RESUMEN
BACKGROUND: The collective cell migration of stratified epithelial cells is considered to be an important phenomenon in wound healing, development, and cancer invasion; however, little is known about the mechanisms involved. Furthermore, whereas Rho family proteins, including RhoA, play important roles in cell migration, the exact role of Rho-associated coiled coil-containing protein kinases (ROCKs) in cell migration is controversial and might be cell-type dependent. Here, we report the development of a novel modified scratch assay that was used to observe the collective cell migration of stratified TE-10 cells derived from a human esophageal cancer specimen. RESULTS: Desmosomes were found between the TE-10 cells and microvilli of the surface of the cell sheet. The leading edge of cells in the cell sheet formed a simple layer and moved forward regularly; these rows were followed by the stratified epithelium. ROCK inhibitors and ROCK small interfering RNAs (siRNAs) disturbed not only the collective migration of the leading edge of this cell sheet, but also the stratified layer in the rear. In contrast, RhoA siRNA treatment resulted in more rapid migration of the leading rows and disturbed movement of the stratified portion. CONCLUSIONS: The data presented in this study suggest that ROCKs play an important role in mediating the collective migration of TE-10 cell sheets. In addition, differences between the effects of siRNAs targeting either RhoA or ROCKs suggested that distinct mechanisms regulate the collective cell migration in the simple epithelium of the wound edge versus the stratified layer of the epithelium.
Asunto(s)
Movimiento Celular/fisiología , ARN Interferente Pequeño/farmacología , Quinasas Asociadas a rho/fisiología , Línea Celular Tumoral , Neoplasias Esofágicas , Humanos , MicroARNs/fisiología , Quinasas Asociadas a rho/antagonistas & inhibidoresRESUMEN
BACKGROUND: The collective cell migration of stratified epithelial cells is considered to be an important phenomenon in wound healing, development, and cancer invasion; however, little is known about the mechanisms involved. Furthermore, whereas Rho family proteins, including RhoA, play important roles in cell migration, the exact role of Rho-associated coiled coil-containing protein kinases (ROCKs) in cell migration is controversial and might be cell-type dependent. Here, we report the development of a novel modified scratch assay that was used to observe the collective cell migration of stratified TE-10 cells derived from a human esophageal cancer specimen. RESULTS: Desmosomes were found between the TE-10 cells and microvilli of the surface of the cell sheet. The leading edge of cells in the cell sheet formed a simple layer and moved forward regularly; these rows were followed by the stratified epithelium. ROCK inhibitors and ROCK small interfering RNAs (siRNAs) disturbed not only the collective migration of the leading edge of this cell sheet, but also the stratified layer in the rear. In contrast, RhoA siRNA treatment resulted in more rapid migration of the leading rows and disturbed movement of the stratified portion. CONCLUSIONS: The data presented in this study suggest that ROCKs play an important role in mediating the collective migration of TE-10 cell sheets. In addition, differences between the effects of siRNAs targeting either RhoA or ROCKs suggested that distinct mechanisms regulate the collective cell migration in the simple epithelium of the wound edge versus the stratified layer of the epithelium.
Asunto(s)
Humanos , Movimiento Celular/fisiología , ARN Interferente Pequeño/farmacología , Quinasas Asociadas a rho/fisiología , Neoplasias Esofágicas , MicroARNs/fisiología , Línea Celular Tumoral , Quinasas Asociadas a rho/antagonistas & inhibidoresRESUMEN
Nanoparticles are currently attracting considerable attention because of their ability to conjugate to various substances. As such, these nanoparticles can assist the transfer of the conjugated substance to target tissues where they are gradually released. In this study, vancomycin-conjugated nanoparticles (VCM NPs) were prepared. The antibacterial activity of VCM NPs was compared with that of VCM alone by exposure to vancomycin-resistant enterococci (VRE). The morphology of the cells was then analyzed by electron microscopy. VCM NPs were found to have more potent antibacterial activity against VRE compared to VCM alone, but the activity against vancomycin-sensitive enterococci (VSE) remained the same. The antibacterial activity of nanoparticles alone was the same against VSE and VRE. The nanoparticles were found to induce characteristic morphological changes in the bacteria based on scanning and transmission electron microscopy. The results suggest that the strong antibacterial activity of VCM NPs against VRE may be attributable not only to the well-known control release carrier property of the nanoparticles but to an additional mechanism that involves VCM NPs avoiding the drug resistant mechanism of VRE.
