ABSTRACT
O objetivo deste estudo foi verificar a capacidade de diferenciação das células-tronco da polpa dentária canina em células progenitoras neurais bem como quantificar obtenção e viabilidade celular, durante três passagens em cultura. As células foram extraídas da polpa dentária de dois cadáveres caninos, com aproximadamente dez meses de idade, que foram a óbito em decorrência de traumatismo automotivo. Após três subculturas, realizou-se avaliação da viabilidade celular por quantificação em câmara de Neubauer. A partir disso, induziu-se diferenciação neural em meio de cultura neurobasal (Gibco™), com células aderidas ao plástico ou suspensas em placas tratadas com agarose. Após sete e 14 dias em cultivo indutor, observou-se morfologia e perfil imunofenotípico utilizando citometria de fluxo e imunocitoquímica fluorescente. Aos 14 dias as células apresentaram alto grau de expressão para marcadores anti-nestina e anti-glial fibrillary acidic protein (anti-GFAP). Anteriormente, obteve-se ao 25º dia, média de 18x106 células viáveis indiferenciadas oriundas do tecido pulpar. Sugere-se que as células-tronco indiferenciadas da polpa dentária canina apresentem índices satisfatórios de diferenciação em células progenitoras neurais, aderidas ou suspensas em cultura. A polpa dentária dos dentes decíduos caninos, fornece células indiferenciadas viáveis em quantidade adequada.(AU)
The objective of this study was to verify the differentiation capacity of canine tooth pulp stem cells in neural progenitor cells as well as to quantify the attainment and viability during three culture passages. The cells were extracted from the dental pulp of two canine cadavers, with approximately ten months of age, which died due to automotive trauma. After three subcultures, cell viability evaluation was performed by Neubauer chamber quantification. Neural differentiation was induced in neurobasal culture medium (Gibco ™), with cells adhered to the plastic or suspended in agarose-treated plates. After seven and 14 days in inducer culture, morphology and immunophenotypic profile were observed using flow cytometry and fluorescent immunocytochemistry. At 14 days the cells had a high degree of expression for anti-nestin and anti-glial fibrillary acidic (anti-GFAP) markers. Previously, an average of 18x106 undifferentiated viable cells from the pulp tissue were obtained on the 25th day. It is suggested that the undifferentiated canine pulp stem cells present satisfactory differentiation indices in neural progenitor cells, adhered or suspended in culture. The dental pulp of deciduous canine teeth provides viable undifferentiated cells in adequate quantity.(AU)
Subject(s)
Animals , Dogs , Dental Pulp/ultrastructure , Neural Stem Cells , Cell- and Tissue-Based Therapy/veterinary , Demyelinating Diseases/veterinary , Flow Cytometry/veterinaryABSTRACT
Objective To investigate the influence of recombinant neuregulin-1 beta (rhNRG-1β) on neural stem cell proliferation through extracellular regulated protein kinases (ERK) signaling pathway in oxygen and glucose deprivation (OGD) environment.Methods Neural stem cells were obtained from embryonic brain of mice pregnant for 14-17 d,cultured and identified by immunochemical staining through detection of the indicator nestin using the SABC-FITC (POD)double standard kit.Neural stem cells were divided into three experiment groups (OGD group,control group and OGD + rhNRG-1β group).Control group:identified neural stem cells,2 × 107,were cultured for 3 h in the 24-hole culture plate with DMEM/F12 complete culture medium;OGD group:neural stem cells,2 × 107,were cultured in the 24-hole culture plate deprived glucose DMEM/F12 in a wet airtight container (37 ℃ constant temperature),cells were cultured with mixed gas of nitrogen (950 ml/L) and oxygen (50 ml/L) for 1 h,and then the culture medium was replaced with complete culture medium and cultured for 3 h;OGD + rhNRG-1β group:before OGD intervention,100 μg/L rhNRG-1β was given for 3 h.Neurospheres formation:the three groups of stem cells were dispersed into single cells,1 × 106/ml cells were inoculated to culture plates containing cover slips coated with poly lysine,and cultivated for 7 d,and neurospheres formation of the 3 groups of neural stem cells was observed under microscope,which was aimed to record neural stem cells proliferation changes.Colony formation:the three groups of stem cells,vaccinated in 60 mm in a petri dish,2 × 107 in number,were cultivated in complete culture medium for 24 h.The colony formation of the three groups of cells was observed under microscope,and neural stem cells proliferation changes were observed.Western blotting:the change of phosphorylation ERK (pERK) protein of the three groups of stem cells was determined,and the effect of pERK protein expression regulated by rhNRG-1β in mice neural stem cells proliferation through ERK signaling pathway was observed.Results Microscopically the primary cultured stem cells grew in single or in pairs,in a round shape;neural stem cell proliferated in clumps or colony;neural stem cells expressed the specificity of nestin protein markers with fluorescent yellow-green color.The differences in the aspects of the average diameter of neurospheres and neurospheres quantity in the neural stem cells between groups were statistically significant (F =693.66,1 002.09,all P ＜ 0.01),and among them the neuropheres formation of OGD group was significantly suppressed.Formation quantity and average diameter in OGD group [(88.78 ± 7.14) numbers,(62.12 ± 2.52) μm] were significantly lower than those in the control group [(246.34 ± 8.67) numbers,(128.45 ± 2.33) μm] and those in OGD + rhNRG-1β group [(237.87 ± 6.61) numbers,(118.37 ± 2.71) μm,all P ＜ 0.01].The difference of colony formation rate of neural stem cell was statistically significant (F =132.03,P ＜ 0.01),and among them colony formation of OGD group significantly suppressed.Formation rate in OGD group [(11.65 ± 0.94)％] was significantly lower than that in the control group [(33.23 ± 2.93)％] and that in OGD + rhNRG-1β group [(31.42 ± 2.61)％,all P ＜ 0.01].Western blotting showed that the difference of pERK protein expression of neural stem cells between groups was statistically significant (F =63.76,P ＜ 0.01).Relative expression of the pERK protein in OGD group (0.487 ± 0.072) was significantly lower than that in the control group (1.013 ± 0.112) and that in OGD + rh-NRG-1β group (1.752 ± 0.278,all P ＜ 0.01).Conclusion rhNRG-1β preserves neural stem cell proliferation with phosphorylation ERK protein expression up-regulated in oxygen and glucose deprivation environment.
ABSTRACT
AIM: To discover specific neural stem cell (NSC) proliferation-promoting factor, which will contribute to study on development of nervous system and treatment of nervous system diseases. METHODS: The extracts of forebrain, midbrain,afterbrain and cerebellum of neonatal mice were prepared, and the NSCs of newborn mice were cultured in vitro. Neurospheres were observed, immunocytochemical staining of characteristic protein, nestin, and MTT assay were performed to identify NSCs and their proliferative properties. RESULTS: A great deal of neurospheres were formed in the presence of the extracts of afterbrain and cerebellum, which were positive for characteristic protein (Nestin) of NSC showed by immunocytochemical staining. CONCLUSION: The afterbrain and cerebellum extracts can increase the total number of NSCs isolated from newborn mice in vitro in a dose-dependent manner.