RESUMEN
<p><b>OBJECTIVE</b>To investigate whether the suppression of Wnt10b by siRNA could prevent the development of hair follicle in the cultured rat embryonic skin.</p><p><b>METHODS</b>siRNA-Wnt10b was synthesized by chemosynthesis method. The dorsal skin of SD rat at embryos were cultured in DMEM in the presence of different percentage of interfering RNA targeting Wnt10b. Wnt10b/beta-catenin expression was analyzed by real-time PCR everyday and by Western blot on the third day. The cultured embryonic skin underwent paraffin embedding, section, HE staining on the third day,in which the number of de novo hair follicle was calculated and statistically analyzed.</p><p><b>RESULTS</b>Wnt10b gene in the cultured embryonic skin could be knocked down with the siRNA-based method. Beta-catenin mRNA was not greatly influenced by the downregulation of Wnt10b mRNA. The number of de novo hair follicle placode in cultured embryonic skin decreased, along with the downregulation of Wnt10b and beta-catenin proteins expression.</p><p><b>CONCLUSIONS</b>The downregulation of Wnt10b mRNA and protein by siRNA reduces the number of de novo hair follicle placode in the cultured rat embryonic skin. Wnt10b may control cytoplasm beta-catenin concentration at the protein level.</p>
Asunto(s)
Animales , Ratas , Folículo Piloso , Embriología , Metabolismo , ARN Mensajero , Genética , ARN Interferente Pequeño , Genética , Piel , Embriología , Metabolismo , Técnicas de Cultivo de Tejidos , Proteínas Wnt , Genética , Metabolismo , beta Catenina , MetabolismoRESUMEN
The mammalian brain exhibits diverse types of neural plasticity, including activity-dependent neurogenesis in the adult hippocampus. How transient activation of mature neurons leads to long-lasting modulation of adult neurogenesis is unknown. Here we identify Gadd45b as a neural activity-induced immediate early gene in mature hippocampal neurons. Mice with Gadd45b deletion exhibit specific deficits in neural activity-induced proliferation of neural progenitors and dendritic growth of newborn neurons in the adult hippocampus. Mechanistically, Gadd45b is required for activity-induced DNA demethylation of specific promoters and expression of corresponding genes critical for adult neurogenesis, including brain-derived neurotrophic factor and fibroblast growth factor. Thus, Gadd45b links neuronal circuit activity to epigenetic DNA modification and expression of secreted factors in mature neurons for extrinsic modulation of neurogenesis in the adult brain.
Asunto(s)
Antígenos de Diferenciación/genética , Antígenos de Diferenciación/metabolismo , Metilación de ADN , Epigénesis Genética , Hipocampo/fisiología , Neurogénesis , Neuronas/fisiología , Animales , Factor Neurotrófico Derivado del Encéfalo/genética , Proliferación Celular , Células Cultivadas , ADN/metabolismo , Dendritas/fisiología , Dendritas/ultraestructura , Giro Dentado/citología , Giro Dentado/fisiología , Electrochoque , Factor 1 de Crecimiento de Fibroblastos/genética , Perfilación de la Expresión Génica , Genes Inmediatos-Precoces , Hipocampo/citología , Ratones , Ratones Noqueados , Esfuerzo Físico , Células Madre/citología , Células Madre/fisiología , Activación TranscripcionalRESUMEN
<p><b>OBJECTIVE</b>To induce hair follicle regeneration in rat ear by microencapsulated dermal papillae (DP) cells.</p><p><b>METHODS</b>Intact dermal papillae were obtained from human scalp follicles which were digested with collagenase I. The human hair DP cells were encapsulated with alginate-polylysine-alginate (APA) by a high-voltage electric field droplet generator. The diameters of the DP cell microcapsules were optimized by regulating the voltage, the distance between the needle head and the solution surface and the injection speed. Then DP cell microencapsulations were xenotransplanted into ears of 20 SD rats with a novel method. One rat was killed every week at the postoperative 2-12 weeks and the implantation sites were biopsied for histological observation.</p><p><b>RESULTS</b>The DP cell microencapsulations were found in a group of round, smooth and transparent microcapsules under a phase-contrast microscope. The optimal combination of parameters to obtain 0.4 mm DP cell microcapsules was voltage 7.0 kV, injection speed 55 mm/h, and distance 10 mm. After 4-12 weeks, 18 of 20 DP cell microcapsule implantations had produced high-density hair. Histological observation indicated that both large follicles and sebaceous gland structures were formed in the rat ear within 3-12 weeks.</p><p><b>CONCLUSIONS</b>These findings show that the DP cell microencapsulation maintain the capacity for initiating the follicle regeneration and can be considered as a substitute for fresh isolated dermal papillae.</p>
