RESUMEN
The cultivated epithelial transplantation is a new surgical modality for treating a variety of severe ocular surface disorders. This type of tissue-engineered epithelial sheet provides a rapid epithelial coverage on the corneal surface that reduces inflammation and postoperative complications. Although cultivated corneal epithelial transplantation is an effective surgical strategy, autologous transplantation is limited to unilateral cases. Autologous cultivated oral mucosal epithelial transplantation (COMET) enables surgeons to reconstruct the ocular surface using autologous, non-ocular surface cells, and has opened a new pathway for treating severe, bilateral ocular surface disorders.
Asunto(s)
Córnea/patología , Enfermedades de la Córnea/cirugía , Células Epiteliales/citología , Epitelio/trasplante , Oftalmopatías/cirugía , Ojo/patología , Mucosa Bucal/trasplante , Procedimientos Quirúrgicos Oftalmológicos/métodos , Ingeniería de Tejidos/métodos , Células Cultivadas , Córnea/citología , Enfermedades de la Córnea/patología , Células Epiteliales/trasplante , Oftalmopatías/terapia , Humanos , Inflamación , Proyectos Piloto , Procedimientos de Cirugía Plástica , Trasplante AutólogoRESUMEN
BACKGROUND: Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are acute severe blistering diseases of the skin and also two of the most devastating ocular surface diseases leading to corneal damage and loss of vision. The extreme rarity of cutaneous and ocular surface reactions to drug therapies led us to suspect individual susceptibility. SJS/TEN patients in the acute stage were reported to manifest increased serum levels of Fas Ligand (FasL). Thus, we performed SNP association analysis of the FasL gene. METHODS: In 76 Japanese SJS/TEN patients with ocular surface complications and 160 Japanese healthy controls, we examined four SNPs of FasL reported in the Japanese Single Nucleotide Polymorphisms (JSNP) database by sequencing. RESULTS: The SNP rs.3830150 A/G showed a significant strong inverse association with SJS/TEN. Analysis of the genotype pattern of SNPs rs.3830150 and rs.2639614 (rs.3830150 A/A-rs.2639614 G/G) also manifested a strong inverse association with SJS/TEN. CONCLUSION: FasL gene polymorphisms might be associated with SJS/TEN.
Asunto(s)
Proteína Ligando Fas/genética , Polimorfismo de Nucleótido Simple , Síndrome de Stevens-Johnson/genética , Adulto , Anciano , Estudios de Casos y Controles , Frecuencia de los Genes , Predisposición Genética a la Enfermedad , Humanos , Persona de Mediana EdadRESUMEN
BACKGROUND/AIMS: To determine outcomes of transplants of cultivated autologous oral epithelial cells in patients with severe ocular surface disorders. METHODS: The eyes (n = 6) of four patients with Stevens-Johnson syndrome (three eyes) or chemical burns (three eyes) were studied. Autologous oral epithelial cells, grown for 2-3 weeks on a denuded amniotic membrane carrier in the presence of 3T3 fibroblasts, were air lifted. The resultant sheet was transplanted onto the damaged eye, and acceptance of the sheet by the corneal surface was confirmed 48 hours after surgery. The success of ocular surface reconstruction, graft survival, changes in visual acuity, and postoperative complications were assessed and the quality of the cultivated oral epithelial sheet was evaluated histologically. RESULTS: At 48 hours after transplant, the entire corneal surface of all six eyes was free of epithelial defects indicating complete survival of the transplanted oral epithelium. Visual acuity was improved in all eyes. During follow up (mean 13.8 (SD 2.9) months), the corneal surface remained stable, although all eyes manifested mild peripheral neovascularisation. CONCLUSIONS: Autologous oral epithelial cells grown on denuded amniotic membrane can be transplanted to treat severe ocular surface disorders.
