Aproximación a los mecanismos moleculares de la acción farmacológica del Factor Estimulador de Colonias de Granulocito Recombinante Humano (GCSFRH) sobre células de sangre de cordón umbilical y mesenquimales en un modelo "In Vitro"
dc.rights.license | Atribución-NoComercial-SinDerivadas 4.0 Internacional |
dc.contributor.author | Ávila Portillo, Luz Mabel |
dc.date.accessioned | 2020-02-19T15:16:06Z |
dc.date.available | 2020-02-19T15:16:06Z |
dc.date.issued | 2019 |
dc.identifier.uri | https://repositorio.unal.edu.co/handle/unal/75647 |
dc.description.abstract | The effect of different types of G-CSF on the transcriptome of umbilical cord blood cells and mesenchymal adipose tissue ADSC. In vitro model. The aim of the study was to evaluate the transcriptome of umbilical cord blood cells (UCB) and adenosine mesenchymal cells of adipose tissue (ADSC) exposed to biosimilar and innovative G-CSF for the comparison of molecular targets and as well as to gain key gene information in the cellular response. Prior to signing the donor informed consent, 101 UCB 3 samples of liposuction were obtained for the ADSC. The ADSC and UCB cells were stimulated with 100 ng / ml G-CSF (Innovative and biosimilar and negative control without stimulation) and cultured for 6 hours. We performed technical and 3 biological replicas, finishing the culture obtained mRNA and performed an analysis of gene expression using Agilent microarrays. We then performed the pre-processing of the data with the Bioconductor package of R, and the evaluation of data quality with the LIMMA package. Next the unsupervised hierarchical clustering analysis, as well as the supervised analysis for the identification of common overexpressed and low expressed genes with RANK test in order to assess the differential expression of genes between replicates with the MultiExperiment program Viewer MeV was carried out. Additionally, the identification of common molecular pathways with the INNATEDB bioinformatic tool, gene ontology with REVIGO, identification of signaling pathways with cytoscape was accomplished. The confirmation of selected targets was performed by flow cytometry, colony forming units, and adipogenic and osteogenic potential. Both biosimilar and innovative G-CSF induced overexpression of 299 genes in UCB cells and under-expression of 2 genes. Functional enrichment analysis with Kegg Pathway showed 10 signaling pathways that had statistical significance: chemokine signaling pathway (p=0.0205); signaling pathway PI3K-Akt (p=0.01214); Hedgehog signaling pathway (p=0.03324); Notch signaling pathway (p=0.01170); Hippo signaling pathway (p= 0.05441); positive regulation of cell growth (p=0.03271); cell cycle (p=0.00210); Assembly of focal adhesions (p=0.01084); and cell migration (p=0.045933). The enrichment analysis of the common genes showed the activation of the transcription factors associated with innate signaling pathways of innate Toll-like receptor (FOS, NFKB1) (p=0.00949), TNF (FOS, NFKB1) pathway (p=0.00983) and differentiation (EP300, TP53) (p=0.0121). Colony forming units (CFU) suggest differentiation into granulocytic colonies. With regard to ADSC cells, 152 common over-regulated and 306 low-regulated genes were identified. Functional enrichment analysis with Kegg Pathway showed 10 statistically significant enriched signaling pathways: JAK-STAT and Wnt (p=0.01669), hyaluronate metabolism (p=0.00163); dissolution of fibrin clot (p=4.78446E-05); MAPK (p=0.02845); arachidonic acid metabolism (p=0.05077); TGF beta (p=0.03249) and within the sub-regulated genes the transcription factors JUN, MYC, ELK1, KRT7, BRF1, STAT3, SMAD2, NFKB1, FOS and TCF12 were found. The gene ontology was related to processes of cellular metabolism, activation of TLRs, induction of the PI3K-Akt signaling pathway and activation of NFkB and MAP. The decrease in CD44+ was demonstrated by cytometry suggesting migratory profile. Both UCB and ADSC cells exposed to G-CSF stimulation in vitro activated intracellular signal cascades. |
dc.description.abstract | Efecto de diferentes tipos de G-CSF en el transcriptoma de células de sangre de cordón umbilical y células mesenquimales de tejido adiposo (ADSC). Modelo in vitro. El objetivo de este estudio fue evaluar el transcriptoma de células de sangre de cordón umbilical (SCU) y ADSC expuestas al factor estimulante de colonias de granulocitos (GCSF) biosimilar e innovador para la comparación de los blancos moleculares y para la identificación de genes claves en la respuesta celular. Previa firma del consentimiento informado de los donantes se obtuvieron 101 muestras de SCU y 3 muestras de lipoaspirado para el caso de las ADSC. Las células de ADSC y de SCU estimuladas con 100 ng/ml G-CSF (Innovador y