RANK, RANKL, OPG, and M-CSF expression in stromal cells during corneal wound healing

PURPOSE: To examine the influx of monocytes into the cornea after epithelial scrape injury and the expression of chemokines that potentially regulate monocyte phenotype in cultured corneal fibroblasts and keratocytes in situ. METHODS: Monocytes were detected by immunocytochemistry for the monocyte-specific antigen CD11b, in unwounded and epithelial scrape-wounded mouse corneas. The receptor activator of NF-kappa B ligand (RANKL), osteoprotegerin (OPG), and monocyte chemotactic and stimulating factor (M-CSF) mRNAs were detected in cultured mouse stromal fibroblasts by RT-PCR and RNase protection assay. RANKL, OPG, and M-CSF proteins were detected in cultured mouse stromal fibroblasts by immunoprecipitation and Western blot analysis. RANKL, RANK, M-CSF, and OPG proteins were detected in unwounded and wounded mouse corneas by immunocytochemistry. Chimeric mice with green fluorescent protein-labeled bone marrow-derived cells underwent corneal scrape injury and were monitored by fluorescence microscopy and immunocytochemistry. RESULTS: A small number of cells expressing the monocyte-specific CD11b antigen were detected in the stromas of unwounded mouse corneas. A larger number of CD11b-positive cells was detected in the stroma at 24 or 48 hours after epithelial scraping injury. Experiments with chimeric mice with fluorescent green protein-labeled, bone marrow-derived cells demonstrated conclusively the origin of these CD11b(+) cells. RANKL, OPG, and M-CSF mRNAs and proteins were detected in cultured mouse stromal fibroblasts. RANKL, M-CSF, and OPG proteins were detected in unwounded corneas, but were expressed at higher levels in stromal cells during the 24- to 48-hour interval after epithelial scrape injury. RANK was detected in stromal cells presumed to be monocytes at 24 and 48 hours after epithelial injury. CONCLUSIONS: Cells expressing the CD11b monocyte-specific antigen appear in the corneal stroma in high numbers by 24 hours after epithelial injury and persist beyond 10 days after wounding. Cultured corneal fibroblasts and keratocytes in situ express RANKL, OPG, and M-CSF cytokines involved in regulating osteoclast differentiation from monocytes in bone. Cells expressing RANK were detected in the stroma at 24 and 48 hours after epithelial injury. The cytokine systems that regulate monocyte transition to osteoclast in bone are upregulated in the cornea in response to epithelial injury and may participate in regulating monocyte phenotype during corneal stromal wound healing.     

Topical interleukin-1 receptor antagonist inhibits inflammatory cell infiltration into the cornea

Interleukin (IL)-1alpha and beta are important modulators of many functions of corneal epithelial and stromal cells that occur following injury to the cornea, including the influx of bone marrow-derived inflammatory cells into the stroma attracted by chemokines released from the stroma and epithelium. In this study, we examined the effect of topical soluble IL-1 receptor antagonist on bone marrow-derived cell influx following corneal epithelial scrape injury in a mouse model. C57BL/6 mice underwent corneal epithelial scrape followed by application of IL-1 receptor antagonist (Amgen, Thousand Oaks, CA) at a concentration of 20 mg/ml or vehicle for 24 h prior to immunocytochemical detection of marker CD11b-positive cells into the stroma. In two experiments, topical IL-1 receptor antagonist had a marked effect in blocking cell influx. For example, in experiment 1, topical IL-1 receptor antagonist markedly reduced detectible CD11b-positive cells into the corneal stroma at 24h after epithelial injury compared with the vehicle control (3.5+/-0.5 (standard error of the mean) cells/400x field and 13.9+/-1.2 cells/400x field, respectively, p<0.01). A second experiment with a different observer performing cell counting had the same result. Thus, the data demonstrate conclusively that topical IL-1 receptor antagonist markedly down-regulates CD-11b-positive monocytic cell appearance in the corneal stroma. Topical IL-1 receptor antagonist could be an effective adjuvant for clinical treatment of corneal conditions in which unwanted inflammation has a role in the pathophysiology of the disorder.     