Asunto(s)
Enfermedades de la Córnea/terapia , Células Epiteliales/trasplante , Lesiones Oculares/terapia , Mucosa Bucal/citología , Adolescente , Adulto , Amnios , Quemaduras Químicas/terapia , Células Cultivadas , Femenino , Supervivencia de Injerto , Humanos , Masculino , Síndrome de Stevens-Johnson/terapia , Resultado del Tratamiento , Agudeza VisualRESUMEN
PURPOSE: To investigate the outcome of cultivated corneal epithelial transplantation for severe stem cell deficiencies using denuded amniotic membrane (AM) as a carrier. DESIGN: Retrospective, noncomparative case series. PARTICIPANTS: Thirteen eyes of 11 patients were studied. These consisted of five eyes with acute Stevens-Johnson syndrome (SJS), two with chronic SJS, one with an acute chemical injury, two with chronic chemical injuries, two with ocular cicatricial pemphigoid, and one with drug-induced pseudopemphigoid. All of these eyes had total stem cell deficiencies. MAIN OUTCOME MEASURES: Adaptation of the cultivated corneal epithelium onto the host corneal surface was confirmed 48 hours after surgery. The reconstruction of the ocular surface and visual acuity were measured. METHODS: Corneal limbal epithelium from donor corneas was cultivated for 4 weeks on a denuded AM carrier, with 3T3 fibroblast coculture and air lifting. The cultivated corneal epithelium showed four to five layers of stratification and was well differentiated. After conjunctival tissue removal from the cornea up to 3 mm outside the limbus and subconjunctival tissue treatment with 0.04% mitomycin C, cultivated allocorneal epithelium, including the AM carrier, was transplanted onto the corneal surface up to the limbus. Lamellar keratoplasty, using preserved donor graft without epithelium, was performed simultaneously for five chronic-phase patients showing corneal stromal scarring. Systemic immunosuppression was used to prevent allograft rejection. RESULTS: In all 13 eyes, the entire corneal surface, on which cultivated allocorneal epithelium had been placed, was free from epithelial defects 48 hours after surgery, indicating complete survival of the transplanted corneal epithelium. Visual acuity improved in all eyes after surgery, and 10 of the 13 eyes were restored to good vision (postoperative visual acuity improved two or more lines) 6 months after the operation. During the follow-up period (mean +/- standard deviation, 11.2 +/- 1.3 months), the corneal surfaces were clear, although three eyes experienced epithelial rejection. CONCLUSIONS: Cultivated corneal epithelial transplantation using denuded AM as a carrier can be used for severe stem cell deficiencies.
Asunto(s)
Enfermedades de la Córnea/cirugía , Epitelio Corneal/citología , Trasplante de Células Madre , Adulto , Anciano , Anciano de 80 o más Años , Amnios , Células Cultivadas , Niño , Técnicas de Cocultivo , Enfermedades de la Córnea/patología , Células Epiteliales/trasplante , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Agudeza VisualAsunto(s)
Enfermedades de la Córnea/cirugía , Células Epiteliales/trasplante , Limbo de la Córnea/citología , Síndrome de Stevens-Johnson/cirugía , Enfermedad Aguda , Adulto , Trasplante de Células , Células Cultivadas , Enfermedades de la Córnea/patología , Células Epiteliales/citología , Fluorofotometría , Humanos , Masculino , Síndrome de Stevens-Johnson/patología , Resultado del TratamientoRESUMEN
PURPOSE: To elucidate the effect of the cotton thread test (CT-T) and Schirmer test (S-T) on the tear reservoir by evaluating the radius of tear meniscus curvature. METHODS: The radii (R) of the central lower tear menisci were measured by a newly developed video meniscometer in 11 eyes of 11 normal volunteers (6 men, 5 women; mean age, 27.7 +/- 3.6 years [SD]) and 9 eyes of 9 patients with tear deficiency and severe dry eye in whom the puncta had been therapeutically occluded (9 women; mean age, 50.6 +/- 10.4 years). In this dry eye group, the absence of reflex tearing, coupled with the absence of lacrimal drainage due to punctal occlusion allowed more precise observation of the removal of tears from the meniscus. A 1-minute CT-T was performed, followed after an interval of 10 minutes by a 1-minute S-T. Tear meniscus curvature was documented before (R:(0)) and during the tests at 30 seconds (R(30)) and 60 seconds (R:(60)). RESULTS: In the normal group, respective R values (CT-T; S-T; mean +/- SD mm) were R(0) (0.26 +/- 0.11; 0.26 +/- 0. 07), R(30) (0.27 +/- 0.16; 0.20 +/- 0.13), and R(60) (0.29 +/- 0.15; 0.23 +/- 0.21); and in the dry eye group, respective R: values (CT-T; S-T) were R(0) (0.59 +/- 0.23; 0.51 +/- 0.19), R(30) (0.52 +/- 0.25; 0.22 +/- 0.09), and R(60) (0.51 +/- 0.19; 0.21 +/- 0.08). It was demonstrated in the dry eye group that R was diminished more by the S-T than by the CT-T in the time course of the measurement (P = 0.01). In the dry eye group alteration of R occurred within the first 30 seconds, and in this group significant correlation was found between R(0) and the S-T result (r = 0.67; P = 0.05), and between R(60)- R(0) and the S-T result (r = -0.81; P = 0.01). Also, there was a significant correlation between R(60)- R(0) and the S-T result in the normal group (r = 0.71; P = 0.02). There were no significant correlations between R(0) or R(60)- R(0) and the CT-T results in either group. CONCLUSIONS: These studies afford some insight into the dynamics of the Schirmer test, suggesting that wetting is influenced by the negative hydrostatic pressure within the tear meniscus. With the protocol used, no conclusion could be drawn about the relation between meniscus radius and wetting of the cotton thread.