Asunto(s)
Antibacterianos/administración & dosificación , Bacteriólisis/efectos de los fármacos , Portadores de Fármacos/química , Enbucrilato/química , Enterococcus faecium/efectos de los fármacos , Nanopartículas/química , Vancomicina/administración & dosificación , Antibacterianos/química , Antibacterianos/farmacología , Composición de Medicamentos , Farmacorresistencia Bacteriana , Enterococcus faecium/crecimiento & desarrollo , Pruebas de Sensibilidad Microbiana , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Tamaño de la Partícula , Propiedades de Superficie , Vancomicina/química , Vancomicina/farmacologíaRESUMEN
We have shown that SU6656, a potent Src family kinase inhibitor, has the ability to induce multinucleation at a high frequency in diverse cells: rat skin fibroblasts, bone marrow adherent cells, 5F9A mesenchymal stem cell-like clones, 2C5 tracheal epithelial cells and MDCK epithelial cells from dog kidney. To gain insight into the mechanism of multinucleation, we observed the process by time-lapse and confocal microscopy. These multinuclei generally seem to exist independently in one cell without any connections with each other. By time-lapse microscopy, multinucleated cells were found to be formed through the mechanism of plasmodium: karyokinesis without cytokinesis. The observation of EGFP-actin transfected cells by time-lapse confocal laser scanning microscopy suggested that plasmodium occurred with deficient contractile ring formation. Although we examined the differentiation of these cells, the multinucleated cells could not be categorized into any type of cell in vivo known to exhibit multinuclei.
Asunto(s)
Citoesqueleto de Actina/ultraestructura , Núcleo Celular/diagnóstico por imagen , Indoles/farmacología , Poliploidía , Inhibidores de Proteínas Quinasas/farmacología , Sulfonamidas/farmacología , Familia-src Quinasas/antagonistas & inhibidores , Animales , Núcleo Celular/metabolismo , Células Cultivadas , Perros , Ratas , Ultrasonografía , Familia-src Quinasas/metabolismoRESUMEN
The evolutionarily conserved polarity proteins PAR-3, atypical protein kinase C (aPKC) and PAR-6 critically regulate the apical membrane development required for epithelial organ development. However, the molecular mechanisms underlying their roles remain to be clarified. We demonstrate that PAR-3 knockdown in MDCK cells retards apical protein delivery to the plasma membrane, and eventually leads to mislocalized apical domain formation at intercellular regions in both two-dimensional and three-dimensional culture systems. The defects in PAR-3 knockdown cells are efficiently rescued by wild-type PAR-3, but not by a point mutant (S827/829A) that lacks the ability to interact with aPKC, indicating that formation of the PAR-3-aPKC-PAR-6 complex is essential for apical membrane development. This is in sharp contrast with tight junction maturation, which does not necessarily depend on the aPKC-PAR-3 interaction, and indicates that the two fundamental processes essential for epithelial polarity are differentially regulated by these polarity proteins. Importantly, highly depolarized cells accumulate aPKC and PAR-6, but not PAR-3, on apical protein-containing vacuoles, which become targeted to PAR-3-positive primordial cell-cell contact sites during the initial stage of the repolarization process. Therefore, formation of the PAR-3-aPKC-PAR-6 complex might be required for targeting of not only the aPKC-PAR-6 complex but also of apical protein carrier vesicles to primordial junction structures.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Moléculas de Adhesión Celular/metabolismo , Comunicación Celular , Polaridad Celular , Células Epiteliales/enzimología , Isoenzimas/metabolismo , Proteína Quinasa C/metabolismo , Vacuolas/enzimología , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Moléculas de Adhesión Celular/genética , Proteínas de Ciclo Celular , Línea Celular , Membrana Celular/enzimología , Colágeno/metabolismo , Quistes/enzimología , Perros , Geles , Humanos , Isoenzimas/genética , Ratones , Mutación , Proteína Quinasa C/genética , Transporte de Proteínas , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Uniones Estrechas/enzimología , Factores de Tiempo , Transfección , beta Carioferinas/metabolismoRESUMEN
We examined changes in the ultrastructure and localization of major extracellular matrix components, including 5 types of collagen (type I, III, IV, VI, and VIII), laminin, fibronectin, and heparan sulfate proteoglycan in Descemet's membrane of the cornea of diabetic GK rats. In the cornea of diabetic GK rats, more long-spacing collagen fibrils were observed in Descemet's membrane than in the membrane of the nondiabetic Wistar rats. Both GK and Wistar rats showed an age-dependent increase in the density of the long-spacing collagen. Immunoelectron microscopy showed that type VIII collagen was localized in the internodal region of the long-spacing collagen, which was not labelled by any of the other antibodies used. The antidiabetic agents nateglinide and glibenclamide significantly suppressed the formation of the long-spacing collagen in the diabetic rats. Long-spacing collagen would thus be a useful indicator for studying diabetic changes in the cornea and the effect of antidiabetic agents.
