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










Base de dados
Tipo de estudo
Intervalo de ano de publicação
1.
Ophthalmology ; 126(11): 1549-1556, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31054281

RESUMO

PURPOSE: To characterize the genetic landscape of patients with suspected retinitis pigmentosa (RP) in the Chinese population. DESIGN: Cohort study. PARTICIPANTS: A total of 1243 patients of Chinese origin with clinically suspected RP and their available family members (n = 2701) were recruited. METHODS: All patients and available family members were screened using multigene panel testing (including 586 eye disease-associated genes), followed by clinical variant interpretation. MAIN OUTCOME MEASURES: Diagnostic yield, the 17 most commonly implicated genes, age at onset, de novo mutations, and clinical usefulness of genetic testing. RESULTS: Overall, 72.08% of patients received a molecular diagnosis, and the 17 top genes covered 75.63% of diagnostic cases. Diagnostic yield was higher among patients in the early-onset subgroup (≤5 years old, 79.58%) than in the childhood or adolescence-onset subgroup (6-16 years old, 73.74%) and late-onset subgroup (≥17 years old, 65.99%). Moreover, different genes associated with different onset ages and subgroups with different onset ages showed a diverse mutation spectrum. Only 11 de novo mutations (3.18%) were identified. Furthermore, 16.84% of the patients who received a molecular diagnosis had refinement of the initial clinical diagnoses, and the remaining 83.16% received definite genetic subtypes of RP. CONCLUSIONS: This large cohort study provides population-based data of the genome landscape of patients with suspected RP in China. The diagnostic yield was significantly higher than that in previous studies, and the mutation spectrum is completely different with other populations. Genetic testing improves the chance to establish a precise diagnosis, identifies features not previously determined, and allows a more accurate refinement of risk to family members. Our results not only expand the existing genotypic spectrum but also serve as an efficient reference for the design of panel-based genetic diagnostic testing and genetic counseling for patients with suspected RP in China.

2.
Cell Death Dis ; 9(5): 540, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29748605

RESUMO

Glaucoma is a neurodegenerative disease characterized by retinal ganglion cell (RGC) loss, optic disc excavation, and progressive visual field loss. Direct or indirect ameliorating retinal neurodegeneration is a promising therapeutic therapy for glaucoma. Circular RNAs (circRNAs) are a class of covalently closed circular RNA transcripts and have emerged as potential regulators in several neurodegenerative diseases. In this study, we show that cZRANB1 expression is significantly upregulated in retinal neurodegeneration induced by glaucoma. cZRANB1 knockdown decreases retinal reactive gliosis, glial cell activation, and facilitates RGC survival in vivo. cZRANB1 knockdown directly regulates Müller cell function and indirectly regulates RGC function in vitro. cZRANB1 acts as miRNA sponge to regulate Müller cell function through cZRANB1/miR-217/RUNX2 network. Intervention of cZRANB1 expression would become an effective strategy for treating retinal neurodegeneration.

3.
Mol Vis ; 23: 605-613, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28867931

RESUMO

PURPOSE: Familial exudative vitreoretinopathy (FEVR) is a genetically and clinically heterogeneous disease, characterized by failure of vascular development of the peripheral retina. The symptoms of FEVR vary widely among patients in the same family, and even between the two eyes of a given patient. This study was designed to identify the genetic defect in a patient cohort of ten Chinese families with a definitive diagnosis of FEVR. METHODS: To identify the causative gene, next-generation sequencing (NGS)-based target capture sequencing was performed. Segregation analysis of the candidate variant was performed in additional family members by using Sanger sequencing and quantitative real-time PCR (QPCR). RESULTS: Of the cohort of ten FEVR families, six pathogenic variants were identified, including four novel and two known heterozygous mutations. Of the variants identified, four were missense variants, and two were novel heterozygous deletion mutations [LRP5, c.4053 DelC (p.Ile1351IlefsX88); TSPAN12, EX8Del]. The two novel heterozygous deletion mutations were not observed in the control subjects and could give rise to a relatively severe FEVR phenotype, which could be explained by the protein function prediction. CONCLUSIONS: We identified two novel heterozygous deletion mutations [LRP5, c.4053 DelC (p.Ile1351IlefsX88); TSPAN12, EX8Del] using targeted NGS as a causative mutation for FEVR. These genetic deletion variations exhibit a severe form of FEVR, with tractional retinal detachments compared with other known point mutations. The data further enrich the mutation spectrum of FEVR and enhance our understanding of genotype-phenotype correlations to provide useful information for disease diagnosis, prognosis, and effective genetic counseling.


