RESUMEN
Cytokeratin 8 (CK8) is a member of the cytokeratins family with multiple functions on the basis of its unique structural hallmark. The aberrant expression of CK8 and its phosphorylation are pertinent with various diseases. We have previously shown that CK8 exists in normal human nucleus pulposus (NP) cells and decreases as the intervertebral disc degenerates. However, the underlying molecular regulatory machinery of CK8 in intervertebral disc degeneration (IDD) has not been clarified. Here, we collected NP samples from patients with idiopathic scoliosis as control and IDD as degenerate groups. We found that CK8 expression decreased in IDD with an increased phosphorylation in degenerate NP cells. Moreover, NP cells were cultured under different compressive load schemes for diverse time duration. We found that compressive loads resulted in phosphorylation and disassembly of CK8 in a time-dependent and degree-dependent manner in vitro. The activation of protein kinase C was a significant molecular factor contributing to this phenomenon. Taken together, this study is the first to address the molecular mechanisms of CK8 downregulation in NP cells. Importantly, our findings provide clues regarding a molecular link between compressive loads and CK8 alterations, which shed a novel light on the etiology of IDD.
Asunto(s)
Degeneración del Disco Intervertebral/enzimología , Queratina-8/metabolismo , Proteína Quinasa C-epsilon/metabolismo , Estrés Mecánico , Adolescente , Adulto , Regulación hacia Abajo/fisiología , Humanos , Fosforilación , Columna Vertebral/fisiología , Regulación hacia Arriba/fisiología , Soporte de Peso , Adulto JovenRESUMEN
To provide a more permissive environment for axonal regeneration, Schwann cells (SCs) were introduced into a collagen-chitosan scaffold with longitudinally oriented micro-channels (L-CCH). The SC-seeded scaffold was then used for reconstruction of a 15-mm-long sciatic nerve defect in rats. The axonal regeneration and functional recovery were examined by a combination of walking track analysis, electrophysiological assessment, Fluoro-Gold retrograde tracing, as well as morphometric analyses to both regenerated axons and target muscles. The findings showed that SCs adhered and migrated into the L-CCH scaffold and displayed a longitudinal arrangement in vitro. Axonal regeneration as well as functional recovery was in the similar range between SCs-seeded scaffold and autograft groups, which were superior to those in L-CCH scaffold alone group. These indicate that the SCs-seeded L-CCH scaffold, which resembles the microstructure as well as the permissive environment of native peripheral nerves, holds great promise in nerve regeneration therapies.
Asunto(s)
Técnicas de Cultivo de Célula/instrumentación , Regeneración Tisular Dirigida , Regeneración Nerviosa/fisiología , Células de Schwann/citología , Nervio Ciático/fisiología , Andamios del Tejido , Animales , Animales Recién Nacidos , Polaridad Celular , Células Cultivadas , Regeneración Tisular Dirigida/instrumentación , Regeneración Tisular Dirigida/métodos , Porosidad , Ratas , Ratas Sprague-Dawley , Recuperación de la Función/fisiología , Células de Schwann/fisiología , Células de Schwann/trasplante , Nervio Ciático/citología , Propiedades de Superficie , Andamios del Tejido/química , Trasplante Autólogo/métodosRESUMEN
Objective To investigate the efficient method which can culture and induce embryonic stem cells to neurocyte in vitro. Methods Isolate the blastula of 3.5 d from BALB/c species mouse. Culture the cells from inner cell mass (ICM) which were isolated by mechanical method on the mouse embryonic fibroblaste cell (MEF) feeder layer or 0.1% gelatin coated dishes. The stem cells were identified by characterized morphology, alkaline phosphatase stain, differential potency in vivo and immunochemistry stain. The isolated cells were differentiated by serial induction method that mimicking the intrinsic developmental process of the neural system. Results The isolated cells were positive for alkaline phosphatatse and SSEA-1 (stage specific embryonic antigen 1). Moreover they were identified pluripotent by differentiation in vivo. Therefore the isolated cells presented the characters of ESCs. Then the isolated cells were able to differentiate into neurocytes in vitro. Conclusion Mouse embryonic stem cells isolation, culture and differentiation system has been established.