Asunto(s)
Colágeno/metabolismo , Ciclohexanos/uso terapéutico , Lámina Limitante Posterior/metabolismo , Diabetes Mellitus Experimental/metabolismo , Gliburida/uso terapéutico , Fenilalanina/análogos & derivados , Animales , Colágeno/efectos de los fármacos , Córnea/efectos de los fármacos , Córnea/patología , Córnea/ultraestructura , Lámina Limitante Posterior/efectos de los fármacos , Lámina Limitante Posterior/ultraestructura , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Experimental/patología , Retinopatía Diabética/patología , Retinopatía Diabética/prevención & control , Hipoglucemiantes/uso terapéutico , Inmunohistoquímica , Masculino , Microscopía Inmunoelectrónica , Nateglinida , Fenilalanina/uso terapéutico , Ratas , Ratas Endogámicas , Ratas Wistar , Valores de ReferenciaRESUMEN
The tracheal epithelium can be induced to move as a cellular sheet by heterotopic transplantation, which offers the opportunity to observe migrating cells as a group in an in vivo environment. We therefor investigated the ultrastructural characteristics of migrating tracheal epithelial cells with special reference to the moving front using this transplantation. The migrating epithelial cells underwent squamous metaplasia and lost their differentiated characteristics such as cilia or secretory granules. Several unique observations were made concerning the mechanism of mobility: one is that epithelial cells in the front were elongated in a direction perpendicular to the course of movement, different from previous reports in vitro. The second is that lamellipodia, which are regarded as the major locomotive machinery in the adult wound epithelium, did not make up the major part of the front; the major portion of the anterior fringe of the moving front was usually smooth and gently curved, and actin cables parallel to the elongated cells were observed by confocal laser microscopy, indicating that the purse-string mechanism of epithelial wound healing takes place. The third finding is that the cells in the front had irregular bleb-like structures on their antero-basal surface, which were formed even in the portion where the cells did not attach to the matrix. Few organelles were recognized in these structures. From their location, one might propose that these bleb-like structures play a role in the recognition of the substrate and thus the movement of the cell sheet.
Asunto(s)
Movimiento Celular , Células Epiteliales/ultraestructura , Tráquea/ultraestructura , Animales , Masculino , Microscopía Electrónica de Rastreo , RatasRESUMEN
We established a mesenchymal stem cell clone, 5F9A, from rat bone marrow substrate adherent cells by repeated limiting dilutions. The cells have a fibroblastic shape and form intimate contacts with adjacent cells with interdigitations and junctions similar to adherence and tight junctions in a semi-confluent culture. Analysis of the phenotypes of these cells by RT-PCR and FACS demonstrated that they resembled mesenchymal stem cells, and the cells could differentiate into adiopocytes and osteoblasts under appropriate conditions in vitro showing their oligopotency. Furthermore, the cells were induced to become multinuclear cells by TPA (12-o-tetradecanoylphorbol 13-acetate) stimulation.