Assuntos
Grupo com Ancestrais do Continente Asiático/genética , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Mutação de Sentido Incorreto , Doenças Retinianas/genética , Deleção de Sequência , Tetraspaninas/genética , Adolescente , Adulto , China/epidemiologia , Estudos de Coortes , Análise Mutacional de DNA , Oftalmopatias Hereditárias , Feminino , Estudos de Associação Genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Linhagem , Fenótipo , Reação em Cadeia da Polimerase em Tempo Real , Adulto Jovem
4.
Circulation ; 136(17): 1629-1642, 2017 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-28860123

RESUMO

BACKGROUND: The vascular complications of diabetes mellitus are the major causes of morbidity and mortality among people with diabetes. Circular RNAs are a class of endogenous noncoding RNAs that regulate gene expression in eukaryotes. In this study, we investigated the role of circular RNA in retinal vascular dysfunction induced by diabetes mellitus. METHODS: Quantitative polymerase chain reactions, Sanger sequencing, and Northern blots were conducted to detect circular HIPK3 (circHIPK3) expression pattern on diabetes mellitus-related stresses. MTT (3-[4,5-dimethythiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assays, EdU (5-ethynyl-2'-deoxyuridine) incorporation assays, Transwell migration assays, and Matrigel assays were conducted to detect the role of circHIPK3 in retinal endothelial cell function in vitro. Retinal trypsin digestion, vascular permeability assays, and ELISA assays were conducted to detect the role of circHIPK3 in retinal vascular dysfunction in vivo. Bioinformatics analysis, luciferase activity assays, RNA pull-down assays, and in vitro studies were conducted to reveal the mechanism of circHIPK3-mediated retinal vascular dysfunction. RESULTS: circHIPK3 expression was significantly upregulated in diabetic retinas and retinal endothelial cells following stressors related to diabetes mellitus. circHIPK3 silencing or overexpressing circHIPK3 changed retinal endothelial cell viability, proliferation, migration, and tube formation in vitro. circHIPK3 silencing in vivo alleviated retinal vascular dysfunction, as shown by decreased retinal acellular capillaries, vascular leakage, and inflammation. circHIPK3 acted as an endogenous miR-30a-3p sponge to sequester and inhibit miR-30a-3p activity, which led to increased vascular endothelial growth factor-C, FZD4, and WNT2 expression. Ectopic expression of miR-30a-3p mimicked the effect of circHIPK3 silencing on vascular endothelial phenotypes in vivo and in vitro. CONCLUSIONS: The circular RNA circHIPK3 plays a role in diabetic retinopathy by blocking miR-30a function, leading to increased endothelial proliferation and vascular dysfunction. These data suggest that circular RNA is a potential target to control diabetic proliferative retinopathy.


Assuntos
Diabetes Mellitus Experimental/metabolismo , Retinopatia Diabética/metabolismo , Células Endoteliais/metabolismo , RNA não Traduzido/metabolismo , Vasos Retinianos/metabolismo , Animais , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patologia , Retinopatia Diabética/genética , Retinopatia Diabética/patologia , Células Endoteliais/fisiologia , Receptores Frizzled/biossíntese , Receptores Frizzled/genética , Regulação da Expressão Gênica , Masculino , Camundongos , MicroRNAs/biossíntese , MicroRNAs/genética , RNA não Traduzido/genética , Vasos Retinianos/patologia , Fator C de Crescimento do Endotélio Vascular/biossíntese , Fator C de Crescimento do Endotélio Vascular/genética , Proteínas Wnt/biossíntese , Proteínas Wnt/genética
5.
Front Genet ; 8: 107, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28890726

