RESUMO
Bone morphogenetic proteins (BMPs) are members of the TGF-ß superfamily, acting as potent regulators during embryogenesis and bone and cartilage formation and repair. Cell and molecular biology approaches have unveiled the great complexity of BMP action, later confirmed by transgenic animal studies. Genetic engineering allows for the production of large amounts of BMPs for clinical use, but they have systematically been associated with a delivery system, such as type I collagen and calcium phosphate ceramics, to ensure controlled release and to maximize their biological activity at the surgical site, avoiding systemic diffusion. Clinical orthopedic studies have shown the benefits of FDA-approved recombinant human BMPs (rhBMPs) 2 and 7, but side effects, such as swelling, seroma, and increased cancer risk, have been reported, probably due to high BMP dosage. Several studies have supported the use of BMPs in periodontal regeneration, sinus lift bone-grafting, and non-unions in oral surgery. However, the clinical use of BMPs is growing mainly in off-label applications, with robust evidence to ascertain rhBMPs' safety and efficacy through well-designed, randomized, and double-blind clinical trials. Here we review and discuss the critical data on BMP structure, mechanisms of action, and possible clinical applications.
Assuntos
Proteínas Morfogenéticas Ósseas/fisiologia , Proteína Morfogenética Óssea 2/uso terapêutico , Proteína Morfogenética Óssea 7/uso terapêutico , Proteínas Morfogenéticas Ósseas/uso terapêutico , Regeneração Óssea/efeitos dos fármacos , Condrogênese/efeitos dos fármacos , Sistemas de Liberação de Medicamentos , Humanos , Osteogênese/efeitos dos fármacos , Proteínas Recombinantes/uso terapêutico , Transdução de Sinais/fisiologia , Relação Estrutura-Atividade , Fator de Crescimento Transformador beta/uso terapêuticoRESUMO
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
Social and economical development is closely associated with technological innovation and a well-developed biotechnological industry. In the last few years, Brazil's scientific production has been steadily increasing; however, the number of patents is lagging behind, with technological and translational research requiring governmental incentive and reinforcement. The Cell and Molecular Therapy Center (NUCEL) was created to develop activities in the translational research field, addressing concrete problems found in biomedical and veterinary areas and actively searching for solutions by employing a genetic engineering approach to generate cell lines over-expressing recombinant proteins to be transferred to local biotech companies, aiming at furthering the development of a national competence for local production of biopharmaceuticals of widespread use and of life-saving importance. To this end, mammalian cell engineering technologies were used to generate cell lines over-expressing several different recombinant proteins of biomedical and biotechnological interest, namely, recombinant human Amylin/IAPP for diabetes treatment, human FVIII and FIX clotting factors for hemophilia, human and bovine FSH for fertility and reproduction, and human bone repair proteins (BMPs). Expression of some of these proteins is also being sought with the baculovirus/insect cell system (BEVS) which, in many cases, is able to deliver high-yield production of recombinant proteins with biological activity comparable to that of mammalian systems, but in a much more cost-effective manner. Transfer of some of these recombinant products to local Biotech companies has been pursued by taking advantage of the São Paulo State Foundation (FAPESP) and Federal Government (FINEP, CNPq) incentives for joint Research Development and Innovation partnership projects.
Assuntos
Biofarmácia , Comunicação Interdisciplinar , Proteínas Recombinantes/biossíntese , Transferência de Tecnologia , Amiloide/biossíntese , Animais , Baculoviridae/metabolismo , Biotecnologia , Proteínas Morfogenéticas Ósseas/biossíntese , Brasil , Linhagem Celular , Fator IX/biossíntese , Fator VIII/biossíntese , Hormônio Foliculoestimulante/biossíntese , Engenharia Genética , Vetores Genéticos/biossíntese , Humanos , Polipeptídeo Amiloide das Ilhotas Pancreáticas , Pesquisa/economia , Pesquisa/organização & administração , Spodoptera/virologiaRESUMO
Ex vivo islet cell culture prior to transplantation appears as an attractive alternative for treatment of type 1 diabetes. Previous results from our laboratory have demonstrated beneficial effects of human prolactin (rhPRL) treatment on human islet primary cultures. In order to probe into the molecular events involved in the intracellular action of rhPRL in these cells, we set out to identify proteins with altered expression levels upon rhPRL cell treatment, using two-dimensional (2D) gel electrophoresis and mass spectrometry (MS). An average of 300 different protein spots were detected, 14 of which were modified upon rhPRL treatment (p<0.01), of which 12 were successfully identified using MS and grouped according to their biological functions. In conclusion, our study provides, for the first time, information about proteins that could be critically involved in PRL's action on human pancreatic islets, and facilitate identification of new and specific targets involved in islet cell function and proliferation.
Assuntos
Proliferação de Células/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Prolactina/farmacologia , Adulto , Eletroforese em Gel Bidimensional , Feminino , Humanos , Ilhotas Pancreáticas/citologia , Transplante das Ilhotas Pancreáticas , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Proteínas Recombinantes de Fusão/farmacologia , Técnicas de Cultura de TecidosRESUMO
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