Asunto(s)
Adipocitos/ultraestructura , Diferenciación Celular/fisiología , Núcleo Celular/ultraestructura , Células Madre Mesenquimatosas/fisiología , Osteoblastos/ultraestructura , Animales , Línea Celular , Uniones Intercelulares/ultraestructura , Células Madre Mesenquimatosas/ultraestructura , Fenotipo , Ésteres del Forbol , RatasRESUMEN
The 5F9A cell, which is a mesenchymal stem cell-like clone established from rat bone marrow substrate adherent cells, can differentiate into adipocytes and osteoblasts in vitro under the appropriate conditions. Multinucleated cells could be also induced by 12-O-tetradecanoylphorbol 13-acetate (TPA) in 5F9A cells. This effect was mediated by protein kinase C. Possible mechanisms of multinucleation by TPA were hypothesized to be either karyokinesis without cytokinesis or cell-cell fusion. By observation using time-lapse phase-contrast microscopy, we determined that the multinucleated cells were generated mainly by karyokinesis without cytokinesis. Cell fusion was studied using time-lapse photography, and confocal laser scanning microscopy using two differentially labeled cells. These techniques demonstrated that multinucleated 5F9A cells could be produced by cell fusion, albeit at a low frequency. We conclude that multinucleated 5F9A cells are formed primarily by karyokinesis without cytokinesis, although some cells are also formed by cell-cell fusion.
Asunto(s)
División del Núcleo Celular/efectos de los fármacos , Citocinesis/efectos de los fármacos , Células Gigantes/citología , Células Gigantes/efectos de los fármacos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacología , Animales , Adhesión Celular/efectos de los fármacos , Fusión Celular , Células Clonales , ADN/análisis , Genoma , Citometría de Barrido por Láser , Proteína Quinasa C/metabolismo , RatasRESUMEN
OBJECTIVE: The urinary bladder has considerable regenerative ability and may enable reconstitution of the urinary bladder if used appropriately. MATERIAL AND METHODS: Rat urinary bladders were allotransplanted onto the inner surface of the abdominal wall with the urothelium facing the mesothelial cells. The ureters, urethra and blood supply were left intact. RESULTS: A week after the operation, the mesothelium of the abdominal wall was replaced by the urothelium from the donor urinary bladder and a cyst was formed, the inner surfaces of which were completely covered with the urothelium. After a few months, the submucosal tissue and muscular layer had also moved to cover the wall, forming an almost complete urinary bladder. The mucosal membrane formed complex folds, which was probably due to overgrowth of the epithelial cells and the submucosal connective tissues. The area derived from the abdominal wall showed only minimal shrinkage, whereas the abdominal wall from which the mesothelium had been removed showed significant shrinkage. CONCLUSION: This method is potentially useful for the reconstruction of urinary bladders.
Asunto(s)
Pared Abdominal/cirugía , Epitelio/trasplante , Procedimientos de Cirugía Plástica , Vejiga Urinaria/cirugía , Procedimientos Quirúrgicos Urológicos/métodos , Animales , Estudios de Seguimiento , Ratas , Ratas Wistar , Vejiga Urinaria/citología , Cicatrización de HeridasRESUMEN
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.
Asunto(s)
Trasplante de Médula Ósea , Regeneración Nerviosa/fisiología , Células de Schwann/trasplante , Nervio Ciático/fisiopatología , Animales , Diferenciación Celular , Estudios de Seguimiento , Masculino , Conducción Nerviosa/fisiología , Ratas , Ratas Wistar , Nervio Ciático/lesiones , Células del Estroma/citología , Factores de Tiempo , Caminata/fisiologíaRESUMEN
Bone marrow stromal cells (MSCs) have the capability under specific conditions of differentiating into various cell types such as osteocytes, chondrocytes, and adipocytes. Here we demonstrate a highly efficient and specific induction of cells with neuronal characteristics, without glial differentiation, from both rat and human MSCs using gene transfection with Notch intracellular domain (NICD) and subsequent treatment with bFGF, forskolin, and ciliary neurotrophic factor. MSCs expressed markers related to neural stem cells after transfection with NICD, and subsequent trophic factor administration induced neuronal cells. Some of them showed voltage-gated fast sodium and delayed rectifier potassium currents and action potentials compatible with characteristics of functional neurons. Further treatment of the induced neuronal cells with glial cell line-derived neurotrophic factor (GDNF) increased the proportion of tyrosine hydroxylase-positive and dopamine-producing cells. Transplantation of these GDNF-treated cells showed improvement in apomorphine-induced rotational behavior and adjusting step and paw-reaching tests following intrastriatal implantation in a 6-hydroxy dopamine rat model of Parkinson disease. This study shows that a population of neuronal cells can be specifically generated from MSCs and that induced cells may allow for a neuroreconstructive approach.