RESUMO

Purpose: To show early, rapid and accurate molecular diagnosis of occult macular dystrophy (OMD) in a four-generation Chinese family with inherited macular dystrophy. Methods: In the current study, we comprehensively screened 130 genes involved in common inherited non-syndromic eye diseases with next-generation sequencing-based target capture sequencing of the proband of a four-generation Chinese family that has suffered from maculopathy without a definitive diagnosis for over 10 years. Variants were filtered and analyzed to identify possible disease-causing variants before validation by Sanger sequencing. Results: Two heterozygous mutations-RP1L1 c.133 C > T (p.Arg45Trp), which is a hot spot for OMD, and ABCA4 c.6119 G > A (p.Arg2040Gln), which was identified in Stargardt's disease were found in three patients, but neither of the mutations was found in the unaffected individuals in the same family, who are phenotypically normal or in the normal control volunteers. Conclusion: These results cannot only confirm the diagnosis of OMD in the proband, but also provide presymptomatic diagnosis of the proband's children before the onset of visual acuity impairment and guidance regarding the prognosis and management of these patients. Heterozygous mutations of RP1L1 c.133 C > T (p.Arg45Trp) and ABCA4 c.6119 G > A (p.Arg2040Gln) are likely responsible for OMD. Our results further extend our current understanding of the genetic basis of OMD, and emphasize the importance of molecular diagnosis and genetic counseling for OMD.

6.
Front Mol Neurosci ; 10: 285, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28936163

RESUMO

Glaucoma is a progressive neuropathy characterized by the loss of retinal ganglion cells (RGCs). Strategies that delay or halt RGC loss have been recognized as potentially beneficial for rescuing vision in glaucoma patients. Quercetin (Qcn) is a natural and important dietary flavonoid compound, widely distributed in fruits and vegetables. Mounting evidence suggests that Qcn has numerous neuroprotective effects. However, whether Qcn exerts neuroprotective effects on RGC in glaucoma is poorly understood. In this study, we investigated the protective effect of Qcn against RGC damage in a rat chronic ocular hypertension (COHT) model invivo and hypoxia-induced primary cultured RGC damage in vitro, and we further explored the underlying neuroprotective mechanisms. We found that Qcn not only improved RGC survival and function from a very early stage of COHT invivo, it promoted the survival of hypoxia-treated primary cultured RGCs invitro via ameliorating mitochondrial function and preventing mitochondria-mediated apoptosis. Our findings suggest that Qcn has direct protective effects on RGCs that are independent of lowering the intraocular pressure (IOP). Qcn may be a promising therapeutic agent for improving RGC survival and function in glaucomatous neurodegeneration.

7.
Sci Rep ; 7(1): 1480, 2017 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-28469203

RESUMO

Our previous studies have demonstrated that activation of group I metabotropic glutamate receptors downregulated Kir channels in chronic ocular hypertension (COH) rats, thus contributing to Müller cell gliosis, characterized by upregulated expression of glial fibrillary acidic protein (GFAP). In the present study, we explored possible signaling pathways linking Kir channel inhibition and GFAP upregulation. In normal retinas, intravitreal injection of BaCl2 significantly increased GFAP expression in Müller cells, which was eliminated by co-injecting mitogen-activated protein kinase (MAPK) inhibitor U0126. The protein levels of phosphorylated extracellular signal-regulated protein kinase1/2 (p-ERK1/2) and its upstream regulator, p-MEK, were significantly increased, while the levels of phosphorylated c-Jun N-terminal kinase (p-JNK) and p38 kinase (p-p38) remained unchanged. Furthermore, the protein levels of phosphorylated cAMP response element binding protein (p-CREB) and c-fos were also increased, which were blocked by co-injecting ERK inhibitor FR180204. In purified cultured rat Müller cells, BaCl2 treatment induced similar changes in these protein levels apart from p-p38 levels and the p-p38:p38 ratio showing significant upregulation. Moreover, intravitreal injection of U0126 eliminated the upregulated GFAP expression in COH retinas. Together, these results suggest that Kir channel inhibition-induced Müller cell gliosis is mediated by the MEK-ERK/p38-CREB/c-fos signaling pathway.