20%乙醇处理兔角膜后上皮增生和细胞凋亡的研究

目的探讨准分子激光角膜上皮瓣下磨镶术(LASEK)中采用20%乙醇浸润兔角膜40s后角膜上皮增生和角膜细胞凋亡情况与机械刮除角膜上皮后的异同。方法实验组42只新西兰大白兔,用直径为8mm的LASEK专用角膜上皮刀切割角膜上皮,20%的乙醇浸润单眼40s,机械刮除对侧眼中央8mm直径的角膜上皮,随机分7组,于术后0、4h,1、3、5、8、30d取材;6只兔眼为空白对照。角膜冰冻切片,行Ki67免疫组化检查和TUNEL检测,计数角膜中央前基质细胞。结果乙醇浸润后5d中央角膜上皮增生达峰值,术后1d周边角膜上皮增生达峰值;术后4h上皮刀口下方局限的角膜基质细胞TUNEL染色阳性,数量最多;各组角膜中央前基质细胞计数和空白对照比差异无统计学意义(P=0.68)。机械刮除角膜上皮后3d周边角膜上皮增生达峰值,其高于乙醇浸润后角膜上皮的增生峰值;术后4h可见大量中央前基质细胞TUNEL阳性;术后1d中央前基质细胞数量最少(P<0.05)。结论与机械刮除角膜上皮相比,20%乙醇浸润40s对角膜损伤轻,恢复快,乙醇浸润后的角膜上皮对基质细胞有保护作用。     

Construction of a complete rabbit cornea substitute using a fibrin-agarose scaffold

PURPOSE: To construct a full-thickness biological substitute of the rabbit cornea by tissue engineering. METHODS: Ten rabbit corneas were surgically excised, and the three main cell types of the cornea (epithelial, stromal, and endothelial cells) were cultured. Genetic profiling of the cultured cells was performed by RT-PCR for the genes COL8 and KRT12. To develop an organotypic rabbit cornea equivalent, we used a sequential culture technique on porous culture inserts. First, endothelial cells were seeded on the base of the inserts. Then, a stroma substitute made of cultured keratocytes entrapped in a gel of human fibrin and 0.1% agarose was developed. Finally, cultured corneal epithelial cells were grown on the surface of the scaffold. Stratification of the epithelial cell layer was promoted by using an air-liquid culture technique. Corneal substitutes were analyzed by light and electron microscopy. RESULTS: All three types of corneal cells were efficiently cultured in the laboratory, expanded, and used to construct a full-thickness cornea substitute. Gene expression analyses confirmed that cultured endothelial cells expressed the COL8 gene, whereas epithelial cells expressed KRT12. Microscopic evaluation of the cornea substitutes demonstrated that epithelial cells tended to form a normal stratified layer and that stromal keratocytes proliferated rapidly in the stromal substitute. The endothelial monolayer exhibited a pattern similar to a normal corneal endothelium. CONCLUSIONS: These findings suggest that development of a full-thickness rabbit cornea model is possible in the laboratory and may open new avenues for research.     

Molecular mechanisms controlling the fibrotic repair phenotype in cornea: implications for surgical outcomes