Asunto(s)
Técnicas de Diagnóstico Oftalmológico , Síndromes de Ojo Seco/metabolismo , Aparato Lagrimal/metabolismo , Lágrimas/metabolismo , Adulto , Anciano , Síndromes de Ojo Seco/diagnóstico , Femenino , Humanos , Masculino , Persona de Mediana Edad , Lágrimas/químicaRESUMEN
PURPOSE: Recently, mutations in the M1S1 gene have been identified as responsible for gelatinous drop-like corneal dystrophy (GDLD). How the abnormal M1S1 gene product causes GDLD is not known, although evidence suggests that it may compromise corneal epithelial function. This investigation attempted to determine the effect of the abnormal M1S1 gene product by assessing epithelial barrier function and epithelial ultrastructure in GDLD corneas. METHODS: Epithelial barrier function was assessed on the basis of fluorescein uptake. The method used a modified slit-lamp fluorophotometer. High-resolution scanning electron and atomic force microscopy was used to investigate the amyloid deposits and epithelial cell structure. RESULTS: Epithelial permeability was orders of magnitude higher in GDLD corneas than normal. The structure of the amyloid deposits was characterized, and clear abnormalities in epithelial morphology and cell junctions were observed. CONCLUSIONS: The high epithelial permeability observed in GDLD corneas was directly correlated with abnormalities in epithelial structure, including irregular cell junctions. This suggests that the abnormal M1S1 gene product may affect epithelial cell junctions resulting in increased cell permeability in GDLD corneas.
Asunto(s)
Permeabilidad de la Membrana Celular/fisiología , Distrofias Hereditarias de la Córnea/patología , Epitelio Corneal/metabolismo , Adulto , Medios de Contraste/administración & dosificación , Medios de Contraste/farmacocinética , Distrofias Hereditarias de la Córnea/metabolismo , Epitelio Corneal/ultraestructura , Fluoresceína/administración & dosificación , Fluoresceína/farmacocinética , Fluorofotometría , Humanos , Masculino , Microscopía de Fuerza Atómica , Microscopía Electrónica de Rastreo , Persona de Mediana Edad , Soluciones Oftálmicas , Índice de Severidad de la EnfermedadRESUMEN
PURPOSE: Surgery to reconstruct the ocular surface is greatly facilitated by the use of amniotic membrane, either as a biologic drape or, more recently, as a substrate for the transplantation of cultivated corneal epithelial cells. This study was designed to compare the usefulness of intact and denuded human amniotic membranes as a substrate for corneal epithelial cell culture. METHODS: Small (3-mm-diameter) biopsy specimens of superficial cornea including epithelium were excised from the central and limbal regions in rabbits. They were cultured on human amniotic membrane with or without amniotic epithelial cells and examined by light, scanning electron, and transmission electron microscopy. RESULTS: Cellular outgrowth from the central explants (n = 10) after 14 days in culture measured 1.82 +/- 2.62 mm2 on intact amniotic membrane and 131.83 +/- 28.31 mm2 on denuded amniotic membrane. In contrast, outgrowths from the limbal explants (n = 10) at the same time measured 4.58 +/- 4.56 and 505.39 +/- 134.20 mm2 on intact and denuded amniotic membranes, respectively. The leading edges of the outgrowths on intact amniotic membrane were much less uniform than those on denuded amniotic membrane, and, in the former, corneal epithelial cells appeared to migrate over the top of amniotic epithelial cells. Limbal cells cultivated on denuded amniotic membrane formed a nicely stratified layer that adhered well to the underlying amniotic membrane. CONCLUSIONS: Denuded amniotic membrane appears to be an excellent substrate for the cultivation of corneal epithelial cells, with a view to transplantation.