Asunto(s)
Células de la Médula Ósea/metabolismo , Trasplante de Células , Neuronas/fisiología , Células del Estroma/fisiología , Trasplante Autólogo , Animales , Biomarcadores , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Células Cultivadas , Factor Neurotrófico Ciliar/farmacología , Colforsina/farmacología , Factor 2 de Crecimiento de Fibroblastos/farmacología , Factor Neurotrófico Derivado de la Línea Celular Glial , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Factores de Crecimiento Nervioso/farmacología , Neuronas/citología , Neuronas/efectos de los fármacos , Enfermedad de Parkinson/metabolismo , Fenotipo , Estructura Terciaria de Proteína , Ratas , Ratas Wistar , Receptores Notch , Células del Estroma/citología , Células del Estroma/efectos de los fármacos , Factores de Transcripción/metabolismo , Transfección , Tirosina 3-Monooxigenasa/metabolismo , Corteza Visual/citología , Corteza Visual/metabolismoRESUMEN
Certain hollow organs are known to form cysts when heterologously transplanted. In order to examine the usefulness of the phenomenon for regenerative medicine, rat urinary bladders and other organs were allo-transplanted under the subcutaneous tissue of the back. These transplanted tissues very often formed cysts covered with epithelia. The epithelia covered an area about twice the original size. In the case of the urinary bladder, the epithelium started moving from the edge of the transplants around day 3 after the operation, and as time proceeded, the tela submucosa and tunica muscularis also moved to encircle the epithelium, and formed the wall of the cyst. The basal laminae were formed under the newly expanded epithelium slightly behind the leading tip. All of the organs tested had the capability of cyst formation and epithelialization, although their rate differed between organs. The results are discussed with reference to the potential use of cyst formation for regenerating damaged organs.
Asunto(s)
Trasplante de Órganos/métodos , Regeneración/fisiología , Vejiga Urinaria/fisiología , Vejiga Urinaria/trasplante , Animales , Quistes/fisiopatología , Fenómenos Fisiológicos del Sistema Digestivo , Procedimientos Quirúrgicos del Sistema Digestivo , Epitelio/fisiología , Ratas , Ratas Wistar , Fenómenos Fisiológicos de la Piel , Tejido Subcutáneo , Tráquea/fisiología , Tráquea/trasplanteRESUMEN
Mouse Prrp (mPrrp)/DAZAP1 is a mouse ortholog of Xenopus Prrp, which is involved in vegetal pole localization of Vg1 mRNA in oocytes and is highly expressed in the testis. The mouse protein has been reported to be a shuttling protein which localizes in the nucleus of pre-meiotic spermatogenic cells and round spermatids, and shifts its location into the cytoplasm in elongating spermatids, suggesting that mPrrp may be involved in mRNA transport as well as that of the Xenopus ortholog. We reexamined immunohistochemical analyses of mPrrp/DAZAP1 during spermatogenesis utilizing a newly established monoclonal antibody and reconfirmed it to be a shuttling protein. We also carried out new observations that included remarkable intranuclear movement during spermatogenesis. In addition, we found that a long amino acid stretch which spanned over the C-terminal half of the protein was required for the nuclear import. These observations demonstrated dynamic changes in subnuclear and subcellular localization which might reflect specific functions during spermatogenesis.
Asunto(s)
Núcleo Celular/metabolismo , Prolina/química , Proteínas de Unión al ARN/metabolismo , Espermatogénesis/fisiología , Fracciones Subcelulares/metabolismo , Transporte Activo de Núcleo Celular , Animales , Anticuerpos Monoclonales/metabolismo , Citoplasma/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Masculino , Ratones , Peso Molecular , Proteínas de Unión al ARN/química , Espermátides/metabolismo , Espermatozoides/metabolismo , Testículo/metabolismo , Proteínas de Xenopus/metabolismoRESUMEN
Skins and hollow organs have been shown to form epithelialized cysts when transplanted into subcutaneous tissue of a recipient animal, expanding their surface areas. This system seems to offer a good potential for regenerating organs. We investigated the functional and structural contribution of epithelia and connective tissue compartments in this regeneration system with two experimental systems.
