RESUMEN
The remarkable regenerative capacity of the zebrafish has made it an important model organism for studying heart regeneration. However, current loss-of-function studies are limited by a lack of conditional-knockout and effective gene-knockdown methods for the adult heart. Here, we report a novel siRNA knockdown method facilitated by poly(ethylene glycol)-b-poly(D,L-lactide) (PEG-PLA) nanoparticles. The siRNA-encapsulated nanoparticles successfully entered cells and resulted in remarkable gene-specific knockdown in the adult heart. This effect was demonstrated by down-regulation of the Aldh1a2 and Dusp6 proteins after intrapleural delivery of nanoparticle-encapsulated siRNAs. Furthermore, siRNA-mediated knockdown of Aldh1a2 was sufficient to inhibit myocardial proliferation and decrease the numbers of Gata4-positive cardiomyocytes after ventricular resection. Therefore, the results of this work demonstrate that nanoparticle-facilitated siRNA delivery provides an alternative tool for loss-of-function studies of genes in the adult heart in particular and other organs in general in the adult zebrafish.
Asunto(s)
Técnicas de Silenciamiento del Gen/métodos , Miocardio/metabolismo , Nanopartículas/metabolismo , Polietilenglicoles/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Pez Cebra/genética , Familia de Aldehído Deshidrogenasa 1 , Animales , Proliferación Celular/genética , Proliferación Celular/fisiología , Fosfatasa 6 de Especificidad Dual/genética , Isoenzimas/genética , Miocardio/citología , ARN Interferente Pequeño/genética , Retinal-Deshidrogenasa/genéticaRESUMEN
A simple and economical CE method has been developed for the analysis of four model basic proteins by employing N-methyl-2-pyrrolidonium methyl sulfonate ionic liquid (IL) as the dynamic coating material based on the interaction of both between electrostatic attraction and hydrogen bond, and between the organic cations of IL and the inner surface of bare fused-silica capillary. The N-methyl-2-pyrrolidonium-based IL modified capillary not only generated a stable suppressed electroosmotic flow, but also effectively eliminated the wall adsorption of proteins. Several important parameters such as the IL concentration, pH values, and concentrations of the background electrolyte were optimized to improve the separation of basic proteins. Consequently, under the optimum separation conditions, a satisfied separation of basic proteins including lysozyme, cytochrome c, ribonuclease A, and α-chymotrypsinogen A with theoretical plates ranging from 2.09 × 10(5) to 4.48 × 10(5) plates/m had been accomplished within 15 min. The proposed method first illustrated the effect of hydrogen bond between coating material and inner capillary surface on the coating, which should be a new strategy to design and select more effective coating materials to form more stable coatings in CE.