Asunto(s)
Amnios , Técnicas de Cultivo de Célula/métodos , Epitelio Corneal/citología , Células 3T3/citología , Amnios/citología , Amnios/ultraestructura , Animales , Técnicas de Cocultivo/métodos , Epitelio Corneal/ultraestructura , Humanos , Ratones , Microscopía Electrónica de Rastreo , ConejosRESUMEN
PURPOSE: To investigate the expression of growth factor mRNA and the level of growth factor protein in preserved human amniotic membrane (AM). METHODS: RT-PCR was used to examine the expression of mRNA for eight growth factors (EGF, TGF-alpha, KGF, HGF, bFGF, TGF-beta1, -beta2, -beta3) and two growth factor receptors (KGFR and HGFR) in human AM preserved at -80 degrees C for one month. In addition, ELISAs were used to measure the protein concentrations of seven growth factors (EGF, TGF-alpha, KGF, HGF, bFGF, TGF-beta1, -beta2) in preserved human corneas and in AM both with and without amniotic epithelium. RESULTS: RT-PCR revealed that human AM expresses mRNA for EGF, TGF-alpha, KGF, HGF, bFGF, TGF-beta1, -beta2, -beta3, KGFR and HGFR, while ELISAs showed that it contains EGF, TGF-alpha, KGF, HGF, bFGF, TGF-beta1, -beta2. AM without amniotic epithelium also contains all seven growth factors examined, however, in this tissue the protein levels of EGF, KGF, HGF and bFGF were found to be significantly lower than in native AM. CONCLUSIONS: Preserved human AM expresses mRNAs for a number of growth factors and contains several growth factor proteins that might benefit epithelialization after AM transplantation. High levels of EGF, KGF, HGF and bFGF in AM with amniotic epithelium as compared to AM without amniotic epithelium suggest an epithelial origin for these growth factors. We feel that EGF, KGF and HGF in particular might play important roles in ocular surface wound healing after AM transplantation.
Asunto(s)
Amnios/metabolismo , Factores de Crecimiento de Fibroblastos , Sustancias de Crecimiento/metabolismo , ARN Mensajero/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos , Criopreservación , Ensayo de Inmunoadsorción Enzimática , Factor de Crecimiento Epidérmico/genética , Factor de Crecimiento Epidérmico/metabolismo , Factor 10 de Crecimiento de Fibroblastos , Factor 2 de Crecimiento de Fibroblastos/genética , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Factor 7 de Crecimiento de Fibroblastos , Sustancias de Crecimiento/genética , Factor de Crecimiento de Hepatocito/genética , Factor de Crecimiento de Hepatocito/metabolismo , Humanos , Proteínas Proto-Oncogénicas c-met/genética , Proteínas Proto-Oncogénicas c-met/metabolismo , ARN Mensajero/genética , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos , Receptores de Factores de Crecimiento/genética , Receptores de Factores de Crecimiento/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Crecimiento Transformador alfa/genética , Factor de Crecimiento Transformador alfa/metabolismo , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
PURPOSE: We studied the difference in severity between primary and secondary Sjögren's syndrome (SS). SUBJECTS AND METHODS: Two groups of patients (all females, mean age: 58 years), 31 with primary SS and 18 with secondary SS were studied. We performed the following dry eye tests: fluorescein score and Rose Bengal staining, grading of tear lipid layer interference patterns, measurement of fluorescein break up time, cotton thread test, and Schirmer-I test. Auto antibodies were also investigated. RESULTS: There was no significant difference between primary and secondary SS with respect to any dry eye tests or auto antibodies. In primary SS, however, the presence of anti SS-A antibody was significantly correlated with Rose Bengal scores (p = 0.044). CONCLUSION: The severity of SS is independent of the primary or secondary type. In primary SS, the presence of anti SS-A antibody may be correlated with the severity.
