RESUMEN
Intracellular delivery of synthetic oligopeptides has the potential to promote the occurrence of various cellular events such as cell death, proliferation, growth inhibition, metabolic changes, and morphological changes. However, the regulation of cellular differentiation by intracellular delivery of synthetic oligopeptides has been little studied. Von Hippel-Lindau protein (pVHL) is one of the proteins that functions to induce the differentiation of neural progenitor cells (NPCs). To function in these cells, pVHL forms a complex composed of itself, elongin BC, Clu-2, and Rbx-1. It is suggested that the binding site of elongin BC in pVHL plays a critical role in pVHL function, i.e., ubiquitination, which is related to neuronal differentiation. So, we synthesized an oligopeptide corresponding to the elongin BC binding site, and delivered the oligopeptide into NPCs by using a mixture of trifluoroacetylated lipopolyamine and diloeoyl phosphatidylethanolamine (BioPorter) to form a peptide-lipid complex. After intracellular delivery of the oligopeptide, induction of differentiation of NPCs was shown in terms of neurite outgrowth and by immunocytochemical and electrophysiological means. The intracellular delivery of the synthetic oligopeptide derived from pVHL may provide a safe and valuable approach for the neuronal differentiation of NPCs.
Asunto(s)
Neurogénesis/efectos de los fármacos , Neuronas/efectos de los fármacos , Péptidos/farmacología , Células Madre/efectos de los fármacos , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/farmacología , Animales , Biomarcadores , Diferenciación Celular/efectos de los fármacos , Electrofisiología , Inmunohistoquímica , Mutación , Neuronas/citología , Neuronas/metabolismo , Péptidos/síntesis química , Péptidos/genética , Péptidos/metabolismo , Ratas , Células Madre/citología , Células Madre/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genética , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismoRESUMEN
OBJECT: Cerebral blood flow (CBF) and cerebrovascular reactivity (CVR) to acetazolamide were investigated prospectively in 162 patients with a proposed diagnosis of idiopathic normal-pressure hydrocephalus (NPH). The aim of this study was to assess the usefulness of the measurement of CBF and CVR in determining which patients would be likely to benefit from shunt placement. METHODS: The mean CBF of the whole brain was measured according to the Patlak plot method by using technetium-99m hexamethylpropyleneamine oxime. The CVR value was obtained from the response to administration of 500 mg acetazolamide and calculated as the percentage change from the baseline mean CBF value. RESULTS: One hundred forty-six patients (90.1%) responded to shunt placement ("responders"), but 16 patients (9.9%) did not ("nonresponders"). No significant difference in preoperative CBF was observed between responders and nonresponders. Preoperative CVR was significantly impaired (p<0.0025) in responders compared with healthy controls, but not in nonresponders. Responders with the incomplete triad had a significant reduction (p<0.001) in preoperative CVR, but not in preoperative CBF, compared with healthy controls. Responders with the complete triad had significantly lower preoperative CBF and CVR than those with the incomplete triad (p<0.01 and p<0.05, respectively). Postoperative CBF and CVR increased significantly (p<0.025 and p<0.001, respectively) in responders. CONCLUSIONS: Both CBF and CVR decrease with the development of NPH, suggesting that hemodynamic ischemia may be responsible for manifestation of the symptoms. Impaired CVR and reduced CBF with the development of symptoms can be proposed as diagnostic criteria for idiopathic NPH.
Asunto(s)
Acetazolamida/farmacología , Circulación Cerebrovascular/efectos de los fármacos , Hidrocéfalo Normotenso/fisiopatología , Hidrocéfalo Normotenso/cirugía , Acetazolamida/uso terapéutico , Anciano , Anciano de 80 o más Años , Derivaciones del Líquido Cefalorraquídeo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios ProspectivosRESUMEN
Skin-derived precursors (SKPs) from mammalian dermis represent neural crest-related stem cells capable of differentiating into both neural and mesodermal progency. SKPs are of clinical interest because they serve as accessible autologous donor cells for neuronal repair for neuronal intractable diseases. However, little is known about the efficient generation of neurons from SKPs, and phenotypes of neurons generated from SKPs have been restricted. In addition, the neuronal repair using their generated neurons as donor cells has not been achieved. The von Hippel-Lindau protein (pVHL) is one of the proteins that play an important role during neuronal differentiation, and recently neuronal differentiation of neural progenitor cells by intracellular delivery of a synthetic VHL peptide derived from elongin BC-binding site has been demonstrated. In the present study, a synthetic VHL peptide derived from elongin BC-binding site was conjugated to the protein transduction domain (PTD) of HIV-TAT protein (TATVHL peptide) to facilitate entry into cells, and we demonstrate the efficient generation of cells with dopaminergic phenotype from SKPs with the intracellular delivery of TATVHL peptide, and characterized the generated cells. The TATVHL peptide-treated SKPs expressed neuronal marker proteins, particularly dopamine neuron markers, and also up-regulated mRNA levels of proneural basic helix-loop-helix factors. After the TATVHL peptide treatment, transplanted SKPs into Parkinson's disease (PD) model rats sufficiently differentiated into dopamine neuron-like cells in PD model rats, and partially but significantly corrected behavior of PD model rats. The generated dopamine neuron-like cells are expected to serve as donor cells for neuronal repair for PD.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Dopamina/metabolismo , Neuronas/citología , Péptidos/farmacología , Piel/citología , Células Madre/citología , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/química , Secuencia de Aminoácidos , Animales , Conducta Animal/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Modelos Animales de Enfermedad , Inmunohistoquímica , Masculino , Datos de Secuencia Molecular , Células Madre Multipotentes/citología , Células Madre Multipotentes/efectos de los fármacos , Células Madre Multipotentes/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Enfermedad de Parkinson/terapia , Péptidos/química , Fenotipo , Ratas , Ratas Wistar , Trasplante de Células Madre , Células Madre/efectos de los fármacos , Células Madre/metabolismoRESUMEN