9.
Front Cell Neurosci ; 10: 254, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27932951

RESUMO

Purpose: Our previous study indicated that mitochondrial DNA (mtDNA) damage and mutations are crucial to the progressive loss of retinal ganglion cells (RGCs) in a glaucomatous rat model. In this study, we examined whether high pressure could directly cause mtDNA alterations and whether the latter could lead to mitochondrial dysfunction and RGC death. Methods: Primary cultured rat RGCs were exposed to 30 mm Hg of hydrostatic pressure (HP) for 12, 24, 48, 72, 96 and 120 h. mtDNA alterations and mtDNA repair/replication enzymes OGG1, MYH and polymerase gamma (POLG) expressions were also analyzed. The RGCs were then infected with a lentiviral small hairpin RNA (shRNA) expression vector targeting POLG (POLG-shRNA), and mtDNA alterations as well as mitochondrial function, including complex I/III activities and ATP production were subsequently studied at appropriate times. Finally, RGC apoptosis and the mitochondrial-apoptosis pathway-related protein cleaved caspase-3 were detected using a Terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) assay and western blotting, respectively. Results: mtDNA damage was observed as early as 48 h after the exposure of RGCs to HP. At 120 h after HP, mtDNA damage and mutations significantly increased, reaching >40% and 4.8 ± 0.3-fold, respectively, compared with the control values. Twelve hours after HP, the expressions of OGG1, MYH and POLG mRNA in the RGCs were obviously increased 5.02 ± 0.6-fold (p < 0.01), 4.3 ± 0.2-fold (p < 0.05), and 0.8 ± 0.09-fold (p < 0.05). Western blot analysis showed that the protein levels of the three enzymes decreased at 72 and 120 h after HP (p < 0.05). After interference with POLG-shRNA, the mtDNA damage and mutations were significantly increased (p < 0.01), while complex I/III activities gradually decreased (p < 0.05). Corresponding decreases in membrane potential and ATP production appeared at 5 and 6 days after POLG-shRNA transfection respectively (p < 0.05). Increases in the apoptosis of RGCs and cleaved caspase-3 protein expression were observed after mtDNA damage and mutations. Conclusions: High pressures could directly cause mtDNA alterations, leading to mitochondrial dysfunction and RGC death.

10.
Front Hum Neurosci ; 10: 686, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-28154531

RESUMO

Mesencephalic astrocyte-derived neurotrophic factor (MANF), otherwise named Arginine-Rich, Mutated in Early-stage Tumors (ARMET), is a secretory endoplasmic reticulum stress (ERS) protein that is widely expressed in mammalian tissues. To date, little is known about the distribution and expression of MANF in the retina and optic nerve (ON). Therefore, we studied the expression and distribution of MANF in the ON and retina by real-time PCR, immunofluorescence staining and western blotting. Results from rat and mouse were highly consistent in the retina. MANF was detected in both tissues in rat, wherein it was principally localized to the ganglion cell layer (GCL), followed by the inner nuclear layer (INL). The MANF protein levels in the rat retina were 3.33-fold higher than in the rat ON. Additionally, MANF was robustly expressed by retinal ganglion cells (RGCs) in the human retina. In human ON, MANF was partially co-localized with glial fibrillary acidic protein (GFAP), suggesting that it was not restricted to astrocytes. In vitro studies confirmed that MANF could be robustly expressed in RGCs and was found principally within the cytoplasm. Hypoxia can stimulate up-regulation by of MANF expression over time, suggesting that MANF may play a vital role in the functional regulation of RGCs both in health and disease. We believe that the present study improves our understanding of the distribution and expression of MANF in the retina and ON and could help in further analysis of its interact and correlate with the relevant ophthalmic diseases.

11.
Neurosci Lett ; 588: 12-7, 2015 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-25549543

RESUMO

Müller cell gliosis is a general response in a variety of pathological alternations of the retina, which is characterized by the upregulated expression of glial fibrillary acidic protein (GFAP) and the downregulation of membrane K(+) conductance. We have demonstrated that downregulation of Kir K(+) currents in Müller cells in an experimental glaucoma model is due to activation of group I metabotropic glutamate receptor (mGluR I) by glutamate, which contributes to Müller cell gliosis. Here, whether and how activation of mGluR I modulate membrane Kir4.1 protein internalization and Kir4.1 mRNA expression were investigated in purified cultured rat retinal Müller cells using immunocytochemistry, Western blot and real-time PCR techniques. DHPG (10µM, a selective mGluR I agonist) treatment induced Müller cell gliosis, as evidenced by enhanced GFAP expression. Although total Kir4.1 proteins extracted from the DHPG-treated cells kept unchanged, Kir4.1 proteins in the cell membrane compartment were significantly decreased, which was prior to the change of GFAP in time course. In addition, DHPG (10 and 100µM) treatment induced a transient decrease in Kir4.1 mRNA expression in the cells. All these results suggest that activation of mGluR I by DHPG may decrease the number of functional Kir4.1 channels in purified cultured rat retinal Müller cells through modulating Kir4.1 protein and mRNA, thus contributing to Müller cell gliosis.


