Avaliação da redução da concentração do soro fetal bovino na expressão de proteínas envolvidas na diferenciação neuronal nas linhagens SH-SY5Y, Neuro2a e PC12

Abstract

Cell differentiation to neurons involves neuritogenesis, i. e., neurites formation. Neurites are protrusions of soma that become dendrites and axons. When this change occurs, cell promotes synapses, establishing neuronal connections. In vitro studies, there are differentiation protocols using neurotrophic factors and/or retinoic acid under fetal bovine serum (FBS) concentration reduction that differ normal condictions. However, the role of FBS reduction still needs to be defined. The aim of this study was to test whether only reduction of 10% to 1% or 0% FBS could alter proteins expression, likewise established protocols already alter. Synaptophysin, protein of synaptic vesicles, was used as classic neuronal marker. This study also analyzed cellular príon protein (PrPc

) and stress inducible protein 1 (STI1) as possible neuronal markers. For this, human neuroblastoma (SH-SY5Y), murine neuroblastoma (Neuro2a) and rat pheochromocytoma (PC12) cell lines were cultived under normal conditions with 10% FBS, FBS reduction conditions of 1% and 0% and differentiation conditions using retinoic acid (RA) for neuroblastomas and nerve growth fator (NGF) for pheochromocytoma. After differentiation time, cells were processed to obtain extracts, which were analized by western blot, using following antibodies: antisynaptophysin, anti-PrPc , anti-STI1 and anti-actin. In SH-SY5Y cells experiments, synaptophysin was not detected by antibody anti-synaptophisin, indicating there was no protein expression. PrPc protein presented conflicting results and it was not possible to say its involvement in differentiation. Neuro2a cell line presented higher expression of synaptophysin and PrPc

in group maintained at 1% FBS for 3 days in relation to the 10% FBS group, both in FBS reduction assay, and in RA differentiation assay, showing to be independent of RA. In FBS reduction assay, PC12 cells maintained at 0% FBS showed an increase in the expression of synaptophysin and PrPc proteins in relation to 10% FBS group. In differentiation assay of PC12 cell line, it was possible to observe increase of synaptophysin expression in groups 1% FBS in relation to group 10% FBS, even without NGF, and it was shown to be time dependent. However, groups 1% FBS did not present an increase in PrPc expression in relation to group 10% FBS. Regarding STI1 protein, it was not possible to observe differences of expression between conditions, neither in cell lines. In conclusion, serum reduction can be sufficient to induce differentiation in studies with PC12 maintained to 0% FBS and in Neuro2a cells under FBS reduction conditions, maintained to 1% FBS, using synaptophysin as neural marker and possibly PrPc as a new marker.