Neurons can be specifically induced from bone marrow stromal cells (MSCs) with extremely high efficiency using gene transfection of the Notch intracellular domain and subsequent treatment with basic-fibroblast growth factor, forskolin, and ciliary neurotrophic factor. We investigated the behavioral and histologic efficacy of such bone marrow stromal cell-derived neuronal cell (MSDNC) transplantation into a focal cerebral infarction model in rats. A left middle cerebral artery occlusion (MCAO) was performed on adult Wistar rats. MSDNC transplantation into the ipsilateral hemisphere was performed on day 7 after MCAO. The behavioral analyses were conducted on days 14, 21, 28, 35, and 36-40, and a histologic evaluation was performed on day 41. MSDNC-transplanted rats showed significant recovery compared with controls (MCAO without cell transplantation) in beam balance, limb placing, and Morris water maze tests. Histologically, transplanted cells migrated from the injection site into the ischemic boundary area, including the cortex, corpus callosum, striatum, and hippocampus. Transplanted MSDNCs were positive for MAP-2 (84% +/- 8.11%), whereas only a small number of cells were positive for GFAP (1.0% +/- 0.23%). The survival rates of MSDNCs and MSCs 1 month after transplantation were approximately 45% and 10%, respectively. These results suggest that use of MSDNCs may be a promising therapeutic strategy for cerebral infarction.
Asunto(s)
Conducta Animal , Células de la Médula Ósea/citología , Infarto Cerebral/patología , Infarto Cerebral/psicología , Neuronas/citología , Neuronas/trasplante , Animales , Encéfalo/patología , Diferenciación Celular , Infarto Cerebral/fisiopatología , Infarto Cerebral/cirugía , Masculino , Aprendizaje por Laberinto , Equilibrio Postural , Desempeño Psicomotor , Ratas , Ratas Wistar , Células del Estroma/citología , NataciónRESUMEN
Von Hippel-Lindau protein (pVHL) normally functions to cause ubiquitin-mediated degradation of hypoxia-inducible factor-1alpha (HIF-1alpha) under normoxic but not under hypoxic conditions, and induces neuronal differentiation of neural progenitor cells. However, the role of pVHL in the differentiation of neural progenitor cells under either condition has not been fully elucidated. Herein, we show that under the anoxic condition the expression of pVHL and neuronal markers in neural progenitor cells was inhibited, while HIF-1alpha was induced. In addition, neural progenitor cells expressing pVHL following gene transfer showed distinct neuronal differentiation and no induction of HIF-1alpha under the normoxic condition but not under the anoxic condition. In conclusion, neuronal differentiation induced by pVHL is associated with degradation of HIF-1alpha and occurs normally under the normoxic condition but not under the anoxic condition. Differentiation of neuronal progenitor cells may thus depend on oxygen density.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Hipoxia/patología , Neuronas/efectos de los fármacos , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/farmacología , Ubiquitina-Proteína Ligasas/farmacología , Animales , Western Blotting/métodos , Recuento de Células/métodos , Diferenciación Celular/fisiología , Células Cultivadas , Embrión de Mamíferos , Femenino , Regulación de la Expresión Génica/fisiología , Proteína Ácida Fibrilar de la Glía/metabolismo , Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia , Inmunohistoquímica/métodos , Inmunoprecipitación/métodos , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas de Neurofilamentos/genética , Proteínas de Neurofilamentos/metabolismo , Neuronas/citología , Embarazo , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Proteínas Supresoras de Tumor/genética , Ubiquitina-Proteína Ligasas/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-LindauRESUMEN
OBJECT: Bone marrow stromal cells (BMSCs) can be induced to form Schwann cells by sequentially treating the cells with beta-mercaptoethanol and retinoic acid, followed by forskolin and neurotrophic factors including heregulin. In this study the authors made artificial grafts filled with BMSC-derived Schwann cells (BMSC-DSCs) and transplanted them into the transected sciatic nerve in adult rats to evaluate the potential of BMSCs as a novel alternative method of peripheral nerve regeneration. METHODS: The BMSC-DSCs were suspended in Matrigel and transferred into hollow fibers (12 mm in length), which were transplanted into the transected sciatic nerve in adult Wistar rats. Six months after cell transplantation, electrophysiological evaluation and walking track analysis were performed. Results of these studies showed significant improvement in motor nerve conduction velocity and sciatic nerve functional index in the BMSC-DSC-transplanted group compared with the control group (Matrigel graft only). Immunohistochemical study data demonstrated that transplanted BMSCs labeled with retrovirus green fluorescent protein were positive for P0 and myelin-associated glycoprotein and had reconstructed nodes of Ranvier and remyelinated regenerated nerve axons. The number of regenerated axons in the axial section of the central portion of the graft was significantly greater in the transplanted group. Although BMSCs can differentiate into several types of cells, tumor formation did not occur 6 months after engraftment. CONCLUSIONS: Results in this study indicate that BMSC-DSCs have great potential to promote regeneration of peripheral nerves. The artificial graft made with BMSC-DSCs represents an alternative method for the difficult reconstruction of a long distance gap in a peripheral nerve.