Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 3 de 3
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
J Biomed Sci ; 23: 34, 2016 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-26956435

RESUMO

BACKGROUND: Parkinson's disease (PD) is a severe neurodegenerative disease associated with loss of dopaminergic neurons. Derivation of dopaminergic neurons from human embryonic stem cells (hESCs) could provide new therapeutic options for PD therapy. Dopaminergic neurons are derived from SOX(-) floor plate (FP) cells during embryonic development in many species and in human cell culture in vitro. Early treatment with sonic hedgehog (Shh) has been reported to efficiently convert hESCs into FP lineages. METHODS: In this study, we attempted to utilize a Shh-free approach in deriving SOX1(-) FP cells from hESCs in vitro. Neuroectoderm conversion from hESCs was achieved with dual inhibition of the BMP4 (LDN193189) and TGF-ß signaling pathways (SB431542) for 24 h under defined culture conditions. RESULTS: Following a further 5 days of treatment with LDN193189 or LDN193189 + SB431542, SOX1(-) FP cells constituted 70-80 % of the entire cell population. Upon treatment with Shh and FGF8, the SOX1(-) FP cells were efficiently converted to functional Nurr1(+) and TH(+) dopaminergic cells (patterning), which constituted more than 98 % of the entire cell population. However, when the same growth factors were applied to SOX1(+) cells, only less than 4 % of the cells became Nurr1(+), indicating that patterning was effective only if SOX1 expression was down-regulated. After transplanting the Nurr1(+) and TH(+) cells into a hemiparkinsonian rat model, significant improvements were observed in amphetamine induced ipslateral rotations, apomorphine induced contra-lateral rotations and Rota rod motor tests over a duration of 8 weeks. CONCLUSIONS: Our findings thus provide a convenient approach to FP development and functional dopaminergic neuron derivation.


Assuntos
Diferenciação Celular , Neurônios Dopaminérgicos/metabolismo , Células Alimentadoras/enzimologia , Células-Tronco Embrionárias Humanas/metabolismo , Fatores de Transcrição SOXB1 , Animais , Linhagem Celular , Humanos , Masculino , Ratos , Ratos Sprague-Dawley
2.
J Cell Physiol ; 230(2): 308-17, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24962522

RESUMO

The growth of primary keratinocytes is improved by culturing them with a feeder layer. The aim of this study was to assess whether the feeder layer increases the lifespan of cultured epithelial cells by maintaining or improving telomerase activity and expression. The addition of an irradiated fibroblast feeder layer of either human or mouse origin (i3T3) helped maintain telomerase activity as well as expression of the transcription factor Sp1 in cultured keratinocytes. In contrast, senescence occurred earlier, together with a reduction of Sp1 expression and telomerase activity, in keratinocytes cultured without a feeder layer. Telomerase activity was consistently higher in keratinocytes grown on the three different feeder layers tested relative to cells grown without them. Suppression of Sp1 expression by RNA inhibition (RNAi) reduced both telomerase expression and activity in keratinocytes and also abolished their long-term growth capacity suggesting that Sp1 is a key regulator of both telomerase gene expression and cell cycle progression of primary cultured human skin keratinocytes. The results of the present study therefore suggest that the beneficial influence of the feeder layer relies on its ability to preserve telomerase activity in cultured human keratinocytes through the maintenance of stable levels of Sp1 expression.


Assuntos
Células Alimentadoras/enzimologia , Fibroblastos/metabolismo , Queratinócitos/enzimologia , Pele/metabolismo , Fator de Transcrição Sp1/metabolismo , Telomerase/metabolismo , Adulto , Idoso de 80 Anos ou mais , Animais , Células Cultivadas , Pré-Escolar , Técnicas de Cocultura , Células Alimentadoras/citologia , Humanos , Queratinócitos/citologia , Pessoa de Meia-Idade , Pele/citologia
3.
Drug Chem Toxicol ; 34(4): 433-9, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21740348

RESUMO

Aminoglycoside antibiotics have been in use since 1944 with the discovery of streptomycin. The aim of this study was to derive a new, highly resistant multicopy neo(R) transgenic mouse strain, named TgN3Ems, by random insertion of the plasmid, pPGKneobpA, and compare the level of drug resistance of wild-type and transgenic mice in vivo and corresponding primary mouse embryonic fibroblasts (MEFs) in vitro to a model neomycin analog, G418. The expression neoR in transgenic animals caused a 5-fold increase in the approximate lethal dose of G418, compared to wild type. No adverse pathological changes were found for the transgenic mice treated with G418, as they all died within minutes after injection. In contrast, the G418 treatment of wild-type mice resulted in a marked liver and kidney toxicity detected microscopically and via increases of serum biomarkers for liver and kidney damage. In addition, there was a mild bone marrow and lymphoid depletion. In in vitro studies, the transgenic MEFs survived 20-fold higher G418 levels, compared to the wild-type MEF cells. Therefore, TgN3Ems transgenic mice could be used as a source of G418-resistant feeder cells for gene targeting. Since the expression of drug-resistance genes in transgenic animals confers resistance to toxicity, the TgN3Ems mice might serve as a tool applicable in drug design.


Assuntos
Resistência a Medicamentos/genética , Células Alimentadoras/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Marcação de Genes , Gentamicinas/toxicidade , Canamicina Quinase/genética , Animais , Southern Blotting , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Células Cultivadas , Células Alimentadoras/citologia , Células Alimentadoras/enzimologia , Fibroblastos/citologia , Fibroblastos/enzimologia , Gentamicinas/farmacologia , Dose Letal Mediana , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Plasmídeos , Regiões Promotoras Genéticas , Testes de Toxicidade Aguda , Transgenes
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...