PURPOSE. Incisional or ablation injury to the corneal stroma is repaired by deposition of a fibrotic tissue produced by activated keratocytes, whereas cells lost from the underlying stroma after epithelial abrasion are simply replaced by keratocyte replication without expression of fibrotic markers. The purpose of this study was to investigate mechanisms that determine this differential keratocyte response. METHODS. A penetrating keratectomy rabbit model was adapted for mice to study the fibrotic repair response. A mouse epithelial abrasion model was applied to study the stromal cell replacement response. A primary rabbit corneal cell culture model and an organotypic culture model were also used. RESULTS. When the epithelium was prevented from resurfacing the cornea after penetrating keratectomy, expression of fibrotic markers was considerably reduced. TGF-beta2 was determined to be a major substance produced by corneal epithelial cells capable of inducing the fibrotic phenotype. In the intact mouse cornea, TGF-beta2 was confined to the uninjured epithelium, but was released into the stroma during fibrotic repair. By contrast, TGF-beta1 was never found in the epithelium. When epithelial cells were cultured on a basement-membrane-like gel or allowed to deposit their own basement membrane in organotypic culture, TGF-beta2 production was reduced. Return of a basement membrane after wounding in vivo correlated with loss of the fibrotic phenotype. In the epithelial debridement injury model in which the basement membrane was left intact, TGF-beta2 remained confined to the corneal epithelium, consistent with the absence of a fibrotic phenotype. CONCLUSIONS. These data suggest that integrity of the basement membrane is a deciding factor in determining the regenerative character of corneal repair.     

Neutrophil interactions with keratocytes during corneal epithelial wound healing: a role for CD18 integrins

PURPOSE: To determine the role of keratocytes and leukocyte beta(2) (CD18) integrins in neutrophil (PMN) migration through the corneal stroma after epithelial scrape injury. METHODS: Using C57BL/6 wild-type and CD18(-/-) mice, corneas were excised at 6 hours (wild-type) or 24 hours (CD18(-/-)) after central corneal epithelial abrasion, time points determined previously to have similar levels of emigrated PMNs. Corneas were prepared for ultrastructural morphometric analysis of PMNs, keratocyte networks, and collagen. RESULTS: Transmission electron microscopy revealed intact keratocyte networks within the paralimbus that were morphometrically similar, regardless of epithelial injury or mouse genotype. Secondary to epithelial abrasion, extravasated PMNs within the paralimbus developed close contacts with keratocytes and collagen. In wild-type mice, 40% of the PMN surface was in contact with the keratocyte surface, and this value decreased to 10% in CD18(-/-) mice. PMN contact with collagen was similar in wild-type and CD18(-/-) mice, with approximately 50% of the PMN surface contacting the collagen fibrils. Since corneal edema resulting from scrape injury was similar, regardless of genotype and did not involve structural changes in collagen fibrils, these data favor a direct role for CD18 in mediating PMN contact with keratocytes. CONCLUSIONS: The data show that in response to epithelial scrape injury, PMN migration in the corneal stroma involves close contact between keratocytes and collagen. Although PMN-keratocyte contacts require CD18 integrins, contact with collagen is CD18 independent. Fundamentally, PMN migration along keratocyte networks constitutes the beginning of a new experimental concept for understanding leukocyte migration within the wounded cornea.     

Localization of collagen (I) and collagenase mRNA by in situ hybridization during corneal wound healing after epikeratophakia or alkali-burn.

The expression and localization of type I collagen and collagenase gene were studied by in situ hybridization using rabbit cornea during wound healing following epikeratophakia or alkali-burn. In corneas 24 days after epikeratophakia, alpha 1(I) collagen mRNA was detected in keratocytes which had migrated from the host cornea into the keratolens. In contrast, collagenase mRNA was detected in cells which seemed to be inflammatory cells around the suture between the host stroma and the keratolens. The increase of alpha 1(I) collagen mRNA in keratocytes was observed in corneas 94 days after epikeratophakia and in alkali-burned corneas 1-2 months after the burn. These results provide evidence that keratocytes synthesize collagen and that this synthesizing activity lasts for a long period during corneal wound healing.     

