RESUMEN
Although the primary cell cultures from dental pulp and other oral tissue are frequently used to study osteogenic potential and stem cell responses, few systematic and comparative studies on stemness for the dentinogenic differentiation of these cells have been conducted. In the present study, to investigate the stemness of oral primary cells during extended culture, human adult dental pulp cells (hDPCs), periodontal ligament stem cells (hPDLSCs) and gingival fibroblasts (hGFs) were obtained and cultured from pulp tissue, periodontal ligaments, and marginal and attached gingival tissue of extracted third molars, respectively. As shown by fluorescence-activated cell sorting analysis and immunophenotyping, the mesenchymal stem cell markers, CD44, CD73, CD90, CD146 and CD166, were highly expressed in early passage hDPCs, hPDLSCs and hGFs. However, when the cells were treated with osteogenic additives, mineralization markedly increased in the hDPCs and hPDLSCs, but not in the hGFs. Moreover, the expression of dentinogenic markers, such as dentin sialophosphoprotein and dentin matrix protein-1, appeared to decrease during extended culture past passage number 8 of the hDPCs and hPDLSCs. These data suggest that hDPCs and hPDLSCs may have differentiation potential during the early passages, and that their progenitor potential is diminished during extended culture. The hGFs did not show differentiation capability during culture, even though they contained general mesenchymal stem cell surface proteins. The transcriptional expression of dentinogenic markers in hDPCs was not affected by co-culture with hPDLSCs and/or hGFs.
Asunto(s)
Diferenciación Celular , Pulpa Dental/citología , Fibroblastos/citología , Encía/citología , Osteogénesis , Ligamento Periodontal/citología , Células Madre/citología , Antígenos de Superficie/metabolismo , Biomarcadores/metabolismo , Calcificación Fisiológica , Diferenciación Celular/genética , Células Cultivadas , Técnicas de Cocultivo , Dentina/metabolismo , Fibroblastos/metabolismo , Humanos , Inmunofenotipificación , Cultivo Primario de Células , Células Madre/metabolismoRESUMEN
A new method to investigate the initial protein folding dynamics is developed based on a pulsed laser light triggering method and a unique transient grating method. The side chain of the cysteine residue of apoplastocyanin (apoPC) was site-specifically modified with a 4,5-dimethoxy-2-nitrobenzyl derivative, where the CD and 2D NMR spectra showed that the modified apoPC was unfolded. The substituent was cleaved with a rate of about 400 ns by photoirradiation, which was monitored by the disappearance of the absorption band at 355 nm and the increase in the transient grating signal. After a sufficient time from the photocleavage reaction, the CD and NMR spectra showed that the native beta-sheet structure was recovered. Protein folding dynamics was monitored in the time domain with the transient grating method from a viewpoint of the molecular volume change and the diffusion coefficient, both of which reflect the global structural change, including the protein-water interaction. The observed volume decrease of apoPC with a time scale of 270 micros is ascribed to the initial hydrophobic collapse. The increase in the diffusion coefficient (23 ms) is considered to indicate a change from an intermolecular to an intramolecular hydrogen bonding network. The initial folding process of apoPC is discussed based on these observations.
Asunto(s)
Apoproteínas/química , Luz , Plastocianina/química , Conformación Proteica/efectos de la radiación , Pliegue de Proteína , Estructura Secundaria de Proteína/efectos de la radiación , Dicroismo Circular , Cisteína/química , Difusión , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Rayos Láser , Espectroscopía de Resonancia Magnética , Nitrobencenos/química , Agua/químicaRESUMEN
The photoreaction of caged ATP, P3-[1-(2-nitrophenyl)ethyl]adenosine-5'-triphosphate, has been investigated using the time-resolved transient grating (TG) method. We found that a feature of the TG signal time profile depends sensitively on the grating wavenumber (q) after the photoexcitation of caged ATP. This q-dependent feature of the TG signal was interpreted based on a model where the ATP release rate is comparable to the molecular diffusion process. We found that the TG signals at various q can be consistently analyzed based on this model and the ATP release rate determined. The enthalpy and volume changes of the reaction have been determined by quantitative measurement of the grating and photoacoustic signals.