RESUMO
The type I herpes simplex virus VP22 tegument protein is abundant and well known for its ability to translocate proteins from one cell to the other. In spite of some reports questioning its ability to translocate proteins by attributing the results observed to fixation artifacts or simple attachment to the cell membrane, VP22 has been used to deliver several proteins into different cell types, triggering the expected cell response. However, the question of the ability of VP22 to enter stem cells has not been addressed. We investigated whether VP22 could be used as a tool to be applied in stem cell research and differentiation due to its capacity to internalize other proteins without altering the cell genome. We generated a VP22.eGFP construct to evaluate whether VP22 could be internalized and carry another protein with it into two different types of stem cells, namely adult human dental pulp stem cells and mouse embryonic stem cells. We generated a VP22.eGFP fusion protein and demonstrated that, in fact, it enters stem cells. Therefore, this system may be used as a tool to deliver various proteins into stem cells, allowing stem cell research, differentiation and the generation of induced pluripotent stem cells in the absence of genome alterations.
Assuntos
Proteínas de Transporte/farmacocinética , Membrana Celular/metabolismo , Células-Tronco Embrionárias/metabolismo , Proteínas de Fluorescência Verde/farmacocinética , Proteínas Estruturais Virais/farmacocinética , Animais , Western Blotting , Polpa Dentária/citologia , Citometria de Fluxo , Proteínas de Fluorescência Verde/genética , Humanos , Camundongos , Microscopia Confocal , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Estruturais Virais/genéticaRESUMO
The type I herpes simplex virus VP22 tegument protein is abundant and well known for its ability to translocate proteins from one cell to the other. In spite of some reports questioning its ability to translocate proteins by attributing the results observed to fixation artifacts or simple attachment to the cell membrane, VP22 has been used to deliver several proteins into different cell types, triggering the expected cell response. However, the question of the ability of VP22 to enter stem cells has not been addressed. We investigated whether VP22 could be used as a tool to be applied in stem cell research and differentiation due to its capacity to internalize other proteins without altering the cell genome. We generated a VP22.eGFP construct to evaluate whether VP22 could be internalized and carry another protein with it into two different types of stem cells, namely adult human dental pulp stem cells and mouse embryonic stem cells. We generated a VP22.eGFP fusion protein and demonstrated that, in fact, it enters stem cells. Therefore, this system may be used as a tool to deliver various proteins into stem cells, allowing stem cell research, differentiation and the generation of induced pluripotent stem cells in the absence of genome alterations.
Assuntos
Animais , Humanos , Camundongos , Proteínas de Transporte/farmacocinética , Membrana Celular/metabolismo , Células-Tronco Embrionárias/metabolismo , Proteínas de Fluorescência Verde/farmacocinética , Proteínas Estruturais Virais/farmacocinética , Western Blotting , Polpa Dentária/citologia , Citometria de Fluxo , Proteínas de Fluorescência Verde/genética , Microscopia Confocal , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Estruturais Virais/genéticaRESUMO
Diabetes mellitus is a widespread disease whose frequency increases constantly and is expected to reach alarming levels by the year 2025. Introduction of insulin therapy represented a major breakthrough; however, a very strict regimen is required to maintain blood glucose levels within the normal range and to prevent or postpone chronic complications associated with this disease. Frequent hyper- and hypoglycemia seriously affect the quality of life of these patients. Reversion of this situation can only be achieved through whole organ (pancreas) transplant or pancreatic islet transplant, the former being a high-risk surgical procedure, while the latter is a much simpler and may be accomplished in only 20-40 min. The advantages and perspectives of islet cell transplantation will be discussed, in the light of tissue engineering and gene therapy. Ongoing research carried out in our laboratory, aimed at developing clinical cell and molecular therapy protocols for diabetes will also be focused