RESUMEN
Whole body gamma-ray irradiation of rats with caesium-137 (137Cs) at embryonic day 20 induced marked reduction of the weight of the testis. Body weight and other tissues, however, seemed to remain normal. By light microscopy, complete loss of germ cells was observed in the testis. Other components, such as Sertoli cells and interstitial cells, seemed to be normal. The testes from day 8 postpartum rats contained very few spermatogonia compared with newborn rats, indicating loss of germ cells between days 0 and 8. In the adult, 137Cs-irradiated testes showed two conspicuous features other than the loss of germ cells: empty vacuolar spaces between Sertoli cells and multilayered seminiferous tubule basal laminae (lamina densa). The junctional structures (ectoplasmic specializations) between Sertoli cells, however, seemed normal. The thickness of each layer of multilayered basal laminae was the same as that of normal rats and electron-lucent layers similar to lamina lucida were interposed between them. Of the empty vacuolar spaces between Sertoli cells, basal laminae bridge the gap. The basal laminae contained laminin, type IV collagen and heparan sulphate proteoglycan evenly distributed among layers, suggesting a normal composition. Rough estimation of the amount of basal laminae deposited in 137Cs-irradiated rats indicates that it is within a range similar to that in normal testis. These features imply that Sertoli cells are, in part, determined perinatally to produce basal laminae for germ-line cells.
Asunto(s)
Testículo/efectos de la radiación , Testículo/ultraestructura , Irradiación Corporal Total , Animales , Animales Recién Nacidos , Membrana Basal/efectos de la radiación , Membrana Basal/ultraestructura , Radioisótopos de Cesio , Femenino , Células Germinativas/efectos de la radiación , Células Germinativas/ultraestructura , Masculino , Microscopía Electrónica , Ratas , Túbulos Seminíferos/efectos de la radiación , Túbulos Seminíferos/ultraestructura , Células de Sertoli/efectos de la radiación , Células de Sertoli/ultraestructura , Testículo/embriologíaRESUMEN
Neuronal progenitor cells (NPCs) may provide dopaminergic neurons for the treatment of Parkinson's disease (PD). However, transplantation of NPCs into the striatum by current methods has had limited success. It is possible to reverse the symptoms of PD in model rats but difficult to reverse them in humans because the number of dopaminergic neurons generated from NPCs is low. We transduced the von Hippel-Lindau (VHL) gene into NPCs isolated from embryonic rat brain. The NPCs with the transduced VHL gene efficiently differentiated into tyrosine hydroxylase-positive neurons in vitro. NPCs with the transduced VHL gene, which were labeled in advance with bromodeoxyuridine, were transplanted into the striatum of a rat model of PD. Numerous bromodeoxyuridine-tyrosine hydroxylase double-labeled cells were seen close to the transplant site, showing that the transplanted cells efficiently generated new dopaminergic neurons within the host striatum. Moreover, all of the animals with NPCs with VHL showed a remarkable decrease in apomorphine-induced rotations. These findings show that NPCs with the VHL gene can efficiently generate dopaminergic neurons and that a sufficient number of dopaminergic neurons can develop from them to reverse the symptoms of PD in humans. VHL gene transduction provides a new therapeutic approach for treatment of PD.
