RESUMEN
The technology of virus-based genetic modification in tissue engineering has provided the opportunity to produce more flexible and versatile biomaterials for transplantation. Localizing the transgene expression with increased efficiency is critical for tissue engineering as well as a challenge for virus-based gene delivery. In this study, we tagged the VP2 protein of type 2 adeno-associated virus (AAV) with a 3×FLAG plasmid at the N-terminus and packaged a FLAG-tagged recombinant AAV2 chimeric mutant. The mutant AAVs were immobilized onto the tissue engineering scaffolds with crosslinked anti-FLAG antibodies by N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP). Cultured cells were seeded to scaffolds to form 3D transplants, and then tested for viral transduction both in vitro and in vivo. The results showed that our FLAG-tagged AAV2 exerted similar transduction efficiency compared with the wild type AAV2 when infected cultured cells. Following immobilization onto the scaffolds of PLGA or gelatin sponge with anti-FLAG antibodies, the viral mediated transgene expression was significantly improved and more localized. Our data demonstrated that the mutation of AAV capsid targeted for antibody-based immobilization could be a practical approach for more efficient and precise transgene delivery. It was also suggested that the immobilization of AAV might have attractive potentials in applications of tissue engineering involving the targeted gene manipulation in 3D tissue cultures.
Asunto(s)
Proteínas de la Cápside/metabolismo , Trasplante de Células , Dependovirus/genética , Vectores Genéticos/administración & dosificación , Oligopéptidos/metabolismo , Ingeniería de Tejidos , Transgenes/genética , Células 3T3-L1 , Animales , Proteínas de la Cápside/genética , Sistemas de Liberación de Medicamentos , Terapia Genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Ratones , Oligopéptidos/genética , Andamios del Tejido , Transducción GenéticaRESUMEN
The aim of this study was to investigate the effect of suppression of nicotinamide phosphoribosyltransferase (NAMPT) expression on imatinib-sensitivity in chronic myelogenous leukemia (CML) cell line K562 and its mechanisms, NAMPT siRNA was synthesized and transfected into K562 cells. PI/Calcein staining technique was used to determine survival rate of transfected K562 cells at 48th hour after exposure to 1 µmol/L imatinib. MTS method was used to determine the proliferation changes of transfected K562 cell at 48th hour after exposure to different doses of imatinib, then half inhibitory concentration (IC(50)) was calculated. Expression of NAMPT at 3rd-48th hour after exposure to 1 µmol/L imatinib was determined by Western blot. To explore the effect of NAMPT-siRNA and imatinib on the expression of apoptosis-related genes, the microarray data from NCBI GEO Data-Sets was analyzed, then the results were confirmed by Western blot. The luciferase reporter assay was used to determine the effect of NAMPT and imatinib on transcriptional activity of NF-κB transcription factors. The results showed that after exposure to 1 µmol/L imatinib for 3 - 48 h, there was no significant change of NAMPT expression in K562 cells. The expression of NAMPT could be effectively inhibited by the NAMPT-siRNA. After exposure to 1 µmol/L of imatinib for 48 h, the survival rate of NAMPT-siRNA interference group was lower than that of negative control group (P < 0.05), indicating that suppression of NAMPT expression can increase the sensitivity of K562 cells to imatinib and enhance the killing effect of imatinib on K562 cells. The IC(50) of imatinib in NAMPT-siRNA interference group was the lowest compared with that of control group (P < 0.05) after exposure to different concentrations of imatinib for 48 h, the fitted survival curves showed that the slope of NAMPT-siRNA interference group was the largest ranging between 0.01 - 0.1 µmol/L of imatinib. Data mining of expression profiling indicated that the anti-apoptotic factor Bcl-2 decreased in K562 cells treated with either NAMPT-siRNA or imatinib, which was confirmed by Western blot. The inhibitory effect was much more significant when both NAMPT-siRNA and imatinib were used. The results of luciferase reporter assay showed that either NAMPT-siRNA or imatinib decreased transcriptional activity of NF-κB. The decreased effect was much more significant when both NAMPT-siRNA and imatinib were used. It is concluded that survival of K562 cells affected by imatinib may not be due to regulation of expression of NAMPT. When expression of NAMPT decreases, the K562 cells are more sensitive to imatinib, this may be related with the decreased transcriptional activity of NF-κB and its downstream effector Bcl-2.
Asunto(s)
Citocinas/antagonistas & inhibidores , Nicotinamida Fosforribosiltransferasa/antagonistas & inhibidores , Piperazinas/farmacología , Pirimidinas/farmacología , Benzamidas , Citocinas/metabolismo , Proteínas de Fusión bcr-abl/metabolismo , Humanos , Mesilato de Imatinib , Células K562 , FN-kappa B/metabolismo , Nicotinamida Fosforribosiltransferasa/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismoRESUMEN
Following the rapid development of genomics, the omics studies on various metabolites have emerged, expanded, and fused into a novel area of metabolomics. The shotgun lipidomics, as an important approach to lipid researches, has demonstrated great potentials. It will play indispensable roles in biomedical research and applications as its associated techniques are further developed and advanced. Shotgun lipidomics utilizes the mass spectrum technologies to systematically analyze the full scope or a specific subset of lipids from biological samples to identify the differences and the associated functions and to explore the underling mechanisms. The bottleneck problem with the traditional lipid analyses was overcome by the strategy to adopt electro-spray mass spectrum principles, therefore led to a new era of lipidomic researches with high-throughput, high-resolution and high efficiency. The bioactive lipids include many species, widely distributed in living organisms, and closely related to human diseases. Shotgun lipidomics will facilitate the identification of disease-associated specific lipid markers, reveal their roles in the complex processes of the geresis and progress of diseases and eventually provide new strategies and approaches for its diagnosis and treatments.