Expression of HGF, KGF, EGF and receptor messenger RNAs following corneal epithelial wounding

Hepatocyte growth factor (HGF), keratinocyte growth factor (KGF), epidermal growth factor (EGF), and their receptors have been associated with homeostasis and wound healing in the cornea. The purpose of this study was to examine the expression of the messenger RNAs for these growth factors and receptors in a wounded series of mouse corneas using in situ hybridization. In situ hybridization was performed with 3H-labeled riboprobes on unwounded corneas and corneas at 30 minutes, 4, 12, 24, 48 and 72 hr, and 7 days after epithelial scrape wounds in Balb/C mice. Qualitative and semi-quantitative analyses were performed. Expression of HGF, KGF and EGF mRNAs in keratocytes in the unwounded cornea was low. EGF mRNA was also expressed in unwounded corneal epithelium. Following wounding, however, these growth factor mRNAs were markedly upregulated in keratocytes. EGF mRNA expression in the epithelium appeared unaffected by wounding. At seven days after wounding and several days following closure of the epithelial defect, HGF mRNA and KGF mRNA were still expressed at higher levels in keratocytes compared with unwounded corneas. No difference in expression of HGF or KGF mRNAs between limbal, peripheral corneal, or central corneal keratocytes was noted in the unwounded cornea, KGF receptor mRNA was prominently expressed throughout the unwounded corneal epithelium. HGF receptor mRNA and EGF receptor mRNAs were expressed at low levels in unwounded cornea epithelium. Following scrape injury, expression of HGF receptor mRNA and KGF receptor mRNA were markedly upregulated in the corneal epithelium, while no significant increase in EGF receptor mRNA expression was noted. These studies suggest a prominent role for HGF and KGF in modulating corneal epithelial wound healing following injury. Less prominent changes in EGF mRNA and EGF receptor mRNA in the corneal epithelium following wounding may suggest that EGF has more of a role in homeostasis in the mouse corneal epithelium.     

Microarray studies reveal macrophage-like function of stromal keratocytes in the cornea

PURPOSE: To elucidate biological processes underlying the keratocyte, fibroblast, and myofibroblast phenotypes of corneal stromal cells, the gene expression patterns of these primary cultures from mouse cornea were compared with those of the adult and 10-day postnatal mouse cornea. METHODS: Murine Genome_U74Av2 arrays (Affymetrix Inc., Santa Clara, CA) were used to elucidate gene expression patterns of adult and postnatal day-10 corneal stroma, keratocytes, fibroblasts, and myofibroblasts. RESULTS: Mobilization of stromal cells by culturing led to a wound-healing cascade in which specific extracellular matrix and cornea-transparency-related genes were turned off, and a repertoire of macrophage genes were switched on. Thus, novel transparency-related crystallins detected in the corneal gene expression patterns were downregulated in culture, whereas macrophage genes, mannose receptor type-1, Cd68, serum amyloid-A3, chemokine ligands (Ccl2, Ccl7, Ccl9), lipocalin-2, and matrix metalloproteinase-3 and -12 of innate immunity were induced in primary keratocyte cultures. Fibroblasts expressed the growth-related genes lymphocyte antigen 6 complex locus-A and preprokephalin-1, and myofibroblasts expressed annexin-A8, WNT1-inducible signaling pathway protein-1, arginosuccinate synthetase-1, and procollagen XI of late-stage wound healing. CONCLUSIONS: The emergent biological process suggests a dual role for resident stromal keratocytes in the avascular cornea: one of cornea maintenance, which involves synthesis of proteins related to the extracellular matrix and corneal transparency, and a second of barrier protection macrophage functions, which are switched on during corneal infection and injury.     

Early keratocyte apoptosis after epithelial scrape injury in the human cornea

Animal studies in mice, rats, rabbits, pigs and hens demonstrated that anterior keratocytes undergo programmed cell death or apoptosis after corneal epithelial injury. Many other wound healing changes subsequently follow the keratocyte apoptosis response. This study evaluated early keratocyte apoptosis after corneal epithelial scrape injury in human eyes scheduled for enucleation for malignancy. Two eyes had corneal epithelial scrape 1 h prior to the enucleation and another eye served as a control and had no corneal scrape prior to enucleation. One additional eye was enucleated, washed with balanced salt solution, and then had the corneal epithelium scraped 1 h prior to processing for analysis. Apoptosis was identified by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay and confirmed by transmission electron microscopy (TEM). Anterior keratocyte apoptosis was detected in the three corneas that had epithelial scrape injury, but not in the control unwounded cornea. This study confirmed that keratocyte apoptosis is also an early response to corneal epithelial injury in humans and showed that tears are not essential for keratocyte apoptosis to occur in response to epithelial injury.     