Assuntos
Células Ependimogliais/efeitos dos fármacos , Glicina/análogos & derivados , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , RNA Mensageiro/metabolismo , Receptores de Glutamato Metabotrópico/agonistas , Resorcinóis/farmacologia , Animais , Animais Recém-Nascidos , Membrana Celular/metabolismo , Células Cultivadas , Células Ependimogliais/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Gliose , Glicina/farmacologia , Canais de Potássio Corretores do Fluxo de Internalização/genética , Ratos Sprague-Dawley
12.
Neurobiol Dis ; 74: 167-179, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25478814

RESUMO

Glaucoma is a chronic neurodegenerative disease characterized by the progressive loss of retinal ganglion cells (RGCs). Mitochondrial DNA (mtDNA) alterations have been documented as a key component of many neurodegenerative disorders. However, whether mtDNA alterations contribute to the progressive loss of RGCs and the mechanism whereby this phenomenon could occur are poorly understood. We investigated mtDNA alterations in RGCs using a rat model of chronic intraocular hypertension and explored the mechanisms underlying progressive RGC loss. We demonstrate that the mtDNA damage and mutations triggered by intraocular pressure (IOP) elevation are initiating, crucial events in a cascade leading to progressive RGC loss. Damage to and mutation of mtDNA, mitochondrial dysfunction, reduced levels of mtDNA repair/replication enzymes, and elevated reactive oxygen species form a positive feedback loop that produces irreversible mtDNA damage and mutation and contributes to progressive RGC loss, which occurs even after a return to normal IOP. Furthermore, we demonstrate that mtDNA damage and mutations increase the vulnerability of RGCs to elevated IOP and glutamate levels, which are among the most common glaucoma insults. This study suggests that therapeutic approaches that target mtDNA maintenance and repair and that promote energy production may prevent the progressive death of RGCs.


Assuntos
Dano ao DNA , DNA Mitocondrial , Glaucoma/genética , Glaucoma/fisiopatologia , Mutação , Células Ganglionares da Retina/fisiologia , Animais , Apoptose/fisiologia , Axônios/patologia , Axônios/fisiologia , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Reparo do DNA , Modelos Animais de Doenças , Progressão da Doença , Glaucoma/patologia , Ácido Glutâmico/metabolismo , Pressão Intraocular/genética , Pressão Intraocular/fisiologia , Masculino , Mitocôndrias/genética , Mitocôndrias/patologia , Mitocôndrias/fisiologia , Nervo Óptico/patologia , Nervo Óptico/fisiopatologia , Ratos Wistar , Células Ganglionares da Retina/patologia , Fatores de Tempo
13.
Arch Pharm Res ; 38(5): 614-9, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25011569

RESUMO

Flavonoid glycosides are metabolized by intestinal bacteria, giving rise to a wide range of phenolic acids that may exert systemic effects in the body. The microbial metabolism of flavonoids extracted from the leaves of Diospyros kaki (FLDK) by intestinal bacteria was investigated in vitro. High-performance liquid chromatography/linear trap quadrupole orbitrap mass spectrometry was performed to analyze the metabolites of flavonoids in vivo using Xcalibur2.1 software. The results showed that the levels of flavonoid glycosides and flavonoid aglycones decreased rapidly in the process of microbial metabolism by intestinal bacteria in vitro, and the metabolic rate may be related to the concentration of intestinal bacteria in the culture solution. In vivo metabolites of FLDK were detected in rat plasma and urine after oral administration of FLDK. Eight flavonoids were identified in the urine, and three were identified in the plasma; however, flavonoid aglycones were not found in the plasma.


