整合素α5及其配体在大鼠角膜创伤愈合中表达的研究

目的 观察创伤愈合中大鼠角膜整合素（integrin)α5及其配体－纤维连接蛋白（fibronectin,FN）表达的变化,探讨二者对角膜创伤愈合的影响。方法 大白鼠27只,随机分实验组和对照组。依左右眼手术不同又分左眼和右眼组。右眼刮除中央3mm直径区域内角膜上皮,保持前弹力膜完整;左眼同上操作后,用RK刀作长3mm,深100μm的非穿透切口。术后即刻、3、6、12、24、72h、1w,2w分别处死每组大鼠,取双眼角膜,用免疫荧光染色法观察角膜整合素α5及FN分布的改变。对照组不手术,处死后角膜标本按实验组同样步骤处理。结果 与对照组相比,术后3h双眼角膜创缘上皮表层及左眼基质切口处出现α5和FN荧光、之后荧光亮度升高,72h后下降,术后2w恢复至术前水平。α5与FN的表达相对应。结论 角膜创伤愈合早期整合素α5及其配体FN增多并相协调。二者能促进角膜创伤愈合早期的上皮迁移。     

Effect of fibronectin on corneal epithelial wound healing in the vitamin A-deficient rat

The authors investigated the in vitro and in vivo effects of fibronectin (Fn) on the migration of corneal epithelium of vitamin A-deficient (A-) and pair-fed control (A+) rats. Groups treated with 50 micrograms/ml Fn showed accelerated healing of epithelium in vitro (P less than 0.05) compared with control groups of A- and A+ rats. However, when 100 micrograms/ml Fn eye drops were administered 14 times over 20 hr, they had no significant effect on A+ rats in vivo, but increased the healing in A- rats (P less than 0.05). In this model, Fn promoted the healing of corneal epithelium under A- conditions where decreased endogenous Fn is seen, whereas A+ corneas in vivo, which have sufficient Fn over the wound surface, did not benefit from topical Fn administration.     

Anti-angiogenic role of angiostatin during corneal wound healing

The purpose of this study is to determine whether angiostatin is involved in maintaining corneal avascularity after wounding. We generated polyclonal rabbit anti-mouse angiostatin antibodies directed against each of the five kringle domains, (K1-5) and anti-mouse plasmin B chain antibodies. Mouse corneas were immunostained with anti-K1 angiostatin antibody after excimer laser keratectomy. Corneal epithelial cell lysate was harvested and angiostatin was isolated using lysine sepharose. Purified plasminogen was incubated with lysate of mouse corneal epithelial cells from wild type mice in the presence or absence of MMP inhibitors. Angiostatin activity was determined using calf pulmonary artery endothelial (CPAE) cell proliferation assay with and without angiostatin immunoprecipitation; and corneal neovascularization was assayed by intrastromal injection of anti-plasminogen, anti-K1-3 or anti-B chain antibodies after corneal wounding. Using the anti-mouse angiostatin antibodies that we generated, we confirmed that angiostatin-like molecules were expressed in the corneal epithelium and in cultured corneal epithelial cells. Western blotting after incubation of scraped corneal epithelial cell lysate with purified plasminogen showed reduction of the plasminogen bands at 6, 12, and 24 hr, respectively. Complete cleavage of plasminogen occurred by 48 hr. Functional assays in which corneal epithelial cell extracts were incubated with CPAE cells resulted in inhibition of vascular endothelial cell proliferation. Depletion experiments using anti-angiostatin (K1) antibodies resulted in a 25 +/- 1.2% increase in vascular endothelial cell proliferation as compared to 12 +/- 1.8% using the protein A control (p < 0.05). Corneal neovascularization was observed after excimer laser keratectomy when anti-angiostatin antibodies were injected into the cornea (65 +/- 13%) which was significantly higher than when plasmin B chain antibodies were injected (10 +/- 2.6%; p < 0.05). Plasminogen and angiostatin are produced in the cornea. They may play a role in preventing vascularization and may contribute to the maintenance of corneal avascularity after excimer laser keratectomy.     