Asunto(s)
Síndrome de Sjögren/diagnóstico , Adulto , Anciano , Anciano de 80 o más Años , Autoanticuerpos/sangre , Diagnóstico Diferencial , Femenino , Humanos , Persona de Mediana EdadRESUMEN
PURPOSE: To examine the viability of using human amniotic membrane as substrate for culturing corneal epithelial cells and transplanting them onto severely injured rabbit eyes. METHODS: An ocular-surface injury was created in the right eye of eight rabbits by a lamellar keratectomy extending 5 mm outside the limbus. Next, from the limbal region of the uninjured left eyes of five of these animals, a small biopsy of corneal epithelial cells was taken and cultured on acellular human amniotic membrane. One month later, the invading conjunctiva that covered the corneal surface of all eight injured eyes was surgically removed. Five of the eyes then received grafts of amniotic membrane containing autologous cultured epithelial cells, whereas the other three received grafts of acellular amniotic membrane alone. RESULTS: A confluent primary culture of limbal corneal epithelial cells was established on acellular human amniotic membrane after 14 days. Cells were partially stratified and fairly well attached to the underlying amniotic membrane, although a fully formed basement membrane was not evident. The three rabbits that received amniotic membrane transplantation alone all had total epithelial defects on the graft in the early postoperative period. Eyes that were grafted with amniotic membrane that contained cultivated epithelial cells, however, were all successfully epithelialized up to 5 days after surgery. CONCLUSION: Autologous transplantation of cultivated corneal epithelium is feasible by using acellular amniotic membrane as a carrier.
Asunto(s)
Amnios/citología , Amnios/trasplante , Trasplante de Córnea , Epitelio Corneal/citología , Limbo de la Córnea/citología , Células Madre/citología , Animales , Células Cultivadas , Córnea/cirugía , Lesiones de la Cornea , Modelos Animales de Enfermedad , Epitelio Corneal/trasplante , Lesiones Oculares/cirugía , Humanos , Conejos , Trasplante de Células Madre , Trasplante AutólogoRESUMEN
PURPOSE: To investigate the long-term prognosis for primary conjunctival malignant melanomas in Japan. MATERIALS & METHODS: We conducted a survey of 61 cases which had been reported in a 38-year period (1959 to 1996). We gathered information regarding the survival of patients, the post-operative follow-up period, the causes of death, and recurrences. Answers were obtained segarding 51 cases (84%). Detailed progress was identified in 23 of these cases. The survival rates were calculated using the Kaplan-Meier method. RESULTS: The survival rates were 95.1% after 1 year, 72.9% after 3 years, and 53.4% after 5 years. These values are relatively low compared with those reported in Europe and the United States.
Asunto(s)
Neoplasias de la Conjuntiva/mortalidad , Melanoma/mortalidad , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Japón/epidemiología , Masculino , Persona de Mediana Edad , Pronóstico , Tasa de SupervivenciaRESUMEN
In summary, we have demonstrated that the ocular surface epithelia express at least three mucin genes. We suggest that the gel-forming mucin MUC5AC is a major mucin forming the mucus gel of the tear film. We further suggest that MUC1 facilitates the spread of the MUC5-containing mucus on the ocular surface and, along with the mucus gel, prevents cell and debris adhesion to the ocular surface. The function of MUC4 at the ocular surface remains to be elucidated.
Asunto(s)
Conjuntiva/fisiología , Epitelio Corneal/fisiología , Mucinas/biosíntesis , Lágrimas/fisiología , Animales , Conjuntiva/citología , Células Epiteliales/citología , Células Epiteliales/fisiología , Glicosilación , Humanos , Mucina 5AC , Mucina 4 , Mucina 5B , Membrana Mucosa/fisiologíaRESUMEN
The human mucin gene MUC5AC codes for a large mucin which has tandem repeat units and cysteine rich regions characteristic of several members of this class of glycoproteins. Human epithelia expressing the mucin include that of stomach, bronchus/trachea, endocervix and conjunctiva. We report here a 3.8 kb partial sequence of a rat homologue for the human MUC5AC gene and compare its tandem repeat sequence and cysteine rich domains to those of the human and mouse gene. Rat and mouse have the same number of amino acids (16) in their Muc5AC tandem repeat units and share 69% sequence similarity, whereas human MUC5AC has only 8 amino acids in its tandem repeat. In rat, the tandem repeat domain is flanked at its 3' end by a non-repeat region coding for 1142 amino acids. Four cysteine rich subdomains were identified in this region; one of these has 64% similarity to a corresponding region in human MUC5AC and 80% similarity to a mouse MUC5AC cysteine rich region. Southern blot analysis revealed cross hybridization of a probe for the rat cysteine rich region, to human, mouse, rabbit, and porcine genomic DNA; the rat tandem repeat probe hybridized with mouse and rabbit only. Unlike humans, rat expressed MUC5AC message detectable by Northern blot and in situ hybridization only in stomach epithelium and conjunctival goblet cells.