Assuntos
Diospyros/metabolismo , Medicamentos de Ervas Chinesas/metabolismo , Flavonoides/metabolismo , Microbioma Gastrointestinal/fisiologia , Folhas de Planta/metabolismo , Animais , Medicamentos de Ervas Chinesas/isolamento & purificação , Flavonoides/isolamento & purificação , Masculino , Ratos , Ratos Sprague-Dawley
14.
Zhongguo Zhong Yao Za Zhi ; 38(16): 2628-32, 2013 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-24228577

RESUMO

The research aimed at investigating the physicochemical properties, stability and skin penetration in vitro of total alkaloids of Sophora flavescens nanoemulsion. Prepare total alkaloids of S. flavescens nanoemulsion and detect the determination of matrine and oxymatrine in the nanoemulsion using HPLC method. Transmission electron microscopy and laser particle size analyzer were utilized to detect the shape and size of the nanoemulsion respectively. And also the stability of nanoemulsion was studied under the conditions of low temperature (4 degrees C), normal temperature (25 degrees C) and high temperature (60 degrees C). Franz diffusion cell was used to research the transdermal absorption of nanoemulsion in vitro. The results found that the nanoemulsion we prepared presented appearance of rounded, uniform; its average diameter was (15.55 +/- 2.24) nm, and particle size distribution value was 0. 161; the appearance, diameter and percentage determination of total alkaloids of S. flavescens had no variations after 15 d under 4, 25, 60 degrees C respectively. The steady-state permeation rate was 4.564 1 microg x cm(-2) x h(-1), 24 h cumulative amount of penetration was 110.7 microg x cm(-2), which was 1.86 fold of 24 h cumulative amount of aqueous solution (59.41 microg x cm(-2)). All the results demonstrated total alkaloids of S. flavescens nanoemulsion had good permeability, and could provide a new preparation for its clinical application.


Assuntos
Alcaloides/química , Alcaloides/metabolismo , Fenômenos Químicos , Portadores de Fármacos/química , Nanoestruturas/química , Absorção Cutânea , Sophora/química , Animais , Emulsões , Masculino , Ratos , Ratos Sprague-Dawley
15.
Neurosci Lett ; 554: 99-104, 2013 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-24025791

RESUMO

Elevated intraocular pressure (IOP) is considered as the major risk factor for the loss of retinal ganglion cells (RGCs) and their axons in glaucoma. Emerging evidence suggests elevated IOP can induce Drp1 upregulation and mitochondrial fission, which is involved in cell death. However, the underlying mechanism for these effects remains unknown. The present study used RNAi screening to investigate the effects of 24 kinases associated with mitochondrial activities on DRP1 expression under hydrostatic pressure. We identified, for the first time, that glycogen synthase kinase 3 beta (GSK3ß) knockdown suppressed the upregulation of DRP1 induced by elevated pressure. Use of the pharmacological inhibitor of GSK3ß inhibitor, lithium chloride (LiCl), confirmed this result. Furthermore, we demonstrated that one of the mechanisms of lithium chloride neuroprotection might be via inhibition of mitochondrial fission through downregulation of Drp1.


Assuntos
Dinaminas/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Cloreto de Lítio/farmacologia , Fármacos Neuroprotetores/farmacologia , Interferência de RNA , Epitélio Pigmentado da Retina/efeitos dos fármacos , Animais , Células Cultivadas , Regulação para Baixo , Dinaminas/genética , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Técnicas de Silenciamento de Genes , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/genética , Glicogênio Sintase Quinase 3 beta , Humanos , Pressão Hidrostática , Dinâmica Mitocondrial , Cultura Primária de Células , Ratos Wistar , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/metabolismo
16.
Zhonghua Yan Ke Za Zhi ; 48(7): 615-8, 2012 Jul.
Artigo em Chinês | MEDLINE | ID: mdl-22943866

RESUMO

OBJECTIVE: To establish a method of purifying and characterizing adult astrocytes from optic nerve head (ONH). METHODS: Experimental study. The lamina cribrosa tissue from ONH of human eye was isolated under anatomic microscopy, and then 4 to 6 little explants were incubated in each culture plate containing culture medium DMEM/F12. After 8 to 10 weeks, the cells were removed by digesting cells with 0.25% trypsogen. Selective astrocyte culture medium is subsequently used. After two passages, astrocytes were identified by the observation of cell morphology and immunofluorescent staining of GFAP and NCAM. RESULTS: After 2 to 3 weeks of explants planting, cells showed an obvious migration procession by crawling in succession from the verge of the explants and rapidly splitting. Most cells displayed a flat star shape or polygon after digested with trypsogen. Several cells are long fusiformis. Almost all cells presented a flat star shape and simultaneously expressed GFAP and NCAM when the cells cultured with selective astrocyte culture medium. CONCLUSIONS: Cultured human ONH astrocytes can be obtained by precisely separating lamina cribrosa and placing the explants on the margin of culture medium, a method that promotes cell adherence. Using selective astrocyte culture medium is very effective and convenient in purifying primary astrocytes.