Promotion of corneal epithelial wound healing by a tetrapeptide (SSSR) derived from IGF-1

PURPOSE: A prior study showed that a tetrapeptide (FGLM-amide) derived from the carboxyl terminus of substance P (SP) and a 12-residue peptide corresponding to the C domain of insulin-like growth factor (IGF)-1 mimic the synergistic effect of the full-length molecules on corneal epithelial wound healing. To develop an effective treatment for persistent corneal epithelial defects, the current study was conducted to investigate the minimal sequence within the C domain of IGF-1 that is required for such synergism with SP or FGLM-amide. METHODS: The effects of IGF-1-derived peptides on corneal epithelial migration were evaluated with a rabbit corneal organ-culture system. RESULTS: A tetrapeptide (SSSR; Ser(33)-Ser-Ser-Arg) derived from the C domain of IGF-1 was sufficient for the synergistic promotion with FGLM-amide both of corneal epithelial migration in vitro and of wound closure in vivo. The activity of the SSSR peptide was sequence specific and its potency was similar to that of IGF-1. The SSSR peptide by itself also promoted corneal epithelial migration in vitro at higher concentrations. It was devoid, however, of both the mitogenic action of IGF-1 and the ability of the full-length molecule to induce neovascularization. CONCLUSIONS: The SSSR sequence mediates the synergistic effect of IGF-1 with SP on corneal epithelial wound healing. Clinical application of the SSSR peptide would be expected to be free of potentially deleterious side effects associated with treatment with full-length IGF-1. Local administration of the SSSR tetrapeptide, alone or in combination with FGLM-amide, is thus a potential new strategy for the treatment of nonhealing epithelial wounds.     

Expression of VSX1 in human corneal keratocytes during differentiation into myofibroblasts in response to wound healing

PURPOSE: To characterize the expression of the visual system homeobox gene (VSX1) in human corneal keratocytes both in vitro and in vivo. METHODS: The expression of VSX1 was evaluated through semiquantitative RT-PCR, immunofluorescence and in situ hybridization both in corneas (either freshly obtained or wounded) and in collagenase/hyaluronidase-isolated keratocytes grown in the absence or presence of serum to promote keratocyte-to-myofibroblast differentiation. RESULTS: Quiescent or resting keratocytes normally residing in the corneal stroma or cultured in vitro in the absence of serum did not express VSX1. In wounded corneas or when cultured in the presence of serum to mimic wound-healing responses, keratocytes underwent fibroblastic transformation (with appearance of alpha-SMA and disappearance of CD-34 and keratocan signals) and started expressing VSX1. CONCLUSIONS: The results show that VSX1 is expressed in vitro and in vivo during human corneal wound healing, a process in which differentiation of corneal keratocytes into myofibroblasts occurs. These data may help to elucidate the role of VSX1 in cornea physiology suggesting a potential involvement in cornea-related diseases such as keratoconus.     

整合素α5亚单位在大鼠角膜碱烧伤愈合中的表达

目的 观察碱烧伤后角膜创面上皮愈合过程中整合素α5亚单位的表达，探讨整合素α5在角膜上皮损伤愈合中的作用。方法 Wistar大鼠18只，随机分为6组，每组3只，其中1眼为碱烧伤模型，另1眼为自身对照眼。对制造模型眼后0，7h,1,3,7,14d的角膜标本，HE染色行组织病理学检查，间接免疫荧光和免疫组织化学染色观察整合素α5亚单位的表达与分布。结果 伤后7h ,邻近上皮细胞开始表达α5亚,伤后1，3，7d，角膜上皮层显示α5表达逐渐增加，差别有显著性（P＜0．05）。伤后14d，实验组表达仍呈上升趋势。结论 整合素α5亚单位积极参与了碱烧伤后角膜上皮损伤愈合的过程。     

Effect of tetra-peptide isolated from interleukin 1 (IL-1) on corneal epithelial wound healing in the rabbit

To develop a new method for wound healing in case of injured corneal epithelium, the effects of the tetrapeptide (Val-Leu-Leu-Lys), showing the consensus sequence between human interleukin (IL)-1alpha and bovine parotid protein (parotin) on epithelial cell proliferation and elongation were analysed in vitro cell culture experiments on epithelial cells obtained from rabbit cornea. The peptide showed dose-dependent stimulatory effects on epithelial cell proliferation and elongation at 10-100 microg ml(-1)compared with the control experiments. Furthermore, the peptide also exhibited a significant wound healing activity for the epithelial cells in an in situ experiment using mechanically injured rabbit cornea, while the higher concentration of the peptide (100 microg ml(-1)) showed greater efficient results than a previously known agent, sodium hyaluronate (0.3%). In addition, no pyrogenic activity of this peptide was detected by the previously established pyrogenicity test using rabbits.These results suggest that the tetrapeptide (Val-Leu-Leu-Lys) is a promising agent for wound healing in the case of injured corneal epithelium.     

