RESUMEN
OBJECTIVE: To determine the effects of TGFβ1 and an anti-scar protein nAG on normal (un-injured) 3-D astrocyte cultures with special emphasis on astrocyte proliferation, and the expression of GFAP, CSPG4, S100B, and IL-6. STUDY DESIGN: An experimental study. PLACE AND DURATION OF STUDY: King Saud University, Riyadh, Saudi Arabia, from March to September 2017. METHODOLOGY: 3-D astrocyte gels were treated differently to create one control group (control, untreated astrocytes) and five experimental groups: nAg-only (treated astrocytes with 1 nM nAG recombinant protein), TGFβ1-only (treated astrocytes with 10 ng/ml TGFβ1), TGFβ1+low nAg concentration, TGFβ1+intermediate nAg concentration, and TGFβ1+high nAg concentration. Astrocyte proliferation, and the expression of GFAP, CSPG4, S100B, and IL-6 were studied and compared in these experimental groups. RESULTS: There were major differences in the responses of normal astrocytes in vitro versus the responses of astrocytes in vivo in the setting of injury or disease. The effects of TGFβ1 were dominant over the effects of nAG with regard to changes in CSPG4 and S100B by Real-Time PCR. There was a synergistic inhibitory effect of TGFï¢1 and nAG on the expression of CSPG4 by Western blot. CONCLUSION: The different responses of normal astrocytes in vitro versus astrocytes in vivo in the setting of injury or disease. The response at the gene level might not coincide with the response at the protein level.
Asunto(s)
Proteínas Anfibias/farmacología , Astrocitos/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Salamandridae , Factor de Crecimiento Transformador beta1/farmacología , Animales , Astrocitos/metabolismo , Astrocitos/fisiología , Técnicas de Cultivo de Célula , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Proteína Ácida Fibrilar de la Glía/metabolismo , Humanos , Interleucina-6/metabolismo , Proteínas de la Membrana/metabolismo , Subunidad beta de la Proteína de Unión al Calcio S100/metabolismoRESUMEN
BACKGROUND: nAG and Prod-1 are proteins responsible for the regeneration of completely amputated limbs in salamanders (which are lower vertebrates). The purpose of this study was to introduce an experimental distal phalanx amputation model in mice (which are higher vertebrates) in which nAG and Prod-1 are expressed in the amputation stumps. METHODS: Sixteen mice with amputation of the distal two-thirds of the distal phalanx were used. One hind limb was used and the central three digits were amputated. Injection of nAG and Prod-1 plasmids was performed in the footpad twice weekly in experimental mice (n = 8), and injection of solution only (without the plasmids) was performed twice weekly in control mice (n = 8). RESULTS: nAG and Prod-1 were expressed in experimental stumps only. This expression results in quicker and more mature bone regeneration in experimental animals, and this was shown using histology and immune stains to osteocalcin (an osteoblast marker). Finally, quantitative mRNA showed a 21-fold increase of osteocalcin in experimental stumps compared with control stumps, and this was statistically significant. CONCLUSION: Injection of nAG and Prod-1 into the footpad will result in their expression in the distal amputation stumps, and this will enhance bone regeneration in the model described.
Asunto(s)
Muñones de Amputación/patología , Regeneración Ósea/efectos de los fármacos , Citocromo P-450 CYP2B1/farmacología , Proteínas/farmacología , Amputación Quirúrgica/métodos , Animales , Biomarcadores/metabolismo , Biopsia con Aguja , Citocromo P-450 CYP2B1/metabolismo , Modelos Animales de Enfermedad , Inmunohistoquímica , Ratones , Ratones Endogámicos , Proteínas/metabolismo , ARN/metabolismo , Distribución Aleatoria , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Sensibilidad y EspecificidadRESUMEN
Unlike humans, salamanders regrow their amputated limbs. Regeneration depends on the presence of regenerating axons which upregulate the expression of newt anterior gradient (nAG) protein. We had the hypothesis that nAG might have an inhibitory effect on collagen production since excessive collagen production results in scarring, which is a major enemy to regeneration. nAG gene was designed, synthesized, and cloned. The cloned vector was then transfected into primary human fibroblasts. The results showed that the expression of nAG protein in primary human fibroblast cells suppresses the expression of collagen I and III, with or without TGF- ß 1 stimulation. This suppression is due to a dual effect of nAG both by decreasing collagen synthesis and by increasing collagen degradation. Furthermore, nAG had an inhibitory effect on proliferation of transfected fibroblasts. It was concluded that nAG suppresses collagen through multiple effects.