Asunto(s)
Animales , Femenino , Humanos , Masculino , Ratas , Dermis , Biología Celular , Fisiología , Oído , Folículo Piloso , Fisiología , Modelos Animales , Ratas Sprague-Dawley , Regeneración , FisiologíaRESUMEN
The aim of this study was to investigate reactive changes of astrocytes and Müller glial cells in rats subjected to kainate treatment, which leads to neuronal degeneration in the ganglion cell layer and the inner border of the inner nuclear layer as confirmed by labelling with Fluoro-Jade B, a marker for degenerating neurons and fibres. Both the astrocytes and the Müller glial cells reacted vigorously to kainate injection as shown by their up-regulated expression of nestin, glial fibrillary acidic protein and glutamine synthetase. A major finding was the induced expression of nestin together with glial fibrillary acidic protein beginning at 1 day post-injection of kainate. The marked nestin expression appeared to be most intense at 1 day and was sustained till 2 weeks as compared with the untreated/normal retina. Western blotting analysis confirmed a marked increase in expression of nestin, glial fibrillary acidic protein and glutamine synthetase as compared with untreated/normal retina. Double labelling study revealed that astrocytes and Müller glial cells expressed the radial glia marker nestin, and incorporated bromodeoxyuridine to re-enter into their cell cycle. The induced expression of these proteins in astrocytes and Müller glial cells indicated an induction of gliotic responses and de-differentiation that may be associated with regenerative efforts after kainate-induced injury. Indeed, with the acquisition of an immature molecular profile as manifested by the induced expression of brain lipid-binding protein and doublecortin in astrocytes and Müller glial cells, the potential of these cells to de-differentiate in retinal neurodegeneration is greatly amplified.
Asunto(s)
Astrocitos/química , Neuroglía/química , Retina/metabolismo , Degeneración Retiniana/metabolismo , Animales , Biomarcadores/análisis , Western Blotting/métodos , Diferenciación Celular , Colorantes , Proteínas de Dominio Doblecortina , Proteína Doblecortina , Proteína de Unión a los Ácidos Grasos 7 , Proteínas de Unión a Ácidos Grasos/análisis , Fluoresceínas , Proteína Ácida Fibrilar de la Glía/análisis , Glutamato-Amoníaco Ligasa/análisis , Inmunohistoquímica , Proteínas de Filamentos Intermediarios/análisis , Ácido Kaínico , Masculino , Proteínas Asociadas a Microtúbulos/análisis , Modelos Animales , Proteínas del Tejido Nervioso/análisis , Nestina , Neuropéptidos/análisis , Compuestos Orgánicos , Ratas , Ratas Wistar , Regeneración , Retina/química , Retina/efectos de los fármacosRESUMEN
The present study was aimed to elucidate how retinal microglia/macrophages would respond to neuronal death after intravitreal kainate injection. An increased expression of the complement receptor type 3 (CR3) and an induction of the major histocompatibility complex (MHC) class II and ED-1 antigens were mainly observed in the inner retina after kainate injection. Prominent cell death revealed by Fluoro Jade B (FJB) staining and ultrastructural examination appeared at the inner border of the inner nuclear layer (INL) at 1 day post-injection. Interestingly, some immunoreactive cells appeared at the outer segment of photoreceptor layer (OSPRL) at different time intervals. Our quantitative analysis further showed that CR3 immunoreactivity was drastically increased peaking at 7 days but subsided thereafter. MHC class II and ED-1 immunoreactivities showed a moderate but steady increase peaking at 3 days and declined thereafter. Double labeling study further revealed that retinal microglia/macrophages expressed concurrently CR3 and ED-1 antigens (OX-42+/ED-1+) or MHC class II molecules (OX-42+/OX-6+) and remained branched in shape at early stage of kainate challenge. By electron microscopy, microglia/macrophages with CR3 immunoreactivity displayed abundant cytoplasm containing a few vesicles and phagosomes. Other cells ultrastructurally similar to Müller cells or astrocytes could also engulf exogenous substances. In conclusion, retinal microglia/macrophages responded vigorously to kainate-induced neuronal cell death that may also trigger the recruitment of macrophages from neighboring tissues and induce the phagocytotic activity of cells other than retinal microglia/macrophages.
