the latent and active forms of the enzyme after corneal swelling. This suggests tissue-specific, developmentally regulated proteolysis of collagen IX as a trigger for corneal matrix swelling.
鸟类角膜发育过程中的一个关键事件发生在无细胞的初级基质肿胀并被从周边迁移而来的间充质细胞占据时。这些细胞随后沉积成熟的基质，该基质展现出角膜功能所需的独特特征。我们之前的研究发现，在第27阶段（5.5 - 6天），IX型胶原蛋白免疫反应性的消失与基质肿胀和侵入相关。为了进一步探究这种消失的机制，我们使用了用非交联固定剂Histochoice固定组织后的免疫组织化学、蛋白质提取物的免疫印迹分析以及凝胶底物层析（酶谱法）来检测内源性蛋白水解活性。我们发现，用Histochoice固定的角膜在角膜肿胀后1 - 2天仍保留IX型胶原蛋白的免疫反应性。然而，在基质肿胀和间充质细胞侵入开始时，这种免疫反应性可从未固定角膜的组织切片中提取出来。免疫印迹分析证实，肿胀后，角膜中IX型胶原蛋白的免疫反应性大幅下降，但作为对照的玻璃体中则没有。对此类提取物的硫酸铵（AS）分级分析表明，在肿胀时，角膜中IX型胶原蛋白的大部分免疫反应性从AS沉淀物（包含高分子量分子）转移到了AS上清液（包含较小片段）。相比之下，在整个研究期间，玻璃体中的IX型胶原蛋白免疫反应性都被硫酸铵沉淀。II型胶原蛋白是角膜基质和玻璃体中的主要纤维状胶原蛋白，在所有检测时间都保留在高分子量部分。酶谱法检测到在角膜肿胀和侵入之前（4天）存在明胶酶A（基质金属蛋白酶-2，MMP - 2）的潜伏（酶原）形式，角膜肿胀后则同时存在该酶的潜伏形式和活性形式。这表明IX型胶原蛋白的组织特异性、发育调控的蛋白水解是角膜基质肿胀的触发因素。
