PURPOSE: To identify and differentiate genes that are up-regulated or down-regulated in human corneal epithelial cells in response to epidermal growth factor(EGF), hepatocyte growth factor(HGF) or keratinocyte growth factor(KGF). METHODS: Primary cultures of human corneal epithelial cell(HCE) were treated with 25 ng/ml of EGF, 25 ng/ml HGF, 25 ng/ml KGF, or vehicle in serum-free medium for 8 hours. Total RNA was isolated with TRIZOL(GIBCO, NY), and treated with DNAse I.P 32-labeled complementary DNA(cDNA) probes were synthesized using 6 ug of total RNA made from HCE cells. Equivalent counts of P 32-labeled cDNA probes were hybridized with the membrane of Atlas human cell cycle array at 68degreesC overnight. After sequential washing, the membranes were exposed to X-ray film for three days. These results were analyzed using Atlas Image TM 1.1 Software. RNAse protection assay was used to confirm one of known genes on the array, which was up-regulated by EGF, KGF, and HGF in the human corneal epithelial cells. RESULTS: Autoradiographic analysis showed that out of 111 genes analyzed, 22 were up- or down-regulated in EGF, 26 in HGF and 7 in KGF compared to untreated corneal epithelial cell. After different signal intensity was normalized more than 2000 by Atlas Image TM 1.1 Software, 12 genes were up-regulated and 10 genes down-regulated in EGF. HGF have 6 up-regulated genes and 1 down-regulated gene and KGF had all up-regulated 7 genes. EGF, HGF and KGF all up-regulated the expression of cyclin D1(BCL-1 oncogene) and serine/threonine-protein kinase PITALRE in the primary cultured human corneal epithelial cells. EGF and KGF both up-regulated E2F-1 pRB-binding protein gene. HGF and KGF up-regulated cyclin D2 gene. Proto-oncogene raf was down-regulated by EGF and HGF. CONCLUSIONS: The three growth factors seemed to have similar effects on the genes that contribute to cell cycle control. Studies to analyze the significance of the differences among these growth factors are ongoing.
Cell Cycle Checkpoints ; Cell Cycle* ; Cyclin D2 ; Cyclins ; Deoxyribonucleases ; DNA, Complementary ; Epidermal Growth Factor ; Epithelial Cells* ; Hepatocyte Growth Factor ; Hepatocytes* ; Humans* ; Intercellular Signaling Peptides and Proteins ; Keratinocytes* ; Membranes ; Phosphotransferases ; Proto-Oncogenes ; Ribonucleases ; RNA ; X-Ray Film

PURPOSE: To investigate that the effects of early wound healing stage after corneal epithelial scrape injury. METHODS: We studied the change of scraped corneal wound like corneal cells, corneal thickness, acelluar zone, and celluar morphology occurring at the time points of 1, 4, 12, 24, 48, 72 hours, and 7 days after corneal epithelial scrape injury by the confocal microscopy and EM findings in 4 each group rabbits. RESULTS: By normal confocal microscopy, the mean cell density was 891 cells/mm2 in the anterior stroma and decreased to 814 cells/mm2 in the middle stroma, 731 cells/mm2 in the posterior stroma, and the endothelial density was 3236 cells/mm2. The change in the morphology of the keratocyte nuclei from an elliptical shape anteriorly, to a more elongated shape posteriorly. Apoptosis revealed like as condensation or fragmentation of chromatin and nuclei, vesicle formation, apoptotic bodies after corneal scraped injury by EM findings. The mean thickness of normal cornea was as follow; 47 micrometer in the epithelium, 334 micrometer in the stroma, and 392 micrometer in total cornea. The thickness of postoperative cornea including stromal thickness and total thickness increased at the early wound healing stage, and then decreased to the postoperative 48 hours(P<0.001). Mean range of acellular zone in the stroma increased at the early wound healing, but significantly decreased at the postoperative 48 hours, 79 micrometer(P<0.001). CONCLUSIONS: Keratocyte cell density and corneal thickness at the three portions of cornea, the thickness of stromal acelluar zone, and the changes of cellular morphology were related with a kind of the early post-inflammatory reaction, especially 24 hours, of corneal scraped injury. It should be needed more studies concerned with control of early post-inflammatory reaction.
Apoptosis ; Cell Count ; Chromatin ; Cornea ; Epithelium ; Microscopy, Confocal ; Rabbits ; Wound Healing ; Wounds and Injuries

PURPOSE: To evaluate the inflammatory response following the insertion of corneal epithelium into rabbit corneal stroma. METHODS: Newzealand white rabbits were underwent corneal flap procedure using Hansatome and corneal epithelium was inserted. We divided the rabbits into three groups: Group A: flap only, Group B: flap with central epithelium insertion, Group C: flap with peripheral epithelium insertion. Eyes are enucleated, 4, 24, 48, 72 hours, 1 week, 1 month. Immunohistochemical stain fro CD4, CD8, and CD11b were used to test frozen section. RESULTS: Cd4 was expressed weakly at 24 hours in group A, B, C. around limbus and peripheral cornea. CD8 was expressed at 24 hours in group a, b, c especially strongly at group 3. In group B, C, CD8 was persistently expressed at 72 housr. CD11b was expressed at 24 hours in all groups. But in group C. CD11b was expressed strongly and persists at 72 hours and 1 week. CONCLUSION: Macrophage and Cytotoxic T cell maybe play an important role in corneal stromal wound healing after iatrogenic corneal epithelial insertion and further evaluation will be needed.
Cornea ; Corneal Stroma* ; Epithelium ; Epithelium, Corneal ; Frozen Sections ; Macrophages ; Rabbits ; Wound Healing