RESUMO
ADAM17 (a disintegrin and metallopeptidase domain 17) is crucial for eye morphogenesis. In this study we analysed the expression pattern of ADAM17 during mouse eye development. ADAM17 expression in adult retina was examined using the reverse transcription-polymerase chain reaction (RT-PCR) and verification of the RT-PCR products by DNA sequencing. Immunohistochemistry was performed to evaluate the ADAM17 expression pattern in mouse eyes at developmental stages of embryonic day (E) 12, E14, E16, E18, postnatal day (P) 0, P1, P4, P7, P14, P 30 and P175 (adult). We detected ADAM17 mRNA in adult retina tissue. ADAM17 protein was expressed in non-pigmented ciliary epithelial cells and in retinal vessels from P7 onwards during eye development. In corneal epithelial cells and endothelium, ADAM17 protein was present from P14 onwards. Although, mice in which the functional ADAM17 gene is significantly reduced develop multiple eye malformations, the expression of ADAM17 is not ubiquitous over the entire eye. Its expression pattern during development suggests that not only TNF-alpha but additional membrane-anchored substrates of ADAM17 play an important role in eye formation.
Assuntos
Proteínas ADAM/biossíntese , Envelhecimento/metabolismo , Desenvolvimento Embrionário/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Retina/embriologia , Retina/metabolismo , Proteína ADAM17 , Animais , Feminino , Masculino , Camundongos , Distribuição TecidualRESUMO
The Rho GTPase activating protein (RhoGAP) Oligophrenin 1 (Ophn1) regulates numerous members of the Rho family that are involved in neuronal morphogenesis of the central and peripheral nervous system. In the present study we investigated the spatial and temporal expression of Ophn1 in the mouse eye. The expression of Ophn1 was analysed on both mRNA and protein level. To identify the Ophn1 transcripts, adult retina and cerebrum (positive control) of postnatal day (P) 158 was subjected to reverse transcription polymerase chain reaction (RT-PCR) and sequencing of the amplified cDNA. The Ophn1 protein was analyzed in adult retina by Western blotting and in developing eyes at embryonic day (E) 12, E14, E16, E18, P0, P3, P7, P14 and P158 by immunohistochemistry. Ophn1 transcripts were detected in adult retina by RT-PCR and confirmed by sequencing. Western blot analysis revealed the expression of Ophn1 protein in the adult retina. Immunohistochemical examination of developing eyes localized the protein to retinal vasculature with an onset of Ophn1 expression from P14 onwards. The specific expression pattern suggests that Ophn1 could have a physiological role in the retinal vasculatures. At P14, the vessel development in the retina is widely completed, implying that Ophn1 has either a function during adulthood or for the generation of the intermediate plexus during the late vessel development of the retina.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Proteínas Nucleares/metabolismo , Retina/metabolismo , Vasos Retinianos/metabolismo , Animais , Cérebro/citologia , Cérebro/metabolismo , Proteínas do Citoesqueleto/genética , DNA Complementar/genética , DNA Complementar/metabolismo , Proteínas Ativadoras de GTPase/genética , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Nucleares/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Retina/citologia , Retina/embriologia , Neurônios Retinianos/citologia , Neurônios Retinianos/metabolismo , Vasos Retinianos/citologia , Vasos Retinianos/embriologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
PURPOSE: The present study was conducted to evaluate the therapeutic effects of topically applied bone marrow (BM) cells and CD117-positive haematopoietic stem (CD117(+)) cells on alkali-induced corneal ulcers. METHODS: Bone marrow cells and CD117(+) cells were isolated from syngenic mice and labelled with an intracellular cell tracer. Defined corneal wounds were produced in 89 eyes of syngenic mice and allowed to partially heal in vivo for 6 hr. The alkali-burned eyes were enucleated 6 hr postinjury and randomly divided into three groups. Control group (33 eyes) was incubated with medium only. The treatment groups received either BM cells (30 eyes) or CD117(+) cells (26 eyes) suspended in medium. Re-epithelialization process of corneal defects was qualitatively and quantitatively assessed and statistically analysed. The corneas were examined by histological and immunohistochemical methods. RESULTS: We found that the re-epithelialization of corneal wounds in both treatment groups was significantly accelerated as compared to the control group. During the follow-up period (85 hr), the corneal transparency was comparable in all groups. Morphological investigations of corneas from control and treatment group showed no evident differences in the phenotype of the regenerated epithelium. Additionally, corneas in the treatment groups were devoid of donor-derived BM cells and CD117(+) cells, respectively. CONCLUSIONS: This study provides evidence that topical application of BM cells or CD117(+) cells can be used to reconstruct corneal surfaces. Because neither BM cells nor CD117(+) cells were integrated into the corneal epithelium, we suggest that soluble factors could be responsible for the positive effect of BM cells and CD117(+) cells on corneal wound healing.