biosimilar) y un control negativo sin estimulo fueron cultivadas por 6 horas. Se realizaron replicas técnicas y 3 biológicas, finalizando el cultivo se obtuvo el ARNm y se realizó un análisis de expresión de genes utilizando microarreglos de Agilent, posteriormente se realizó un pre-procesamiento de los datos con el paquete Bioconductor de R, evaluación de la calidad de los datos con el paquete LIMMA, análisis de agrupamiento jerárquico no supervisado, análisis supervisado para la identificación de los genes comunes sobre-expresados y bajo expresados con test de RANK para valorar la expresión diferencial de los genes entre las réplicas con el programa MultiExperiment Viewer MeV, identificación de las vías moleculares comunes con la herramienta bioinformática INNATEDB, ontología de genes con REVIGO, identificación de vías de señalización con Cytoscape. La confirmación de blancos seleccionados se realizó por citometría de flujo, cuantificación de unidades formadoras de colonias e Identificación de la molécula CD44 en el experimento con ADSC Resultados: Los G-CSF biosimilar e innovador indujeron la sobreexpresión de 299 genes en las células de SCU y la sub-expresión de 2 genes. El análisis de enriquecimiento funcional con Kegg Pathway evidenció 10 vías de señalización enriquecidas con significancia estadística: vía de señalización de quimiocinas (p=0,0205); vía de señalización PI3K-Akt (p=0,01214); vía de señalización Hedgehog (p=0,03324); vía de señalización Notch (p=0,01170); vía de señalización Hippo (p=0,05441); regulación positiva de crecimiento celular (p=0,03271); ciclo celular (p=0,00210); ensamblaje de adhesiones focales (p=0,01084); y migración celular (p 0,045933). El análisis de enriquecimiento de los genes comunes mostró la activación de los factores de transcripción asociados a vías de señalización de inmunidad innata de receptor Toll-like (FOS, NFKB1) p=0,00949, vía de TNF (FOS, NFKB1) p=0,00983 y de diferenciación (EP300, TP53) p=0,0121. Las unidades formadoras de colonias (UFC) sugieren diferenciación a colonias granulocíticas. Con respecto a las células ADSC se identificaron 152 genes comunes sobre-regulados y 306 bajo-regulados. El análisis de enriquecimiento funcional con Kegg Pathway evidenció 10 vías de señalización enriquecidas estadísticamente significativa: JAK-STAT y Wnt (p=0,01669); metabolismo del hialuronato (p=0,00163); disolución del coágulo de fibrina (p=4,78446E-05); MAPK (p=0,02845); metabolismo del ácido araquidónico (p= 0,05077); TGF beta (p=0,03249) y dentro de los genes sub-regulados se encontraron factores de transcripción JUN, MYC, ELK1, KRT7, BRF1, STAT3, SMAD2, NFKB1, FOS y TCF12. La ontología de los genes se relacionó con procesos de metabolismo celular, activación de TLRs, inducción de la vía de señalización PI3K-Akt y activación de NFkB y MAP. La disminución del marcador CD44+ fue evidenciado por citometría de flujo sugiriendo perfil migratorio. Tanto las células de SCU como de ADSC expuestas al estímulo de G-CSF in vitro activan cascadas de señales intracelulares. |
dc.format.extent | 256 |
dc.format.mimetype | application/pdf |
dc.language.iso | spa |
dc.rights | Derechos reservados - Universidad Nacional de Colombia |
dc.rights.uri | http://creativecommons.org/licenses/by-nc-nd/4.0/ |
dc.subject.ddc | Medicina y salud::Farmacología y terapéutica |
dc.title | Aproximación a los mecanismos moleculares de la acción farmacológica del Factor Estimulador de Colonias de Granulocito Recombinante Humano (GCSFRH) sobre células de sangre de cordón umbilical y mesenquimales en un modelo "In Vitro" |
dc.type | Otro |
dc.rights.spa | Acceso abierto |
dc.description.additional | Doctorado en Ciencias Farmacéuticas. |
dc.type.driver | info:eu-repo/semantics/other |
dc.type.version | info:eu-repo/semantics/acceptedVersion |
dc.contributor.researchgroup | Farmacogenética del Cáncer |
dc.description.degreelevel | Doctorado |
dc.publisher.department | Departamento de Farmacia |
dc.publisher.branch | Universidad Nacional de Colombia - Sede Bogotá |
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dc.rights.accessrights | info:eu-repo/semantics/openAccess |
dc.subject.proposal | Human Recombinant Granulocyte |
dc.subject.proposal | Cáncer |
dc.subject.proposal | Cancer |
dc.subject.proposal | Células mesenquimales |
dc.subject.proposal | Cordón umbilical |
dc.subject.proposal | Mesenchymal cells |
dc.subject.proposal | Granulocito Recombinante Humano |
dc.subject.proposal | Umbilical cord |
dc.type.coar | http://purl.org/coar/resource_type/c_1843 |
dc.type.coarversion | http://purl.org/coar/version/c_ab4af688f83e57aa |
dc.type.content | Text |
oaire.accessrights | http://purl.org/coar/access_right/c_abf2 |
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