Caspase inhibitor z-VAD-FMK inhibits keratocyte apoptosis, but promotes keratocyte necrosis, after corneal epithelial scrape

The purpose of this study was to determine whether the caspase inhibitor z-VAD-FMK could be applied topically prior to epithelial scrape injury to inhibit keratocyte apoptosis. Rabbit corneas were treated with z-VAD-FMK or vehicle alone prior to epithelial scrape injury. Cell fate was analysed at 4 hr after epithelial scrape using quantitative TUNEL assay, propidium iodide staining, and transmission electron microscopy. Less stained anterior stromal keratocytes were detected with the quantitative TUNEL assay in corneas pre-treated with z-VAD-FMK than in corneas pretreated with vehicle at 4 hr after epithelial scrape. This difference appeared to be confirmed by propidium iodide staining of keratocyte nuclei. It was observed that fewer nuclei were stained with propidium iodide in the DMSO vehicle treated corneas compared to the z-VAD-FMK treated corneas. Analysis of corneas with transmission electron microscopy, however, indicated that many anterior stromal keratocytes in corneas pretreated with z-VAD-FMK, but not vehicle, had cell morphologic changes more consistent with necrosis. Although pretreatment of corneas with the caspase inhibitor z-VAD-FMK inhibited keratocyte apoptosis detected with the TUNEL assay, transmission electron microscopy revealed that many anterior stromal keratocytes in z-VAD-FMK-treated corneas instead died by necrosis. Thus, z-VAD-FMK is unlikely to be useful to modulate corneal would healing through inhibition of keratocyte apoptosis induced by epithelial injury. The TUNEL assay should not be used to monitor cell fate without confirmation using analyses that also detect necrosis.     

Thy-1 distinguishes human corneal fibroblasts and myofibroblasts from keratocytes

After corneal injury, keratocytes become activated and transform into repair phenotypes-corneal fibroblasts or myofibroblasts, however, these important cells are difficult to identify histologically, compromising studies of stromal wound healing. Recent studies indicate that expression of the cell surface protein, Thy-1, is induced in fibroblast populations associated with wound healing and fibrosis in other tissues. We investigated whether keratocyte transformation to either repair-associated phenotype induced Thy-1 expression. Human corneal keratocytes were isolated by collagenase digestion. The cells were either processed immediately (i.e. freshly isolated keratocytes) or were cultured in the presence of 10% fetal bovine serum or transforming growth factor-beta to induce transformation to the corneal fibroblast and myofibroblast phenotypes, respectively. Thy-1 mRNA and protein expression by freshly isolated keratocytes and corneal fibroblasts were assessed by RT-PCR and Western blotting. mRNA also was extracted from the whole intact stroma and assessed by RT-PCR. Thy-1 was localised immunocytochemically in cultured human corneal fibroblasts, myofibroblasts, and in keratocytes in normal human corneal tissue sections. Thy-1 mRNA and protein were detectable in cultured human corneal fibroblasts, but not freshly isolated keratocytes. Whole uninjured stroma showed no detectable Thy-1 mRNA expression. Cultured human corneal fibroblasts and myofibroblasts both labelled for Thy-1, but keratocytes in the stroma of normal human cornea did not. We conclude that Thy-1 expression is induced by transformation of keratocytes to corneal fibroblasts and myofibroblasts, suggesting a potential functional role for Thy-1 in stromal wound healing and providing a surface marker to distinguish the normal keratocyte from its repair phenotypes.     