Phosphorylation of MAP kinase in corneal epithelial cells during wound healing

PURPOSE: To investigate the role of mitogen-activated protein kinase (MAPK), such as p44/42 MAPK, p38 MAPK and stress-activated protein kinase (SAPK), in corneal epithelial cells during the wound healing process. METHODS: A single non-penetrating incision was produced on rat cornea. Then the corneal wound healing process was observed with an immunocytochemical technique using specific antibodies reacting only with phosphorylated p44/42 MAPK, p38 MAPK or SAPK. Cell lysates of corneal epithelial cells in rabbits stimulated with keratinocyte growth factor (KGF) and hepatocyte growth factor (HGF) were processed for Western blot using antibodies to phosphorylated p44/42 MAPK. RESULTS: Maximum activation of p44/42 MAPK was observed in wing and basal cells at wounded regions in rat cornea at 1 hour after the incision. Activation of p44/42 MAPK was still detected in all basal and wing cells at wounded regions at up to 24 hours when the incisions were completely closed, and then receded to normal intensity after 7 days. Neither p38 MAPK nor SAPK were activated during the wound healing process. Western blot analysis of cultured corneal epithelial cells in rabbits showed phosphorylation of p44/42 MAPK after 30 minutes in response to KGF and HGF, whereas non-activated p44/42 MAPK was ordinarily detected even at the absence of KGF or HGF. CONCLUSIONS: These results demonstrate that p44/42 MAPK is activated during the corneal wound healing process and suggest that KGF and HGF play an important role in initiation of cell migration and proliferation in the initial wound healing process by activating p44/42 MAPK.     

Disappearance of desmosomal components in rat corneal epithelium during wound healing

PURPOSE: Epithelial migration is essential for healing of the ablated corneal epithelium. To reveal the mechanism which enables the corneal epithelial cells to dissociate during migration, we investigated the immunolocalization of the components of the desmosome, which is the main constituent of the intercellular junction attaching the intermediate filaments to the cell surface, desmoplakin 1, desmoglein and plakoglobin, in the corneal epithelium during wound healing in rats. METHODS: Under general anesthesia with ether inhalation, Wistar rats (n = 48) underwent removal of the central corneal epithelium of one eye with a small trephine and a scalpel. After various intervals of healing (5 min; 1, 3, 6, 9, 12, 24, 48 h; 1 and 2 weeks), the animals were killed and the affected eye was excised. Cryosections of each anterior segment of the eye were fixed with cold acetone and treated with primary antibodies against desmoplakin 1, desmoglein and plakoglobin. Immunolocalization of these substances was visualized by the peroxidase-diaminobenzidine reaction. RESULTS: A marked immunoreactivity for these desmosome components was detected at the intercellular junction in the normal corneal and conjunctival epithelium. At 6-24 h after epithelial ablation, a weak immunoreactivity for desmoplakin 1 and plakoglobin was observed in the migrating epithelium. At 48 h after epithelial ablation, a marked immunoreactivity for these desmosome components was seen again. At 6-48 h after epithelial ablation, a weak immunoreactivity for desmoglein was observed in the migrating epithelium. At 1 week after epithelial ablation, a marked immunoreactivity for this desmosome component reappeared. The regenerated epithelium gradually exhibited normal immunolocalization of the proteins. CONCLUSIONS: The desmosome components were considered to be degraded or destroyed prior to epithelial migration and reconstructed during healing of the squamous epithelium.     

Alteration of epithelial paracellular permeability during corneal epithelial wound healing.

We studied the paracellular permeability to mannitol of corneas with epithelium of corneal, limbal, or conjunctival origin. Corneas with epithelial defects reepithelialized by corneal or limbal epithelium were nonvascularized; the corneal permeability was initially increased and returned to normal 3 days later. When epithelial defects extended beyond the limbus, they were healed by conjunctival epithelium. If corneas remained avascular or minimally vascularized, the conjunctiva-derived epithelium underwent a transdifferentiation process into a cornealike morphology in which the corneal permeability was initially increased upon complete reepithelialization, and gradually decreased to a level similar to that of normal cornea, 4 weeks after healing. However, when corneas became vascularized, the conjunctiva-derived epithelium retained its original phenotype, and corneal permeability remained increased throughout the 8-month period of study. The deranged barrier functions noted in the above vascularized cornea were demonstrated further by horseradish peroxidase tracer, which was found in the intercellular spaces of conjunctiva-derived epithelium of vascularized corneas but not in the avascular corneas with epithelia of corneal or limbal origin, or transdifferentiated conjunctival epithelium. To study further the effect of subsequent ocular surface trauma, conjunctival biopsy was performed on transdifferentiated avascular corneas 3 months after initial wounding. The biopsy resulted in extensive vascularization in three of eight previously nonvascularized corneas. Two weeks later, the corneal permeability was increased to a level similar to that of conjunctiva. These results indicate that corneal epithelial paracellular permeability correlates well with the status of the epithelial phenotype.