Asunto(s)
Macrófagos/metabolismo , Microglía/metabolismo , Retina/inmunología , Animales , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Agonistas de Aminoácidos Excitadores/toxicidad , Antígenos de Histocompatibilidad Clase II/efectos de los fármacos , Antígenos de Histocompatibilidad Clase II/metabolismo , Inmunohistoquímica , Ácido Kaínico/toxicidad , Activación de Macrófagos/inmunología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Masculino , Microglía/efectos de los fármacos , Microglía/inmunología , Microscopía Inmunoelectrónica , Neuronas/efectos de los fármacos , Neuronas/patología , Ratas , Ratas Wistar , Receptores de Complemento/efectos de los fármacos , Receptores de Complemento/metabolismo , Retina/citología , Retina/lesionesRESUMEN
<p><b>OBJECTIVE</b>To induce the hair follicle regeneration in mice ear by microencapsulated dermal papillae cells (DPs) and to investigate the permeability of fluorescein in APA microencapsulation to search the ideal diameter of microencapsulation.</p><p><b>METHODS</b>The DPs were encapsulated with alginate-polylysine-alginate by a high-voltage electric field droplet generator. The microencapsulated dermal papilla cells were xenotransplanted into the mice ears. After 6 week, the histological examination was made by microscopy. The diffusion way and speed of fluorescein into the microencapsulations were observed by confocal laser scanning microscopy. The comparison of fluorescein intensity was made in APA microencapsulations with different diameters.</p><p><b>RESULTS</b>Fully developed hair follicles could be easily identified in the skin of implanted site following xenotransplantation of microencapsulation DPs, which were different from the control groups in configuration, number, size and differentiation degree. The fluorescein was diffused gradually into the microencapsulations with a shape of concentric circularity. The fluorescein intensity inside three groups of APA microencapsulations was: small > middle > big.</p><p><b>CONCLUSIONS</b>The microencapsulated DPs retain the physiological function to induce the follicle regeneration. The APA microencapsulations with 400um diameter could ensure the nutrition and metabolite to pass in and out freely, and isolate the immunocompetent substance absolutely.</p>
Asunto(s)
Animales , Femenino , Humanos , Masculino , Ratones , Alginatos , Química , Diferenciación Celular , Trasplante de Células , Células Cultivadas , Oído , Fluoresceína , Química , Folículo Piloso , Biología Celular , Fisiología , Ratones Endogámicos BALB C , Polilisina , Química , Cuero Cabelludo , Biología Celular , TrasplanteRESUMEN
An increase in incidence and severity of gram-positive infections has emerged in the past decade. In this regard, attention has been focused recently on immune responses of microglial cells in the central nervous system to gram-positive bacteria. The underlying immunological and cellular events in microglial activation induced by specific bacterial toxin of gram-positive bacteria, however, have not yet been clarified fully. This study reports that a simple cell wall product, lipoteichoic acid (LTA), derived from gram-positive bacteria (Staphylococcus aureus) could trigger microglial activation in vitro. Microglia challenged with LTA showed intense ruffling of plasma membrane in the form of lamellipodia or rounded up forming cell aggregates. MTT assay and Western blot analysis with anti-proliferating cell nuclear antigen antibody showed a significant microglial proliferation that may be induced at the later phases of LTA treatment with low doses but at the early period with a high dose. Concentrated LTA also caused apoptotic death of cultured microglia showing fragmented nuclei and increased expression of annexin V or caspase 3. In response to LTA, isolated microglia increased the expression of inducible nitric oxide synthase and major histocompatibility complex class II antigen. Microglial LTA receptors such as CD14 molecule, complement receptor type 3, and macrophage scavenger receptor were upregulated concurrently. In conclusion, staphylococcal LTA can exert an immunomodulatory effect on microglial morphology, cell cycle, and immunomolecules, including its receptors.