Multifocal corneal argyrosis after an explosion injury.

PURPOSE: To document the clinical and histopathologic corneal features of a patient who developed multifocal corneal argyrosis after a chemical explosion injury with unusual involvement of the corneal stroma and keratocytes. METHODS: The corneal button was investigated by light and transmission electron microscopy and scanning electron microscopy combined with energy-dispersive x-ray microanalysis. RESULTS: Clinically, the patient showed dark discoloration of the lids, periocular skin, episclera, and conjunctiva and had multiple brown dots in the superficial layers of the cornea. Microscopic examination of the cornea showed diffuse deposition of silver particles in the epithelial basement membrane, Bowman's layer, and Descemet's membrane. In the corneal stroma, silver granules accumulated intracellularly within lysosomal structures of degenerative keratocytes and extracellularly in association with collagen fibers and cellular debris. Energy-dispersive x-ray analysis showed peaks of silver and sulfur. CONCLUSION: The toxic influence of intracellular accumulation of silver in stromal keratocytes may lead to cell damage and necrosis and result in visual impairment.     

Loss of alpha3(IV) collagen expression associated with corneal keratocyte activation

PURPOSE: To determine whether changes in the expression of type IV alpha1, alpha2, or alpha3 collagen isoforms are stringently associated with corneal stromal cell activation. METHODS: Keratocytes isolated from rabbit corneal stroma by collagenase digestion were plated in serum-free or insulin-, bFGF/heparin sulfate (HS)-, TGF-beta1-, or fetal bovine serum (FBS)-supplemented DMEM/F12 medium. Expression of type IV collagen isoforms and keratan sulfate proteoglycans (KSPGs) was evaluated by immunocytochemical analysis, Western blot analysis, or both. Concentrations of mRNAs were estimated by quantitative RT-PCR using SYBR Green RT-PCR reagents. RESULTS: Immunohistochemical analysis indicated that type IV alpha1, alpha2, and alpha3 collagens were expressed in normal rabbit corneal stroma and in keratocytes cultured in serum-free and insulin-supplemented media. However, alpha3(IV) collagen was not detectable in the regenerating stroma after photorefractive keratectomy (PRK) in rabbit or in corneal stromal cells cultured in media supplemented with FBS, bFGF/HS, or TGF-beta1. alpha3(IV) collagen mRNA levels were also diminished in the stromal cells cultured in these growth factor-supplemented media. KSPGs (lumican and keratocan) were expressed and secreted in serum-free medium. Although the expression of KSPGs was promoted by insulin, the expression and intracellular levels of lumican and keratocan mRNAs were downregulated by TGF-beta1 and FBS. bFGF/HS promoted the downregulation of intracellular keratocan but not lumican mRNA levels. CONCLUSIONS: The loss in the expression of alpha3(IV) collagen is a stringent phenotypic change associated with activation of keratocytes in vivo and in vitro. This phenotypic change in activated corneal stromal cells is induced by bFGF/HS and by TGF-beta1, and it accompanies the downregulation of keratocan expression.     

Corneal cells: chatty in development,homeostasis, wound healing,and disease

PURPOSE: To provide an overview of cell-cell interactions in the cornea that have a critical role in corneal development, homeostasis, wound healing, and disease. DESIGN: Review. METHODS: Review of the literature. RESULTS; Cell-cell interactions make critical contributions to development, homeostasis, and wound healing in the cornea. Many of these interactions are mediated by cytokines, growth factors, and chemokines. The best characterized are stromal-epithelial interactions between epithelial cells and stromal cells such as keratocytes, keratoblasts, and myofibroblasts. However, interactions also occur between corneal nerves and epithelial cells and between corneal cells (epithelial cells and stromal cells) and corneal immune cells. Although investigations are limited, it is likely that there are interactions between corneal endothelial cells and keratocytes in the posterior stroma. CONCLUSIONS: Cellular communications in the cornea are critical during development, homeostasis, and wound healing. Disorders of cellular communication likely contribute to many corneal diseases.     

