RESUMEN
A method for fast delivery of proteins conjugated to superparamagnetic iron oxide nanoparticles (SPION) into mammalian cells by applying a strong magnetic field in pulses was proposed. Firstly, SPION were prepared from an alkaline solution of divalent and trivalent iron ions and covalently bound with protein through the activation of N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide (EDC). After fluorescently labelling, the protein-nanoparticle conjugate was mixed with mammalian cell line and exposed to a pulsed magnetic field for short durations of few milliseconds. Results suggested that superparamagnetic nanoparticles were able to carry proteins into living cells immediately. Cellular internalization of the fluorescently labelled protein-nanoparticle conjugate was proved by the observation of cell fluorescence in a fluorescent microscopy, as well as cell analysis by a flow cytometer. We found that the cellular uptake was accomplished dominantly by the process of bombardment of magnetic nanoparticles.
Asunto(s)
Endocitosis , Nanopartículas de Magnetita/química , Nanoconjugados/química , Proteínas/química , Proteínas/farmacocinética , Campos Electromagnéticos , Citometría de Flujo , Fluoresceína/química , Células HeLa , Humanos , Microscopía Confocal , Albúmina Sérica Bovina/química , Albúmina Sérica Bovina/farmacocinética , Espectrofotometría AtómicaRESUMEN
Through the use of superparamagnetic iron oxide nanoparticles (SPIONs), fast delivery of DNA into adherent and suspended cells could be achieved by the mediation of a strong impulsed magnetic field. Mammalian cells were well transfected with enhanced green fluorescent protein gene. To mediate the cellular uptake, cells and nucleic acid complexes were mixed together and exposed once or several times to impulsed magnetic field for short durations of few milliseconds. In the transfection of adherent cells, most complexes of plasmid DNA and polyethylenimine (PEI)-coated SPIONs were internalized immediately. In comparison with no magnetic pulsing, the enhancement in transfection efficiency was about two fold on average by pulsing in magnetic field of 0.6 Tesla three times. The transfection yield increased with the strength of magnetic field and the number of pulsing. Disregarding the cytotocixity of internalized PEI, the loss of cell viability by magnetic pulsing was not evidenced.
Asunto(s)
ADN Circular/metabolismo , Compuestos Férricos/química , Magnetismo , Nanopartículas/química , Transfección/métodos , Adhesión Celular , Supervivencia Celular , ADN Circular/genética , Compuestos Férricos/metabolismo , Células HeLa , Humanos , Polietileneimina/química , Polietileneimina/metabolismo , Polilisina/química , Polilisina/metabolismoRESUMEN
BACKGROUND: The discovery of molecular biomarkers is crucial to the diagnosis of head and neck squamous cell cancer (HNSCC). METHODS: Proteins from pre-surgery serum samples of patients with HNSCC and healthy individuals were analyzed by 2-dimensional gel electrophoresis (2-DE) using a 17 cm-long immobilized pH gradient gel strip (large gel). The differentially expressed protein spots were detected by statistical analysis. Because 2 haptoglobin (Hp) alpha chains were found to be differentially expressed, the genotypic distribution of Hp alpha chains in patients and healthy individuals was assayed by polymerase chain reaction. The protein expression levels of Hp alpha chains in individuals carrying different Hp alleles were analyzed by 2-DE with a small gel. RESULTS: Two isoforms of haptoglobin alpha2 chain (Hp alpha2) in patients' sera were found from 2-DE analysis to be up-regulated, while the isoforms of haptoglobin alpha1 chain (Hp alpha1) were significantly down-regulated. Apolipoprotein AII and 2 isoforms of apolipoprotein CII were also differentially expressed in the sera of patients with HNSCC. The Hp alpha2 chain was significantly up-regulated in the patients carrying at least one haptoglobin 2 allele, according to the spot intensities from scanned images of small-gel 2-DE. CONCLUSIONS: The expression pattern of seven differentially expressed polypeptides and the up-regulation of Hp alpha2 in individuals with the Hp 2 allele are potential biomarkers.
Asunto(s)
Haptoglobinas/metabolismo , Neoplasias de Cabeza y Cuello/diagnóstico , Neoplasias de Células Escamosas/diagnóstico , Adolescente , Anciano , Alelos , Apolipoproteína A-II/biosíntesis , Apolipoproteína A-II/genética , Apolipoproteínas C/biosíntesis , Apolipoproteínas C/genética , Biomarcadores , Electroforesis en Gel Bidimensional , Electroforesis en Gel de Poliacrilamida , Femenino , Genómica , Haptoglobinas/análisis , Haptoglobinas/genética , Neoplasias de Cabeza y Cuello/sangre , Neoplasias de Cabeza y Cuello/genética , Humanos , Concentración de Iones de Hidrógeno , Focalización Isoeléctrica , Isomerismo , Masculino , Persona de Mediana Edad , Neoplasias de Células Escamosas/sangre , Neoplasias de Células Escamosas/genética , Proteoma/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tinción con Nitrato de Plata , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Adulto JovenRESUMEN
Previous studies indicated that region 11.2 of the long arm of chromosome 22 (22q11.2) might be a locus encoding a tumor suppressor gene, since its deletion is a recurrent genetic characteristic of aggressive pediatric cancer. This region is found in the human immunodeficiency virus integrase interactor 1 (hSNF5/INI1) gene. To investigate whether the hSNF5/INI1 gene is involved in leukemogenesis, mutation analysis of the hSNF5/INI1 gene was performed in the present study using 5 hematopoietic cell lines, acute myeloid leukemia (AML) specimen and normal control. We found two single nucleotide polymorphisms at the hSNF5/INI1 gene in exon 4 and exon 9. The results of this study suggest that the hSNF5/INI1 gene does not play an important role in the leukemogenesis of AML.