Assuntos
Astrócitos/citologia , Técnicas de Cultura de Células , Disco Óptico/citologia , Adulto , Células Cultivadas , Humanos
17.
Ophthalmic Res ; 47(2): 87-97, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-21865765

RESUMO

AIM: To determine whether a diet containing excessive amounts of milk aggravates naphthalene-initiated cataracts in a common animal model of age-related human cataract. METHODS: Ninety Sprague-Dawley rats were fed a natural diet supplemented with either water (group A), normal amounts of milk (group B), excessive amounts of milk (group C), naphthalene plus water (group D), naphthalene plus normal amounts of milk (group E), naphthalene plus excessive amounts of milk (group F). Cataract development was monitored weekly using a slit lamp and lens gray value analysis. Concentrations of reactive oxygen species (ROS), reduced glutathione (GSH) and malondialdehyde (MDA) in rat lenses were measured to determine the role of oxidative stress in cataract induction. RESULTS: By week 4, the cortical gray value was significantly higher in group F than that in group D, and the cortical gray value was significantly higher in group D than in group A. However, by week 8, no significant differences were observed among groups C, F, B, E and A. ROS concentrations in lenses of rats of groups C and F were slightly higher than in those of groups B, E and A, but ROS concentrations in group F were significantly higher than in the other groups receiving naphthalene (i.e. groups D and E). GSH concentrations in group F were significantly lower than in the other groups. MDA concentrations in group F were significantly higher than in the other groups receiving naphthalene, indicating increased lipid peroxidation induced by naphthalene plus excessive intake of milk. CONCLUSIONS: Our results provide quantitative evidence that excessive intake of milk aggravates naphthalene-initiated cataracts, which is probably due to oxidative damage caused by increased ROS.


Assuntos
Catarata/induzido quimicamente , Leite/efeitos adversos , Estresse Oxidativo/fisiologia , Animais , Catarata/metabolismo , Catarata/fisiopatologia , Dieta , Modelos Animais de Doenças , Feminino , Glutationa/metabolismo , Cristalino/metabolismo , Masculino , Malondialdeído/metabolismo , Naftalenos , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Fatores de Risco
18.
Zhong Yao Cai ; 34(11): 1776-80, 2011 Nov.
Artigo em Chinês | MEDLINE | ID: mdl-22506405

RESUMO

OBJECTIVE: To investigate the optimization of extraction conditions of saikosaponins a, c and d from Bupleurum falcatum. METHODS: Experimental factors and levels were firstly selected by one-factor test. According to the central composite experimental design principle, the response surface methodology with 3 factors and 3 levels was adopted. Used HPLC to determine the contents of saikosaponins a, c and d. Used column symmetry C18 (150 mm x 4.6 mm, 5 microm); Mobile phase was acetonitrile-water gradient elution, flow rate was 1.0 mL/min, the detection wavelength was 200 nm, detecting temperature was 25 degrees C. RESULTS: The optimum conditions of aikosaponins extraction were as follows:extraction time was 93 min, power was 150 W, solvent to solid ratio was 60. The predicted total saikosaponin extraction yield was 13.66 mg/g,while the actual extraction yield was 13.64 mg/g, with relative error of 0.15%. CONCLUSION: The optimum extraction process is reasonable, reliable and high yield of extracting effective content.