In vivo confocal microscopy in the patients with cornea farinata

PURPOSE: To report in vivo corneal confocal microscopic findings of patients with cornea farinata. PATIENTS AND METHODS: Two unrelated patients, a 47-year-old man and a 77-year-old woman, with cornea farinata were studied. Examination with a confocal microscope was performed in addition to routine slit-lamp biomicroscopy. RESULTS: In both cases, slit-lamp biomicroscopy showed numerous small, faint opacities in the deep stroma in both eyes. Using confocal microscopy, highly reflective small particles were observed in the cytoplasm of keratocytes in the deep stroma adjacent to the corneal endothelial layer. No abnormalities could be detected in the epithelial layer, in the mid-stromal layer, at the level of Descemet's membrane, and in the endothelial layer. CONCLUSIONS: In vivo corneal confocal microscopy is useful for observing stromal abnormalities in cornea farinata. Further investigation of posterior stromal opacities using confocal microscopy may be useful to understand and differentiate various corneal conditions involving primarily deep stromal layers.     

组织工程角膜基质的体外构建及移植的实验研究

目的探讨利用组织工程技术体外构建角膜基质进行板层角膜移植的可行性和有效性。方法将猪角膜基质去细胞处理后制备成组织工程角膜基质载体;取幼兔角膜基质细胞体外培养,将其种植在载体上,体外构建成组织工程角膜基质,用PKH26荧光标记兔角膜基质细胞示踪角膜基质的构建;将16只兔的角膜基质内植入壳聚糖膜使之形成无菌性角膜溃疡,随机从16只兔中选8只,进行组织工程角膜基质移植;另外8只作为对照组,进行新鲜的同种异体兔板层角膜移植。术后对角膜进行裂隙灯、光学显微镜、透射电镜观察。结果体外构建的组织工程角膜的基质细胞具有活性,其结构与正常角膜基质相近。移植治疗无菌性角膜溃疡术后,1~2周有新生血管侵入组织工程角膜基质植片边缘,植片为灰白色半透明状;3~4周随着新生血管减退,组织工程角膜基质植片局部开始透明变薄;术后8~10周角膜溃疡完全修复,角膜恢复透明性,角膜神经可再生;观察最长达10月,角膜仍保持透明,无免疫排斥发生,与对照组疗效相同。结论体外构建的组织工程角膜基质无免疫原性、具有良好的生物相容性,可作为临床治疗角膜溃疡的移植材料。     

Protein expression pattern of collagen type XV in mouse cornea

Purpose To examine the alteration of protein expression pattern of collagen type XV in cornea during embryonic development and adult tissue repair. Collagen type XV is a basement membrane collagen of a subfamily of multiplexins (multiple triple helix domains and interruptions). Its COOH-terminal peptide has an anti-angiogenic effect and its distribution in avascular tissue of cornea is of interest.Methods Eyes of mouse embryos [day (E) 10.5–18.5] and healing adult mouse corneas following either débridement injury or incision were embedded in paraffin. Deparaffinized sections were processed for immunofluorescent staining with anti-collagen XV antibody.Results At E14.5 embryonic corneal epithelium, as well as fibroblasts in eyelids, began to express this collagen type very faintly, and at E18.5, besides corneal epithelial expression, epidermis, palpebral conjunctiva, and keratocytes started to express collagen type XV. In adult mouse cornea, collagen type XV was observed in basal and suprabasal epithelial cells and stroma, but not in the subepithelial basement membrane. Healing epithelial cells following débridement or incision injury down-regulated its protein expression.Conclusions Mouse embryonic corneal epithelium and keratocytes begin to express collagen type XV before birth. Healing murine corneal epithelium down-regulates collagen XV expression. The presence of collagen XV in corneal stroma may play a part in avascularity.