RESUMO
Sphingosine-1-phosphate (S1P) and ceramide-1-phosphate (C1P) belong to a family of bioactive sphingolipids that act as important extracellular signaling molecules and chemoattractants. This study investigated the influence of S1P and C1P on the morphology, proliferation activity and osteogenic properties of rat multipotent stromal cells derived from bone marrow (BMSCs) and subcutaneous adipose tissue (ASCs). We show that S1P and C1P can influence mesenchymal stem cells (MSCs), each in a different manner. S1P stimulation promoted the formation of cellular aggregates of BMSCs and ASCs, while C1P had an effect on the regular growth pattern and expanded intercellular connections, thereby increasing the proliferative activity. Although osteogenic differentiation of MSCs was enhanced by the addition of S1P, the effectiveness of osteoblast differentiation was more evident in BMSCs, particularly when biochemical and molecular marker levels were considered. The results of the functional osteogenic differentiation assay, which includes an evaluation of the efficiency of extracellular matrix mineralization (SEM-EDX), revealed the formation of numerous mineral aggregates in BMSC cultures stimulated with S1P. Our data demonstrated that in an appropriate combination, the bioactive sphingolipids S1P and C1P may find wide application in regenerative medicine, particularly in bone regeneration with the use of MSCs.
Assuntos
Ceramidas/farmacologia , Lisofosfolipídeos/farmacologia , Células-Tronco Multipotentes/efeitos dos fármacos , Medicina Regenerativa/métodos , Esfingosina/análogos & derivados , Células Estromais/efeitos dos fármacos , Tecido Adiposo/citologia , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Células Cultivadas , Expressão Gênica/efeitos dos fármacos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/ultraestrutura , Microscopia Eletrônica de Varredura , Microscopia de Fluorescência , Células-Tronco Multipotentes/citologia , Células-Tronco Multipotentes/ultraestrutura , Osteocalcina/genética , Osteocalcina/metabolismo , Osteogênese/efeitos dos fármacos , Osteogênese/genética , Osteopontina/genética , Osteopontina/metabolismo , Ratos Wistar , Medicina Regenerativa/tendências , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Esfingosina/farmacologia , Células Estromais/citologia , Células Estromais/ultraestruturaRESUMO
The recent spread of Zika virus stimulated extensive research on its structure, pathogenesis, and immunology, but mechanistic study of entry has lagged behind, in part due to the lack of a defined reconstituted system. Here, we report Zika membrane fusion measured using a platform that bypasses these barriers, enabling observation of single-virus fusion kinetics without receptor reconstitution. Surprisingly, target membrane binding and low pH are sufficient to trigger viral hemifusion to liposomes containing only neutral lipids. Second, although the extent of hemifusion strongly depends on pH, hemifusion rates are relatively insensitive to pH. Kinetic analysis shows that an off-pathway state is required to capture this pH-dependence and suggests this may be related to viral inactivation. Our surrogate-receptor approach thus yields new understanding of flaviviral entry mechanisms and should be applicable to many emerging viruses.