Assuntos
Bupleurum/química , Ácido Oleanólico/análogos & derivados , Plantas Medicinais/química , Saponinas/isolamento & purificação , Ultrassom , Cromatografia Líquida de Alta Pressão/métodos , Etanol/química , Ácido Oleanólico/análise , Ácido Oleanólico/isolamento & purificação , Raízes de Plantas/química , Saponinas/análise , Solventes/química , Temperatura Ambiente , Fatores de Tempo
19.
Curr Eye Res ; 33(1): 81-90, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18214745

RESUMO

The transduction efficiency and cell tropism of viral vectors rAAV2/1, rAAV2, Ad5, Ad5/F35, and Lentivirus were evaluated in retina. All viral vectors achieved efficient transduction in living rat retina. However, each vector showed distinctive efficiency in vitro especially for rAAV2/1, which displayed poor transduction in cultured retinal cells. Distinctive cell-specific GFP expression was observed in vivo and in vitro for the same viral vector. The cell-specific tropism was not strictly correlated with the correspondent distribution of viral receptors in retina. These results provided important insights into the selection of appropriate vectors when specific retinal diseases are considered for gene therapy.


Assuntos
Adenoviridae/genética , Dependovirus/genética , Vetores Genéticos , HIV-1/genética , Retina/metabolismo , Transdução Genética , Adulto , Animais , Células Cultivadas , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus , Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteoglicanas de Heparan Sulfato/metabolismo , Humanos , Técnicas Imunoenzimáticas , Integrinas/metabolismo , Proteína Cofatora de Membrana/metabolismo , Epitélio Pigmentado Ocular/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores Virais/metabolismo , Receptores de Vitronectina/metabolismo , Tropismo
20.
Zhonghua Yi Xue Za Zhi ; 86(40): 2841-6, 2006 Oct 31.
Artigo em Chinês | MEDLINE | ID: mdl-17200021

RESUMO

OBJECTIVE: To investigate the transduction and gene expression of the recombinant adeno-associated viruses (rAAV) of the serotypes 1 and 2 in the retinal cells. METHODS: rAAV vectors of type 1 and type 2 encoding EGFP were infected into the cultured retinal pigmentary epithelium (RPE) cells of the line CRL-2302 and primarily cultured retinal neural cells from normal SD rats, and primarily cultured RPE cells from an adult cornea donor. The cultured RPE cells transduced by rAAV2-EGFP or rAAV2/1-EGFP were harvested at the 7 th and 14 th day after infection to be detected by fluorescence-activated cell sorter. The onset of EGFP gene expression and EGFP positive rate were detected by flow cytometry and fluorescence microscopy. Then, rAAV2/1-EGFP and rAAV2-EGFP were injected into the subretinal spaces of 32 SD rats to investigate the onset of EGFP fluorescence and its distribution in the fundus in vivo via fluorescence stereoscope. HE staining and immunohistochemistry were used to observe the infected cell type and immune response in the retina. RESULTS: The percentage of EGFP positive cells and mean intensity of EGFP fluorescence in the cells transduced by rAAV2-EGFP 7 and 14 days after transduction were 13.50% +/- 1.70% and 15.60% +/- 0.82%, and 2.75 +/- 0.12 and 3.80 +/- 0.72 respectively; and the EGFP positive cells and mean intensity of EGFP fluorescence in the cells transduced by rAAV2/1-EGFP were 1.09% +/- 0.5% and 1.98% +/- 0.45%, and 1.12 +/- 0.09 and 1.75 +/- 0.2 respectively. The EGFP fluorescence area in the retina were (5389 +/- 211) microm(2), (9832 +/- 364) microm(2), (14 454 +/- 446) microm(2), (20 528 +/- 648) microm(2), and (20 264 +/- 683) microm(2) respectively 3, 7, 14, and 75 days, and 4 month after transduction by rAAV2-EGFP in vivo; In the rat retina transduced by rAAV2/1-EGFP, the EGFP fluorescence areas were (9666 +/- 348) microm(2), (12 160 +/- 439) microm(2), (19 794 +/- 621) microm(2), (26 172 +/- 923) microm(2), and (26 022 +/- 965) microm(2) respectively 3, 7, 14, and 75 days, and 4 month after infection. CONCLUSION: rAAV2 efficiently transduces retinal cells both in vitro and in vivo. rAAV2/1 is a more effective gene-transferring vector to be used in retinal cells in vivo than rAAV2.


Assuntos
Dependovirus/genética , Epitélio Pigmentado Ocular/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Citometria de Fluxo , Vetores Genéticos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Microscopia de Fluorescência , Epitélio Pigmentado Ocular/citologia , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Retina/citologia , Transfecção
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA