RESUMO
Different approaches of engineering cartilage to treat defects in the articulating surfaces of the joints have been designed, which mainly use mesenchymal stem cells or autologous chondrocytes for in situ transplantation. However, these cells are poorly characterized with respect to viability, degree of differentiation and morphology or production of extracellular matrix. At present, one of the key approaches to generate chondrocytes is the stimulation of stem cells with transforming growth factor (TGF) ß1. To characterize the molecular alterations occurring during the cellular transformation induced by TGF-ß1 exposure, the differentiation process of bone marrow-derived stem cells into chondrocytes was investigated using an in vitro chondrogenesis model and the RNA arbitrarily primed PCR (RAP-PCR) fingerprinting technique. Distinct genes were found to be differentially regulated during chondrocyte development beginning on day 1: collagen type I, non-muscle myosin MYH9, followed by manganese superoxide dismutase and sodium-potassium ATPase on day 7. The results suggest that using RAP-PCR for differential display fingerprinting is a useful tool to investigate the differentiation process of bone marrow-derived stem cells following TGF-ß1-stimulation.
Assuntos
Condrogênese , Impressões Digitais de DNA , Matriz Extracelular , Regulação da Expressão Gênica , Reação em Cadeia da Polimerase , Células-Tronco , Fator de Crescimento Transformador beta1/farmacologia , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Células Cultivadas , Condrogênese/efeitos dos fármacos , Condrogênese/genética , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Perfilação da Expressão Gênica , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase/métodos , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Fatores de TempoRESUMO
Changes in the expression of repellent factors, i.e., Netrins and their receptors, may be responsible for the invasive behavior of the synovial tissue cells in patients with rheumatoid arthritis (RA) and osteoarthritis (OA). This study was carried out to analyze the expression of Netrins and their receptors in synovial cells of patients with RA, OA, and control subjects without synovial inflammation. Quantitative RT-PCR was performed to measure the expression of Netrin-1, -3, -4, Neogenin, DCC, UNC5A-D. The influence of Netrin-1 on synovial fibroblasts (SF) was analyzed by determining proliferation, migration, and their ability to organize collagen. SF expressed all repellent factors of the Netrin family. When comparing SF of healthy donors to patients with RA and OA, a stronger expression of UNC5B (4 fold) and UNC5C (769 fold) in RA and OA was found, whereas expression of the other molecules revealed no significant differences. Treating the SF-cells with recombinant Netrin-1 resulted in inhibition of migration of RA- and OA-SFs whereas control cells were not affected. The stronger expression of UNC5B and UNC5C receptors might contribute to the disordered phenotype of RA- and OA-SFs. Addition of Netrin-1 reduces the migratory ability of SFs, potentially by repulsion, as seen in neuronal cells in embryonic development. Due to its function, Netrin-1 may constitute a novel target in the treatment of OA and RA.
Assuntos
Artrite Reumatoide/metabolismo , Movimento Celular , Fibroblastos/metabolismo , Fatores de Crescimento Neural/metabolismo , Osteoartrite/metabolismo , Receptores de Superfície Celular/metabolismo , Membrana Sinovial/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Adulto , Idoso , Artrite Reumatoide/genética , Artrite Reumatoide/patologia , Cartilagem/metabolismo , Estudos de Casos e Controles , Células Cultivadas , Colágeno/metabolismo , Receptor DCC , Feminino , Fibroblastos/patologia , Glicosaminoglicanos/metabolismo , Humanos , Masculino , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Fatores de Crescimento Neural/genética , Proteínas do Tecido Nervoso/genética , Receptores de Netrina , Netrina-1 , Netrinas , Osteoartrite/genética , Osteoartrite/patologia , Receptores de Superfície Celular/genética , Proteínas Recombinantes/metabolismo , Membrana Sinovial/patologia , Proteínas Supressoras de Tumor/genéticaRESUMO
The capsids of human parvovirus B19 consist of two structural proteins, the minor-capsid protein VP1 and the major-capsid protein VP2. The latter which constitutes for 95% of the capsid are able to form virus-like particles (VLPs) in yeast without the presence of VP1-proteins. VP2-proteins produced in Saccharomyces cerevisiae have the capacity to form VLPs in the absence of VP1-proteins. These yeast-derived VLPs resemble native virus or recombinant VP2-VLPs produced by baculovirus systems in respect of size, molecular weight and of antigenicity as shown by antigen-capture ELISA and T-cell proliferation tests. Regarding costs, yield and ease of handling particle production in yeast represents an alternative to the recombinant baculovirus expression system which is so far the source for VP2-VLPs of human parvovirus B19.
