The main objective of the present work was the development of new nanoparticulate carrier systems for the delivery of plasmid DNA. These new carriers consist of a blend matrix formed by a poly(lactic-co-glycolic acid) (PLGA) copolymer and polyoxyethylene derivatives. More specifically, we have prepared nanostructures with different PLGA:poloxamer and PLGA:poloxamine compositions by an optimized emulsification-solvent diffusion technique and studied the potential of these carriers for the encapsulation and controlled release of plasmid DNA. Depending on the particle composition, the encapsulation efficiency of the model plasmid pEGFP-C1 varied between 30% and 45%. All formulations provided continuous and controlled release of the plasmid with minimal burst effect. In addition, the release rate and duration was dependent on the composition of the particle matrix. Moreover, gel electrophoresis and cell culture (MCF-7 cell line) assays allowed us to confirm that the biologically active form of the plasmid was preserved during the particle preparation process and also during its release. Cell culture experiments also indicated that the new nanoparticles do not exhibit toxic effects on these cells at concentrations up to 5 mg/mL. Altogether, these results indicate that these composite nanostructures present a promising approach for the delivery of plasmid DNA.
BackgroundNeural stem cells (NSC) have been extensively used as a tool to investigate the mechanisms responsible for neural repair, and they have been also considered as the source for a series of promising replacement therapies in various neurodegenerative diseases. However, their use is limited by their relative rarity and anatomical localization, and also because, the methods for isolation and characterization are usually time consuming and have some technical limitations.ResultsIn this study, we describe a resource and method for obtaining immortalized cells with NSC characteristics obtained from mouse brain embryo.ConclusionsBecause these cells can be maintained indefinitely in culture, they may constitute a permanent source of NSC that can be used for research studies on neural development and regeneration.
We show in this work that the inhibition of Cdk4 (6) in Rb ؊/؊ 3T3 cells enhances the accumulation of the p27 kip1 cyclin-dependent kinase inhibitor when these cells are induced into quiescence. Two different forms of inhibition of Cdk4 (6), namely overexpression of the Cdk4 (6) inhibitor p16 and overexpression of a dominant negative mutant of Cdk4 (Cdk4 N158 ), result in this effect. This suggests that the relevant activity of Cdk4 (6) that has to be inactivated in this setting is its kinase activity. The accumulation of p27 kip1 is due to enhanced translation of the protein, mediated by the 3-untranslated region of the p27 mRNA. Moreover, the cells that overexpress p16 ink4a or Cdk4 N158 show a delay in G 1 when made quiescent and restimulated to proliferate. This delay is overcome by transfection of a plasmid expressing antisense p27 kip1 , which shows that the accumulation of p27 kip1 in these cells is related to their G 1 delay. In summary, we report a new functional link between two important cell cycle regulators, Cdk4 and p27 kip1 , and provide a mechanistic explanation to the previously reported epistatic relations between these two proteins.
Due to its aggressive and invasive nature glioblastoma (GBM), the most common and aggressive primary brain tumour in adults, remains almost invariably lethal.Significant advances in the last several years have elucidated much of the molecular and genetic complexities of GBM. However, GBM exhibits a vast genetic variation and a wide diversity of phenotypes that have complicated the development of effective therapeutic strategies. This complex pathogenesis makes necessary the development of experimental models that could be used to further understand the disease, and also to provide a more realistic testing ground for potential therapies.In this report, we describe the process of transformation of primary mouse embryo astrocytes into immortalized cultures with neural stem cell characteristics, that are able to generate GBM when injected into the brain of C57BL/6 mice, or heterotopic tumours when injected IV. Overall, our results show that oncogenic transformation is the fate of NSC if cultured for long periods in vitro. In addition, as no additional hit is necessary to induce the oncogenic transformation, our model may be used to investigate the pathogenesis of gliomagenesis and to test the effectiveness of different drugs throughout the natural history of GBM.
Due to its aggressive and invasive nature glioblastoma (GBM), the most common and aggressive primary brain tumour in adults, remains almost invariably lethal. Significant advances in the last several years have elucidated much of the molecular and genetic complexities of GBM. However, GBM exhibits a vast genetic variation and a wide diversity of phenotypes that has complicated the development of effective therapeutic strategies. This complex pathogenesis makes it necessary the development of experimental models that could be used to further understand the disease, and also to provide a more realistic testing ground for potential therapies. In this report, we describe the process of transformation of primary mouse embryo astrocytes into immortalized cultures with neural stem cell characteristics, that are able to generate of GBM when injected in the brain of C57BL/6 mice, or heterotopic tumours when injected iv. Overall, our results show that oncogenic transformation is a fate for NSC if cultured for long periods in vitro. In addition, since no additional hit is necessary to induce the oncogenic transformation, our model may be used to investigate the pathogenesis of gliomagenesis and to test the effectiveness of different drugs throughout the natural history of GBM.
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