Asunto(s)
Clonación Molecular , Mucinas/análisis , Secuencia de Aminoácidos , Animales , Southern Blotting , Secuencia de Consenso , ADN Complementario/química , ADN Complementario/aislamiento & purificación , Expresión Génica , Humanos , Ratones , Datos de Secuencia Molecular , Mucina 5AC , Mucinas/química , Mucinas/genética , Ratas , Ratas Sprague-Dawley , Secuencias Repetitivas de Ácidos Nucleicos , Análisis de Secuencia de ADN , Homología de Secuencia , Distribución TisularRESUMEN
PURPOSE: To determine whether human conjunctival epithelium expresses any of the human mucin genes designated MUC2 through MUC7. METHOD: Northern blot analysis was performed using total RNA isolated from surgically removed conjunctival tissues. Complementary DNA or oligonucleotides to the tandem repeat region of each mucin gene were labeled and hybridized to conjunctival RNA. In situ hybridization also was performed to determine the distribution of mucin mRNA. RESULTS: Only MUC4 and MUC5 probes hybridized to conjunctival RNA by Northern blot analysis. Both probes bound in a polydispersed pattern, which is characteristic of mucin genes. Using in situ hybridization, MUC4 mRNA was detected in the cells of the stratified conjunctival epithelium, whereas MUC5 mRNA expression was limited to goblet cells MUC4 or MUC5 probes did not hybridize to sections of corneal epithelium. CONCLUSIONS: The mucins MUC4 and MUC5 are expressed by the human conjunctiva. These mucins may play an important role in forming the tear-film layer at the air and ocular surface epithelium interface.
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Conjuntiva/metabolismo , Regulación de la Expresión Génica , Mucinas/biosíntesis , Mucinas/genética , Adulto , Anciano , Secuencia de Bases , Northern Blotting , Epitelio/metabolismo , Femenino , Humanos , Hibridación in Situ , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Mucinas/metabolismo , Sondas de Oligonucleótidos/química , ARN/aislamiento & purificación , ARN Mensajero/metabolismoRESUMEN
Daily profiles of somatostatin mRNA expression were investigated in the rat suprachiasmatic nucleus (SCN) by semiquantitative in situ hybridization histochemistry. Under 12 h light/12 h dark conditions, somatostatin mRNA signals were higher during the day time (Zeitgeber time (ZT) 1) than during the night time (ZT 16). This day-night difference was still maintained in constant darkness where the somatostatin mRNA was higher in the subjective day (circadian time (CT) 1) than in the subjective night (CT 16). Together with previous Northern blot hybridization studies, the present observation suggests that the level of somatostatin mRNA in SCN neurons is controlled by the circadian clock, independent of photic environment.
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Oscuridad , Fotoperiodo , ARN Mensajero/metabolismo , Somatostatina/genética , Núcleo Supraquiasmático/metabolismo , Animales , Secuencia de Bases , Northern Blotting , Ritmo Circadiano , Hibridación in Situ , Masculino , Datos de Secuencia Molecular , Sondas de Oligonucleótidos/genética , Ratas , Ratas WistarRESUMEN
PURPOSE: To determine if human corneal and conjunctival epithelial synthesize MUC1 mucin, a membrane-spanning mucin present in a variety of simple epithelia. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) was carried out to examine the expression of MUC1 mRNA by epithelial cells, using total cellular RNA prepared from cultured corneal epithelial cells and conjunctival epithelial cells stripped from the ocular surface with nitrocellulose filter paper. Northern blot analysis was performed to examine the transcription of MUC1 gene in cultured corneal epithelial cells. In situ hybridization histochemistry was performed to determine distribution of MUC1 mRNA in ocular surface epithelium. Human milk fat globule antibody (HMFG-1) and monoclonal antibody 139H2, which are specific for MUC1 core protein, were used in immunoblot analysis, immunohistochemistry, or both, to determine the presence and distribution of MUC1. RESULTS: MUC1 mRNA was detected in cultured corneal and ex vivo conjunctival epithelial cells by RT-PCR. Northern blot analysis showed production of two different sizes of transcripts in the cultured corneal epithelium. Expression of MUC1 mRNA was observed in all layers of corneal epithelium. By immunoblot analysis, HMFG-1 binding (> 200 kd) was detected in human cultured corneal epithelium, and its binding was enhanced by neuraminidase pretreatment. Immunohistochemically, HMFG-1 binding was observed along the apical membranes of corneal epithelium after neuraminidase pretreatment. In the conjunctiva, HMFG-1 and 139H2 binding were detected in the basal region and sporadically on the apical surface, but not in the goblet cells. CONCLUSIONS: The stratified epithelia of the cornea and conjunctiva synthesize MUC1 mucin. This transmembrane mucin may have a role in tear film spread and may prevent adhesion of foreign debris, cells, or pathogens to the ocular surface.
