RESUMEN
Since aptamers bind their targets with high affinity and specificity, they are promising alternative ligands in protein affinity purification. As aptamers are chemically synthesized oligonucleotides, they can be easily produced in large quantities regarding GMP conditions allowing their application in protein production for therapeutic purposes. Several advantages of aptamers compared to antibodies are described in general within this paper. Here, an aptamer directed against the human Vascular Endothelial Growth Factor (VEGF) was used as affinity ligand for establishing a purification platform for VEGF in small scale. The aptamer was covalently immobilized on magnetic beads in a controlled orientation resulting in a functional active affinity matrix. Target binding was optimized by introduction of spacer molecules and variation of aptamer density. Further, salt-induced target elution was demonstrated as well as VEGF purification from a complex protein mixture proving the specificity of protein-aptamer binding.
RESUMEN
Aptamers are short oligonucleotides that are capable of selectively binding to their corresponding target. Therefore, they can be thought of as a nucleic acid-based alternative to antibodies and can substitute for their amino acid-based counterparts in analytical applications, including as receptors in biosensors. Here they offer several advantages because their nucleic acid nature and their binding via an induced fit mechanism enable novel sensing strategies. In this article, the utilization of aptamers as novel bio-receptors in combination with nanoparticles as transducer elements is reviewed. In addition to these analytical applications, the medical relevance of aptamer-modified nanoparticles is described.
Asunto(s)
Aptámeros de Nucleótidos/química , Aptámeros de Nucleótidos/genética , Técnicas Biosensibles/métodos , Mediciones Luminiscentes/métodos , Nanopartículas/química , Técnica SELEX de Producción de Aptámeros/métodos , Resonancia por Plasmón de Superficie/métodos , AdsorciónRESUMEN
Efficient cell expansion is a basic requirement for obtaining clinically relevant numbers of mesenchymal stem cells designed for cell-based therapies or tissue-engineering application. Previous studies have demonstrated that mesenchymal stem cells (MSC) cultivated under reduced atmospheric oxygen concentrations (2.5% O2) possess enhanced proliferation potential and can maintain their differentiation properties. We have analyzed the oxygen-dependent cytokine expression of human MSC derived from umbilical cord and attempted to link the results to the proliferation and differentiation capacities of these cells. By quantitative reverse transcription plus the polymerase chain reaction and by protein microarray, we measured the gene expression and intracellular protein concentration of several growth factors and growth factor receptors. Fibroblast growth factor-7, two growth factor receptors (vascular endothelial growth factor receptor 2 and stem cell factor receptor), and two growth-factor-binding proteins (insulin-like growth-factor-binding proteins 3 and 6) were over-expressed under hypoxic conditions, indicating that their signaling pathways participate in cell proliferation. On the other hand, typical differentiation factors such as bone morphogenetic protein-4, endothelial growth factor, and tissue growth factor-ß1 were absent in cells cultivated under hypoxic and normoxic conditions. The absolute concentration of some intracellular cytokines was also measured for the first time under hypoxia and normoxia. Our results in combination with previous findings indicate that enhanced proliferation potential and a maintained undifferentiated cell state can be ascribed to the oxygen-dependent expression of a set of cytokines. This knowledge might help in the understanding of MSC physiology and in the achievement of directed cell fate of MSC for clinical application.
Asunto(s)
Hipoxia de la Célula/fisiología , Células Madre Mesenquimatosas/metabolismo , Oxígeno/metabolismo , Cordón Umbilical/metabolismo , Proteína Morfogenética Ósea 4/deficiencia , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Factores de Crecimiento Endotelial/deficiencia , Factor 7 de Crecimiento de Fibroblastos/biosíntesis , Factor 7 de Crecimiento de Fibroblastos/metabolismo , Expresión Génica , Humanos , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/biosíntesis , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Proteína 6 de Unión a Factor de Crecimiento Similar a la Insulina/biosíntesis , Proteína 6 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Proteínas Proto-Oncogénicas c-kit/biosíntesis , Proteínas Proto-Oncogénicas c-kit/metabolismo , Factor de Crecimiento Transformador beta1/biosíntesis , Cordón Umbilical/citología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/biosíntesis , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Up to date research in biology, biotechnology, and medicine requires fast genome and transcriptome analysis technologies for the investigation of cellular state, physiology, and activity. Here, microarray technology and next generation sequencing of transcripts (RNA-Seq) are state of the art. Since microarray technology is limited towards the amount of RNA, the quantification of transcript levels and the sequence information, RNA-Seq provides nearly unlimited possibilities in modern bioanalysis. This chapter presents a detailed description of next-generation sequencing (NGS), describes the impact of this technology on transcriptome analysis and explains its possibilities to explore the modern RNA world.