Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 6 de 6
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Mol Vis ; 16: 2867-72, 2010 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-21203402

RESUMO

PURPOSE: To establish a robust method to isolate primary retinal ganglion cells (RGCs) from human fetal retina for long-term culture while maintaining neuronal morphology and marker protein expression. METHODS: A total of six human retinas were obtained from aborted fetuses at 10 to 12 weeks of gestation with informed consent from mothers. RGCs were isolated and purified by a modified two-step immunopanning procedure. The cells were maintained in a serum-free defined medium supplemented with brain-derived neurotrophic factor, ciliary neutrophic factor, and forskolin. The viable RGCs and the extent of neurite outgrowth were examined by calcein-acetoxymethylester assay. Expression of RGC markers was studied by immunocytochemistry. RESULTS: Primary RGCs from human fetal retinas were isolated and maintained in vitro for one month with substantial neurite elongation. In cell culture, almost 70% of the isolated cells attached, spread, and displayed numerous dendrites. They were immunoreactive to RGC-specific markers (Thy-1, TUJ-1, and Brn3a) and negative for glial fibrillary acidic protein and amacrine cells marker HPC-1. CONCLUSIONS: Human RGCs were successfully isolated and maintained in long-term culture. This can serve as an ideal model for biologic, toxicological, and genomic assays of human RGCs in vitro.


Assuntos
Técnicas de Cultura de Células/métodos , Separação Celular/métodos , Células Ganglionares da Retina/citologia , Células Ganglionares da Retina/imunologia , Biomarcadores/metabolismo , Células Cultivadas , Feto/citologia , Humanos , Especificidade de Órgãos , Fatores de Tempo
2.
Mol Vis ; 15: 2239-48, 2009 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-19907666

RESUMO

PURPOSE: The PAX6 gene, located at the reported myopia locus MYP7 on chromosome 11p13, was postulated to be associated with myopia development. This study investigated the association of PAX6 with high myopia in 379 high myopia patients and 349 controls. METHODS: High myopia patients had refractive errors of -6.00 diopters or greater and axial length longer than 26 mm. Control subjects had refractive errors less than -1.00 diopter and axial length shorter than 24 mm. The P1 promoter, all coding sequences, and adjacent splice-site regions of the PAX6 gene were screened in all study subjects by polymerase chain reaction and direct sequencing. PAX6 P1 promoter-luciferase constructs with variable AC and AG repeat lengths were prepared and transfected into human ARPE-19 cells prior to assaying for their transcriptional activities. RESULTS: No sequence alterations in the coding or splicing regions showed an association with high myopia. Two dinucleotide repeats, (AC)(m) and (AG)(n), in the P1 promoter region were found to be highly polymorphic and significantly associated with high myopia. Higher repeat numbers were observed in high myopia patients for both (AC)(m) (empirical p = 0.013) and (AG)(n) (empirical p = 0.012) dinucleotide polymorphisms, with a 1.327-fold increased risk associated with the (AG)(n) repeat (empirical p = 0.016; 95% confidence interval: 1.059-1.663). Luciferase-reporter analysis showed elevated transcription activity with increasing individual (AC)(m) and (AG)(n) and combined (AC)(m)(AG)(n) repeat lengths. CONCLUSIONS: Our results revealed an association between high myopia and AC and AG dinucleotide repeat lengths in the PAX6 P1 promoter, indicating the involvement of PAX6 in the pathogenesis of high myopia.


Assuntos
Repetições de Dinucleotídeos/genética , Proteínas do Olho/genética , Proteínas de Homeodomínio/genética , Miopia/genética , Fatores de Transcrição Box Pareados/genética , Regiões Promotoras Genéticas , Proteínas Repressoras/genética , Sequência de Bases , Sítios de Ligação , Estudos de Casos e Controles , Frequência do Gene/genética , Humanos , Dados de Sequência Molecular , Fator de Transcrição PAX6 , Fatores de Transcrição/metabolismo , Transcrição Gênica
3.
Mol Vis ; 15: 1521-9, 2009 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-19668596