Asunto(s)
Bacterias Grampositivas/química , Lipopolisacáridos/farmacología , Microglía/efectos de los fármacos , Ácidos Teicoicos/farmacología , Animales , Animales Recién Nacidos , Anexina A5/metabolismo , Anticuerpos/farmacología , Apoptosis/efectos de los fármacos , Western Blotting/métodos , Encéfalo/citología , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Medio de Cultivo Libre de Suero/farmacología , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Técnica del Anticuerpo Fluorescente/métodos , Regulación de la Expresión Génica/efectos de los fármacos , Antígenos de Histocompatibilidad Clase II/metabolismo , Antígeno Ki-67/metabolismo , Receptores de Lipopolisacáridos/inmunología , Antígeno de Macrófago-1/inmunología , Microglía/metabolismo , Microglía/patología , Microglía/ultraestructura , Microscopía Electrónica de Rastreo/métodos , Óxido Nítrico Sintasa de Tipo II/metabolismo , Proteínas Nucleares/inmunología , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteínas de Unión al ARN , Ratas , Ratas Wistar , Sales de Tetrazolio , Tiazoles , Factores de TiempoRESUMEN
Lipopolysaccharide (LPS), the major proinflammatory component of gram-negative bacteria, is well known to induce sepsis and microglial activation in the CNS. On the contrary, the effect of products from gram-positive bacteria especially in areas devoid of blood-brain barrier remains to be explored. In the present study, a panel of antibodies, namely, OX-6, OX-42 and ED-1 was used to study the response of microglia/macrophages in the pineal gland of rats given an intravenous LPS or lipoteichoic acid (LTA). These antibodies recognize MHC class II antigens, complement type 3 receptors and unknown lysosomal proteins in macrophages, respectively. In rats given LPS (50 microg/kg) injection and killed 48 h later, the cell density and immunoexpression of OX-6, OX-42 and ED-1 in pineal microglia/macrophages were markedly increased. In rats receiving a high dose (20 mg/kg) of LTA, OX-42 and OX-6, immunoreactivities in pineal microglia/macrophages were also enhanced, but that of ED-1 was not. In addition, both bacterial toxins induced an increase in astrocytic profiles labelled by glial fibrillary acid protein. An interesting feature following LPS or LTA treatment was the lowering effect on serum melatonin, enhanced serotonin immunolabelling and cellular vacuolation as studied by electron microscopy in pinealocytes. The LPS- or LTA-induced vacuoles appeared to originate from the granular endoplasmic reticulum as well as the Golgi saccules. The present results suggest that LPS and LTA could induce immune responses of microglia/macrophages and astroglial activation in the pineal gland. Furthermore, the metabolic and secretory activity of pinealocytes was modified by products from both gram-positive and -negative bacteria.
Asunto(s)
Pared Celular , Bacterias Gramnegativas/química , Bacterias Grampositivas/química , Neuroglía/microbiología , Glándula Pineal/microbiología , Animales , Ensayo de Inmunoadsorción Enzimática , Masculino , Melatonina/sangre , Microscopía Electrónica , Neuroglía/citología , Glándula Pineal/citología , Ratas , Ratas WistarRESUMEN
UNLABELLED: Kikuchi-Fujimoto disease (KFD) is a histiocytic necrotising lymphadenitis, which is a benign disease of unknown aetiology. Misdiagnosing KFD as lymphoma or systemic lupus erythematosus is not uncommon due to the similarity of clinical and histopathological features of these diseases. A 12-year-old female suffered from cervical lymphadenopathy, leukocytopenia, fever and especially skin rash. The biopsy of the lymph node was compatible with KFD. The skin biopsy showed interface alterations with vacuolar degeneration of the basal cells suggesting lupus erythematosus; however, the patient did not fulfill the diagnostic criteria for systemic lupus erythematosus. After treatment with acetaminophen, fever subsided and the skin rashes disappeared without relapse during a 10-month follow-up period. CONCLUSION: The histopathological findings of cutaneous lesions in KFD are usually similar to those observed in the involved lymph nodes. This report suggests that interface change might be one of the pathological features of the cutaneous manifestations of KFD.
Asunto(s)
Linfadenitis Necrotizante Histiocítica/patología , Piel/patología , Biopsia , Niño , Femenino , Linfadenitis Necrotizante Histiocítica/diagnóstico , Humanos , Ganglios Linfáticos/patologíaRESUMEN
Using specific macrophage antibodies (OX-42, OX-6, ED-1 and ED-2), this study examined the distribution of macrophages/microglia in the pineal gland of adult rats. Except for ED-2, all antibodies labeled distinct subpopulations of macrophages/microglia in the gland; ED-2 labeling was hardly detectable. The quantitative study showed that the pineal macrophages/microglia (PMM) expressing complement type 3 receptors (OX-42) were more numerous than those expressing the major histocompatibility complex class II antigen (OX-6) or unknown cytoplasmic/lysosomal antigens (ED-1). The PMM were ubiquitous, especially the OX-42 labeled cells which were distributed from the dorsal to the ventral aspect of the gland. The macrophages/microglia labeled with OX-6 or ED-1 were localized mainly in the intermediate portion of the pineal gland. Immunolabeled cells were sparsely distributed in the distal portion of the pineal gland. A notable feature was that the OX-6 labeled macrophages/microglia showed a proximal-distal gradient in cell density. Another interesting feature was the occurrence of prominent cell aggregations around the larger blood vessels. These cells were mostly round and exhibited different immunoreactivity. Confocal microscopic study with triple immunolabeling further revealed that individual PMM cell possessed two or more different antigens (ED-1+/OX-6+, OX-42+/OX-6+ or OX-42+/ED-1+). Remarkably, a large population co-expressed ED-1+/OX-6+/OX-42+. The present results show that the expression of immunoreactive molecules in PMM varies in topographical distribution of the cells. It is suggested that this may be linked to their immunoregulatory functions in the gland.