RESUMO
A composite of marrow mesenchymal stem cells (MSCs) and porous hydroxyapatite (HA) has bone-forming capability. To promote the capability, we added recombinant human bone morphogenetic protein-2 (BMP) to the composite. The bone formation was assessed by rat subcutaneous implantation of 4 different kinds of implants, i.e., HA alone, BMP/HA composites, MSCs/HA composites, and the composites containing BMP (MSCs/BMP/HA). Both HA and the BMP/HA composites did not show bone formation at any time after implantation. The MSCs/HA composites showed moderate bone formation at 4 weeks and extensive bone formation at 8 weeks. The MSCs/BMP/HA composites showed obvious bone formation together with active osteoblasts at 2 weeks and more bone formation at 4 and 8 weeks. The MSCs/BMP/HA composites demonstrated high alkaline phosphatase and osteocalcin expression at both the protein and gene levels. These results indicate that the combination of MSCs, porous HA, and BMP synergistically enhances osteogenic potential and provides a rational basis for their clinical application in bone reconstruction surgery.
Assuntos
Células da Medula Óssea , Proteínas Morfogenéticas Ósseas/farmacologia , Durapatita , Implantes Experimentais , Osteogênese/efeitos dos fármacos , Células-Tronco , Fator de Crescimento Transformador beta , Fosfatase Alcalina/análise , Animais , Materiais Biocompatíveis , Proteína Morfogenética Óssea 2 , Osso e Ossos/química , Osso e Ossos/citologia , Cerâmica , Gliceraldeído-3-Fosfato Desidrogenases/análise , Humanos , Masculino , Osteoblastos/citologia , Osteocalcina/análise , Ratos , Ratos Endogâmicos F344 , Proteínas Recombinantes/farmacologiaRESUMO
The IFN-induced double-stranded RNA (dsRNA)-activated protein kinase PKR is one of the key molecules in the antiviral effects of IFN. To clarify the effects of hepatitis C virus nonstructural protein 5A (NS5A) on antiviral activity of IFN, in particular on PKR kinase activity, in mammalian cells, we established inducible NS5A-expressing cell lines derived from human osteosarcoma (Saos-2). The cells expressing NS5A derived from an IFN-resistant clone (NS5A-lb) that interacted with endogenous PKR in vitro, showed a suppressive effect on IFN function as determined by interference with vesicular stomatitis virus (VSV) infection, whereas NS5A (NS5A-2a) from an IFN-sensitive clone did not block the antiviral effect of IFN. A mutant with deletion of the IFN sensitivity determining region (ISDR) in NS5A-1b (NS5A-AISDR) also interacted with PKR and suppressed its activity in vitro. However, neither NS5A-2a nor the C-terminal truncated mutant of NS5A-1b (NS5A-deltaC) blocked PKR activity. These observations confirmed the previous report that the inhibitory effect of NS5A on IFN activity is mediated at least in part by the repression of PKR. In addition, we showed that IFN sensitivity was determined not only by the ISDR but that the involvement of the C-terminal region of NS5A-1b is important for the suppression of PKR activity.
Assuntos
Hepacivirus/imunologia , Hepatite C/imunologia , Interferon-alfa/farmacologia , Proteínas não Estruturais Virais/imunologia , eIF-2 Quinase/imunologia , Sequência de Aminoácidos , Western Blotting , Farmacorresistência Viral/imunologia , Escherichia coli/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Hepacivirus/genética , Hepatite C/tratamento farmacológico , Hepatite C/virologia , Humanos , Interferon-alfa/imunologia , Dados de Sequência Molecular , Mutação , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Células Tumorais Cultivadas , Proteínas não Estruturais Virais/genética , eIF-2 Quinase/antagonistas & inibidoresRESUMO
A composite of marrow mesenchymal stem cells and porous hydroxyapatite (HA) has in vivo osteogenic potential. To investigate factors enhancing the osteogenic potential of marrow/HA composites, we prepared a bone morphogenetic protein (BMP) fraction from the 4M guanidine extract of bovine bone by heparin-sepharose affinity chromatography. Marrow/HA composites or composites containing marrow mesenchymal stem cells, BMP, and HA (marrow/BMP/HA composites) were implanted subcutaneously in 7-week-old male Fischer rats. BMP/HA composites and HA alone were also implanted. The implants were harvested after 2, 4, or 8 weeks and were prepared for histological and biochemical studies. Histological examination showed obvious de novo bone formation together with active osteoblasts at 2 weeks, as well as more extensive bone formation at 4 and 8 weeks in many pores of the marrow/BMP/HA composites. The marrow/HA composites did not induce bone formation at 2 weeks, but there was moderate bone formation at 4 weeks. At 2 weeks, only marrow/BMP/HA composites resulted in intensive osteogenic activity, judging from alkaline phosphatase and osteocalcin expression at both the protein and gene levels. These results indicate that the combination of marrow mesenchymal stem cells, porous HA, and BMP synergistically enhances osteogenic potential, and may provide a rational basis for their clinical application, although further in vivo experiment is needed.