RESUMO

PURPOSE: To identify the genetic lesions for congenital coralliform cataract. METHODS: Two Chinese families with autosomal dominant coralliform cataract, 12 affected and 14 unaffected individuals, were recruited. Fifteen known genes associated with autosomal dominant congenital cataract were screened by two-point linkage analysis with gene based single nucleotide polymorphisms and microsatellite markers. Sequence variations were identified. Recombinant FLAG-tagged wild type or mutant gammaD-crystallin was expressed in human lens epithelial cells and COS-7 cells. Protein solubility and intracellular distribution were analyzed by western blotting and immunofluorescence, respectively. RESULTS: A novel heterozygous change, c.43C>A (R15S) of gammaD-crystallin (CRYGD) co-segregated with coralliform cataract in one family and a known substitution, c.70C>A (P24T), in the other family. Unaffected family members and 103 unrelated control subjects did not carry these mutations. Similar to the wild type protein, R15S gammaD-crystallin was detergent soluble and was located in the cytoplasm. ProtScale and ScanProsite analyses revealed raised local hydrophobicity and the creation of a hypothetical casein kinase II phosphorylation site. CONCLUSIONS: A novel R15S mutation caused congenital coralliform cataract in a Chinese family. R15S possessed similar properties to the wild type gammaD-crystallin, but its predicted increase of hydrophobicity and putative phosphorylation site could lead to protein aggregation, subsequently causing opacification in lens.


Assuntos
Catarata/congênito , Catarata/genética , Mutação/genética , gama-Cristalinas/genética , gama-Cristalinas/metabolismo , Adulto , Sequência de Aminoácidos , Arginina/genética , Povo Asiático , Sequência de Bases , Criança , Pré-Escolar , Biologia Computacional , Análise Mutacional de DNA , Família , Feminino , Ligação Genética , Humanos , Interações Hidrofóbicas e Hidrofílicas , Lactente , Masculino , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/genética , Especificidade de Órgãos , Linhagem , Transporte Proteico , Serina/genética , Solubilidade , gama-Cristalinas/química
4.
Mol Vis ; 15: 89-98, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19145250

RESUMO

PURPOSE: To evaluate the individual and interactive effects of polymorphisms in the myocilin (MYOC),optineurin (OPTN), WD repeat domain 36 (WDR36), and apolipoprotein E (APOE) genes on primary open-angle glaucoma (POAG) in northern Chinese. METHODS: Northern Chinese study subjects, 176 POAG patients and 200 controls, were recruited for screening of the coding exons and splicing regions of MYOC. Five single nucleotide polymorphisms (SNPs) in OPTN (M98K, R545Q, IVS5+38T>G, IVS8-53T>C, and IVS15+10G>A), one SNP in WDR36 (IVS5+30C>T) as well as the APOE promoter and epsilon2/epsilon3/epsilon4 polymorphisms were also examined. Association analysis was performed by using chi(2) analysis. High-order gene-gene interaction was also analyzed using the multifactor dimensionality reduction (MDR) method. RESULTS: In MYOC, 22 variants were identified. Four of them were novel but found in controls only. The missense mutation, Val53Ala, is likely a glaucoma causing mutation, accounting for 0.6% of cases. No individual polymorphism in OPTN, WDR36, or APOE was associated with POAG. MDR analysis identified a best 6-factor model for POAG: MYOC IVS2+35A>G, OPTN Met98Lys, OPTN IVS5+38T>G, OPTN IVS8-53T>C, WDR36 IVS5+30C>T, and APOE -491A>T. CONCLUSIONS: The association pattern between the genes, MYOC, OPTN, WDR36, and APOE, and POAG in northern Chinese is different from that of southern Chinese. Disease-causing mutations in MYOC accounted for a small proportion of northern Chinese POAG patients. Common polymorphisms in these genes were not associated with POAG individually but might interactively contribute to the disorder, supporting a polygenic etiology.


Assuntos
Povo Asiático/genética , Glaucoma de Ângulo Aberto/genética , Polimorfismo Genético , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Apolipoproteínas E/genética , Proteínas de Ciclo Celular , Criança , China , Proteínas do Citoesqueleto/genética , Epistasia Genética , Proteínas do Olho/genética , Feminino , Predisposição Genética para Doença , Glicoproteínas/genética , Haplótipos , Humanos , Masculino , Proteínas de Membrana Transportadoras , Pessoa de Meia-Idade , Modelos Genéticos , Polimorfismo de Nucleotídeo Único/genética , Fator de Transcrição TFIIIA/genética
5.
Eur J Neurosci ; 26(4): 828-42, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17714182

