RESUMO
Surgical intervention with the use of autografts is considered the gold standard to treat peripheral nerve injuries. However, a biomaterial that supports and guides nerve growth would be an attractive alternative to overcome problems with limited availability, morbidity at the site of harvest, and nerve mismatches related to autografts. Native spider silk is a promising material for construction of nerve guidance conduit (NGC), as it enables regeneration of cm-long nerve injuries in sheep, but regulatory requirements for medical devices demand synthetic materials. Here, we use a recombinant spider silk protein (NT2RepCT) and a functionalized variant carrying a peptide derived from vitronectin (VN-NT2RepCT) as substrates for nerve growth support and neurite extension, using a dorsal root ganglion cell line, ND7/23. Two-dimensional coatings were benchmarked against poly-d-lysine and recombinant laminins. Both spider silk coatings performed as the control substrates with regards to proliferation, survival, and neurite growth. Furthermore, NT2RepCT and VN-NT2RepCT spun into continuous fibers in a biomimetic spinning set-up support cell survival, neurite growth, and guidance to an even larger extent than native spider silk. Thus, artificial spider silk is a promising biomaterial for development of NGCs.
Assuntos
Proliferação de Células/efeitos dos fármacos , Regeneração Nervosa/efeitos dos fármacos , Neuritos/metabolismo , Seda/farmacologia , Aranhas/metabolismo , Vitronectina/farmacologia , Animais , Autoenxertos , Materiais Biocompatíveis/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Gânglios Espinais/citologia , Humanos , Laminina/farmacologia , Camundongos , Neuritos/efeitos dos fármacos , Traumatismos dos Nervos Periféricos/cirurgia , Engenharia de Proteínas/métodos , Ratos , Proteínas Recombinantes/farmacologia , Seda/genética , Vitronectina/genéticaRESUMO
Gene- and cell-based therapies are promising strategies for the treatment of degenerative retinal diseases such as age-related macular degeneration, Stargardt disease, and retinitis pigmentosa. Cellular engineering before transplantation may allow the delivery of cellular factors that can promote functional improvements, such as increased engraftment or survival of transplanted cells. A current challenge in traditional DNA-based vector transfection is to find a delivery system that is both safe and efficient, but using mRNA as an alternative to DNA can circumvent these major roadblocks. In this study, we show that both unmodified and modified mRNA can be delivered to retinal pigmented epithelial (RPE) cells with a high efficiency compared with conventional plasmid delivery systems. On the other hand, administration of unmodified mRNA induced a strong innate immune response that was almost absent when using modified mRNA. Importantly, transfection of mRNA encoding a key regulator of RPE gene expression, microphthalmia-associated transcription factor (MITF), confirmed the functionality of the delivered mRNA. Immunostaining showed that transfection with either type of mRNA led to the expression of roughly equal levels of MITF, primarily localized in the nucleus. Despite these findings, quantitative RT-PCR analyses showed that the activation of the expression of MITF target genes was higher following transfection with modified mRNA compared with unmodified mRNA. Our findings, therefore, show that modified mRNA transfection can be applied to human embryonic stem cell-derived RPE cells and that the method is safe, efficient, and functional.
Assuntos
Células-Tronco Embrionárias/metabolismo , Células Epiteliais/metabolismo , Expressão Gênica , RNA Mensageiro/genética , Transfecção/métodos , Transporte Ativo do Núcleo Celular , Western Blotting , Diferenciação Celular/genética , Linhagem Celular , Núcleo Celular/metabolismo , Células-Tronco Embrionárias/citologia , Células Epiteliais/ultraestrutura , Células HEK293 , Humanos , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/metabolismo , Fator de Transcrição Associado à Microftalmia/genética , Fator de Transcrição Associado à Microftalmia/metabolismo , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Fatores de Transcrição Otx/genética , Fatores de Transcrição Otx/metabolismo , RNA Mensageiro/metabolismo , Epitélio Pigmentado da Retina/citologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismo , cis-trans-Isomerases/genética , cis-trans-Isomerases/metabolismoRESUMO
Mastermind-like 1 (MAML1) is a transcriptional coregulator of activators in various signaling pathways, such as Notch, p53, myocyte enhancer factor 2C (MEF2C) and beta-catenin. In earlier studies, we demonstrated that MAML1 enhanced p300 acetyltransferase activity, which increased the acetylation of Notch by p300. In this study, we show that MAML1 strongly induced acetylation of the transcription factor early growth response-1 (EGR1) by p300, and increased EGR1 protein expression in embryonic kidney cells. EGR1 mRNA transcripts were also upregulated in the presence of MAML1. We show that MAML1 physically interacted with, and acted cooperatively with EGR1 to increase transcriptional activity of the EGR1 and p300 promoters, which both contain EGR1 binding sites. Bioinformatics assessment revealed a correlation between p300, EGR1 and MAML1 copy number and mRNA alterations in renal clear cell carcinoma and p300, EGR1 and MAML1 gene alterations were associated with increased overall survival. Our findings suggest MAML1 may be a component of the transcriptional networks which regulate EGR1 target genes during nephrogenesis and could also have implications for the development of renal cell carcinoma.