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

• Autores: Luz Mabel Ávila Portillo
• Directores de la Tesis: Fabio Ancízar Aristizábal Gutiérrez (dir. tes.)
• Lectura: En la Universidad Nacional de Colombia (UNAL) ( Colombia ) en 2019
• Idioma: español
• Enlaces
  • Tesis en acceso abierto en: repositorio.unal.edu.co
• Resumen

  • 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.