RESUMO

Recently we unexpectedly found that PI3K/akt, JAK/STAT and MEK/ERK pathway inhibitors enhanced retinal ganglion cell (RGC) survival after optic nerve (ON) axotomy in adult rat, a phenomenon contradictory to conventional belief that these pathways are pro-survival. In this study we showed that: (i) the RGC protection was pathway inhibition-dependent; (ii) inhibition of PI3K/akt and JAK/STAT, but not MEK/ERK, activated macrophages in the eye, (iii) macrophage removal from the eye using clodronate liposomes significantly impeded PI3K/akt and JAK/STAT inhibition-induced RGC survival and axon regeneration whereas it only slightly affected MEK/ERK inhibition-dependent protection; (iv) in the absence of recruited macrophages in the eye, inhibition of PI3K/akt or JAK/STAT did not influence RGC survival; and (v) strong PI3K/akt, JAK/STAT and MEK/ERK pathway activities were located in RGCs but not macrophages after ON injury. In retinal explants, in which supply of blood-derived macrophages is absent, MEK/ERK inhibition promoted RGC survival whereas PI3K/akt or JAK/STAT inhibition had no effect on RGC viability. However, MEK/ERK inhibition exerted opposite effects on the viability of purified adult RGCs at different concentrations in vitro, suggesting that this pathway may be bifunctional depending on the level of pathway activity. Our data thus demonstrate that inhibition of the PI3K/akt or JAK/STAT pathway activated macrophages to facilitate RGC protection after ON injury whereas the two pathways per se did not modulate RGC viability under the injury conditions (in the absence of the pathway activators). In contrast, the MEK/ERK pathway inhibition protected RGCs via macrophage-independent mechanism(s).


Assuntos
MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Janus Quinases/fisiologia , Fármacos Neuroprotetores/farmacologia , Traumatismos do Nervo Óptico/patologia , Fosfatidilinositol 3-Quinases/fisiologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Células Ganglionares da Retina/fisiologia , Fatores de Transcrição STAT/fisiologia , Transdução de Sinais/fisiologia , Analgésicos não Narcóticos/farmacologia , Animais , Axônios/patologia , Axotomia , Western Blotting , Butadienos/farmacologia , Cromonas/farmacologia , Ácido Clodrônico/farmacologia , Inibidores Enzimáticos/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Flavonoides/farmacologia , Imuno-Histoquímica , Janus Quinases/antagonistas & inibidores , Macrófagos/efeitos dos fármacos , Morfolinas/farmacologia , Nitrilas/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Ratos , Ratos Endogâmicos F344 , Fatores de Transcrição STAT/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Tirfostinas/farmacologia
6.
Cell Transplant ; 13(5): 585-94, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15565870

RESUMO

The present study was designed to evaluate the use of collagen gel loaded with human retinal pigment epithelium (ARPE19) in cellular transfer and to assess its viability within the gel. Collagen solution was prepared by dissolving calfskin in hydrochloric acid to make a final concentration of 2.0 mg/ml and this was mixed with 10,000 ARPE19 cells/ml. The cell viability in gel was determined using MTT assay. van Gieson stain and proliferating cell nuclear antigen (PCNA) were used to identify the location of collagen and to localize the site of cell proliferation, respectively. The ARPE19 cells in gel appeared to be healthy with a rounded morphology. The optimal collagen concentration was 1.9 mg/ml. When this concentration was used to hold cells for over 12 days, it could be seen that the growth rate was the same between day 2 and day 8 in gel and on plastic. When the cell-loaded gels were transferred onto standard tissue culture plastics, progressive cell migrations over time resembling cell migrations in organotypic explant cultures were observed. Upon intravitreal injection of cell-containing collagen suspension into a rabbit's eye, the gel became suspended within the vitreous a few hours after injection (day 0). However, it became obvious that the gel dispersed and spread around the vitreous even after just 24 h. These cells inside the vitreous were PCNA positive, indicating that the human ARPE19 cells have the capacity to proliferate even after 11 days. The present study demonstrated the potential use of collagen gel as a tool in the transfer of cellular matrix onto other substrates. The results show that the cell seeding number must be critically balanced with the concentration of gel for it to be used as transplant material.


Assuntos
Transplante de Células/métodos , Colágeno/farmacologia , Olho/citologia , Transplante de Tecidos/métodos , Animais , Linhagem Celular , Movimento Celular , Proliferação de Células , Sobrevivência Celular , Colágeno/química , Meios de Cultura , Humanos , Ácido Clorídrico/química , Ácido Clorídrico/farmacologia , Imuno-Histoquímica , Microscopia de Contraste de Fase , Epitélio Pigmentado Ocular/citologia , Antígeno Nuclear de Célula em Proliferação/biossíntese , Coelhos , Fatores de Tempo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...