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Conjuntiva/metabolismo , Córnea/metabolismo , Glicoproteínas de Membrana/biosíntesis , Mucinas/biosíntesis , Secuencia de Bases , Northern Blotting , Neoplasias de la Mama/metabolismo , Células Cultivadas , Conjuntiva/citología , Córnea/citología , Cartilla de ADN/química , Epitelio/metabolismo , Técnica del Anticuerpo Fluorescente , Expresión Génica , Humanos , Hibridación in Situ , Glicoproteínas de Membrana/genética , Datos de Secuencia Molecular , Mucina-1 , Mucinas/genética , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Reacción en Cadena de la Polimerasa , ARN Mensajero/biosíntesis , Transcripción Genética , Células Tumorales CultivadasRESUMEN
PURPOSE: To examine whether the topical application of keratinocyte growth factor (KGF) can enhance corneal epithelial healing in vivo. In addition, the distribution of S-phase cells in KGF-treated and control corneas was investigated during regeneration and under normal conditions. METHODS: A 10-mm diameter epithelial defect was made in the center of rabbit corneas. A 50-microliters aliquot of 10 micrograms/ml human keratinocyte growth factor (hKGF) was then applied topically five times a day. The same volume of phosphate-buffered saline (PBS) vehicle was applied to the contralateral eye as a control. Each corneal epithelial defect was subsequently photographed every 12 hours and was measured by a computer-assisted digitizer. For the S-phase cell analysis, entire corneas were labeled with 3H-thymidine and were subjected to autoradiography at 24 hours after wounding or in the normal cornea at 24 hours after the application of KGF or PBS. RESULTS: Topical application of 10 micrograms/ml hKGF significantly accelerated corneal epithelial wound healing when compared with controls. In the S-phase cell analysis, KGF did not have any effect on normal corneal epithelial cells. However, in the regenerating cornea, the number of S-phase cells in the KGF-treated limbal epithelium was twofold higher than in the controls. CONCLUSIONS: Topical application of KGF accelerated corneal epithelial wound healing in vivo and increased cell proliferation in the limbal epithelium of the regenerating cornea.
Asunto(s)
Córnea/fisiología , Factores de Crecimiento de Fibroblastos , Sustancias de Crecimiento/farmacología , Cicatrización de Heridas/efectos de los fármacos , Administración Tópica , Animales , Autorradiografía , División Celular , Córnea/citología , Córnea/efectos de los fármacos , ADN/biosíntesis , Replicación del ADN/efectos de los fármacos , Replicación del ADN/fisiología , Epitelio/efectos de los fármacos , Epitelio/fisiología , Factor 10 de Crecimiento de Fibroblastos , Factor 7 de Crecimiento de Fibroblastos , Soluciones Oftálmicas , Conejos , Proteínas Recombinantes , Fase S/efectos de los fármacos , Fase S/fisiologíaRESUMEN
Healing of corneal wounds is a complex process involving epithelial, keratocyte, and endothelial interactions that are affected by their associations with wound bed matrix and by cytokine availability and activation. The spectrum of possible cellular-matrix-growth factor interactions is indeed great and growing. Several of the significant contributions made during the past year include development of an organotypic organ culture model of the cornea that allows in vitro assembly of the epithelial extracellular matrix-anchoring complex, demonstration of epithelial synthesis of Bowman's layer collagens, demonstration of transforming growth factor-beta 2's inhibition of stromal cell collagenase synthesis, and demonstration of the paracrine pathway of keratinocyte growth factor action in the cornea.