Ishizaki M, Zhu G, Haseba T, Shafer S S, Kao W W
Department of Ophthalmology, University of Cincinnati, OH 45267-0527.
Invest Ophthalmol Vis Sci. 1993 Nov;34(12):3320-8.
Alkali-burned corneas can seldom heal properly to restore corneal transparency. To provide a better understanding of this devastating corneal injury, we compared the expression of collagen I, smooth muscle alpha-actin (alpha-SMA), and vimentin in lacerated and alkali-burned rabbit corneas.
A radiolabeled cDNA probe of alpha 1(I) chain was used in slot-blot hybridization to determine the levels of alpha 1(I) mRNA in alkali-burned corneas. In situ hybridization was used to identify the cell types that express the alpha 1(I) chain. Antibodies against collagen I, alpha-SMA, and vimentin were used in immunohistochemical studies to determine the tissue distribution of collagen I and to identify cells expressing alpha-SMA and vimentin.
The levels of alpha 1(I) mRNA in alkali-burned corneas increased steadily after the alkali burn and reached a plateau within 2 weeks. One day after alkali burn, specific in situ hybridization signals were detected in stromal cells immediately surrounding the edge of the corneal injury. As the healing proceeded, the fibroblastic cells migrated into the injured stroma, and they showed positive reactions by in situ hybridization and by immunostaining with anti-collagen I probes. In alkali-burned corneas, retrocorneal membranes were formed 1 week after injury. This fibrillar membrane was stained by anti-collagen I antibody, and the fibroblastic cells in the membrane were hybridized by the 3H-labeled alpha 1(I) cDNA probe. No retrocorneal membrane was formed in the lacerated corneas, even after the injured corneas were allowed to heal for 3 weeks. The epithelial cells in the epithelial plug of lacerated corneas were positive by in situ hybridization, whereas the epithelial cells in the regenerated epithelium of alkali-burned cornea was not. Antibodies against alpha-SMA reacted with the migrating fibroblastic cells but did not react with epithelial cells or endothelial cells in the injured corneas. Anti-vimentin antibody reacted with fibroblastic cells, endothelial cells, and keratocytes in normal and injured corneas, and with the basal epithelial cells of injured corneas.
During wound healing, the keratocytes that migrate to injured stroma transform into myofibroblasts. These myofibroblasts express high levels of alpha 1(I) mRNA, alpha-SMA, and vimentin. The healing of alkali-burned corneas differ from that of lacerated corneas in that the retrocorneal membranes are formed in the former but not in the latter. In addition, the epithelial cells of alkali-burned corneas lack alpha 1(I) mRNA, whereas it is found in the epithelium of lacerated corneas. These differences may result from the persistence of inflammatory cells in the alkali-burned corneas.
碱烧伤的角膜很少能正常愈合以恢复角膜透明度。为了更好地理解这种毁灭性的角膜损伤，我们比较了兔角膜裂伤和碱烧伤中Ⅰ型胶原蛋白、平滑肌α - 肌动蛋白（α - SMA）和波形蛋白的表达。
使用α1（Ⅰ）链的放射性标记cDNA探针进行狭缝印迹杂交，以测定碱烧伤角膜中α1（Ⅰ）mRNA的水平。原位杂交用于鉴定表达α1（Ⅰ）链的细胞类型。抗Ⅰ型胶原蛋白、α - SMA和波形蛋白的抗体用于免疫组织化学研究，以确定Ⅰ型胶原蛋白的组织分布，并鉴定表达α - SMA和波形蛋白的细胞。
碱烧伤角膜中α1（Ⅰ）mRNA的水平在碱烧伤后稳步上升，并在2周内达到平台期。碱烧伤后1天，在角膜损伤边缘紧邻的基质细胞中检测到特异性原位杂交信号。随着愈合过程的进行，成纤维细胞迁移到受损基质中，它们通过原位杂交和用抗Ⅰ型胶原蛋白探针免疫染色显示出阳性反应。在碱烧伤角膜中，损伤后1周形成角膜后膜。这种纤维状膜用抗Ⅰ型胶原蛋白抗体染色，膜中的成纤维细胞用3H标记的α1（Ⅰ）cDNA探针杂交。即使受伤角膜愈合3周后，裂伤角膜中也未形成角膜后膜。裂伤角膜上皮栓中的上皮细胞通过原位杂交呈阳性，而碱烧伤角膜再生上皮中的上皮细胞则否。抗α - SMA抗体与迁移的成纤维细胞反应，但不与受伤角膜中的上皮细胞或内皮细胞反应。抗波形蛋白抗体与正常和受伤角膜中的成纤维细胞、内皮细胞和角膜细胞反应，并与受伤角膜的基底上皮细胞反应。
在伤口愈合过程中，迁移到受损基质的角膜细胞转化为肌成纤维细胞。这些肌成纤维细胞高水平表达α1（Ⅰ）mRNA、α - SMA和波形蛋白。碱烧伤角膜的愈合与裂伤角膜不同，前者形成角膜后膜而后者不形成。此外，碱烧伤角膜的上皮细胞缺乏α1（Ⅰ）mRNA，而在裂伤角膜的上皮中则可发现。这些差异可能是由于碱烧伤角膜中炎症细胞的持续存在所致。