Asunto(s)
Cuerpo Estriado/trasplante , Ligasas/genética , Neuronas/trasplante , Trastornos Parkinsonianos/terapia , Trasplante de Células Madre , Proteínas Supresoras de Tumor , Ubiquitina-Proteína Ligasas , Animales , Trasplante de Tejido Encefálico , Diferenciación Celular/fisiología , Células Cultivadas , Cuerpo Estriado/citología , Cuerpo Estriado/fisiología , Dopamina/metabolismo , Embrión de Mamíferos , Trasplante de Tejido Fetal , Supervivencia de Injerto , Humanos , Inmunohistoquímica , Masculino , Modelos Animales , Actividad Motora/fisiología , Neuronas/citología , Neuronas/fisiología , Oxidopamina , Trastornos Parkinsonianos/inducido químicamente , Ratas , Ratas Sprague-Dawley , Células Madre/citología , Células Madre/metabolismo , Transducción Genética , Proteína Supresora de Tumores del Síndrome de Von Hippel-LindauRESUMEN
Recent studies have demonstrated that GnRH-analogues can stimulate regeneration of spermatogenesis of rats when administered after testicular damages. Although the mechanism of this phenomenon has not been elucidated yet, stem cell factor (SCF) produced by Sertoli cells was proposed to mediate the effects of GnRH-analogues on spermatogonial proliferation and/or survival. In the present study, we quantitatively evaluated the proliferation of spermatogonia and addressed whether SCF mediates the effect of GnRH-analogue on spermatogonial proliferation, using a novel approach combining spermatogonial transplantation and laser confocal microscopic observation. In the first experiment, using wild-type mice as recipients for spermatogonial transplantation, the number of donor spermatogonia per 100 Sertoli cells in each spermatogenic colony was significantly higher in the experimental group of mice treated with leuprorelin, a GnRH-agonist, than that of the control group at 4 and 5 wk after transplantation. In the second experiment, Steel/Steeldickie (Sl/Sld) mutant mice, which lack expression of membrane bound form SCF, were used as recipients. As seen in the first experiment, the number of undifferentiated spermatogonia was significantly higher in leuprorelin-treated than in the control group. Since undifferentiated spermatogonia do not express the receptor of SCF, the present study clearly demonstrates that neither membrane-bound nor secreted forms of SCF are involved in the mechanism of GnRH-analogue's effect on spermatogonial proliferation and/or survival.
Asunto(s)
Hormona Liberadora de Gonadotropina/análogos & derivados , Hormona Liberadora de Gonadotropina/farmacología , Proteínas Proto-Oncogénicas c-kit/fisiología , Transducción de Señal/fisiología , Espermatogonias/efectos de los fármacos , Factor de Células Madre/genética , Factor de Células Madre/fisiología , Animales , Peso Corporal/efectos de los fármacos , Células Germinativas/efectos de los fármacos , Proteínas Fluorescentes Verdes , Inmunohistoquímica , Leuprolida/farmacología , Proteínas Luminiscentes/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , Tamaño de los Órganos/efectos de los fármacos , Túbulos Seminíferos/fisiología , Recuento de Espermatozoides , Espermatogénesis/genética , Espermatogonias/trasplanteRESUMEN
Mammalian male germ cells might be generally thought to have infinite proliferative potential based on their life-long production of huge numbers of sperm. However, there has been little substantial evidence that supports this assumption. In the present study, we performed serial transplantation of spermatogonial stem cells to investigate if they expand by self-renewing division following transplantation. The transgenic mouse carrying the Green fluorescent protein gene was used as the donor cell source that facilitated identification and recollection of colonized donor germ cells in the recipient testes. The established colonies of germ cells in the recipient testes were collected and transplanted to new recipients. This serial transplantation of spermatogonial stem cells repopulated the recipient testes, which were successfully performed sequentially up to four times from one recipient to the next. The incubation periods between two sequential transplantations ranged from 55 to 373 days. During these passages, the spermatogonial stem cells showed constant activity to form spermatogenic colonies in the recipient testis. They continued to increase in number for more than a year following transplantation. Colonization efficiency of spermatogonial stem cells was determined to be 4.25% by using Sl/Sl(d) mice as recipients that propagated only undifferentiated type A spermatogonia in their testes. Based on the colonization efficiency, one colony-forming activity was assessed to equate to about 20 spermatogonial stem cells. The spermatogonial stem cells were estimated to expand over 50-fold in 100 days in this experiment.
Asunto(s)
Espermatogonias/trasplante , Trasplante de Células Madre , Animales , División Celular , Genes Reporteros , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Espermatogénesis , Espermatogonias/citología , Células Madre/citología , Factores de TiempoRESUMEN
During student dissecting practice, a rare developmental anomaly showing persistent sciatic artery (PSA) was found in the bilateral lower limbs of a 74-year-old Japanese male cadaver. The PSA was a continuation of the internal iliac artery on both sides, did not anastomose with the perforating arteries and ended by anastomosing with the popliteal artery on both sides. The course and distribution of the PSA were relatively consistent with previous reports. In the present case, however, the PSA and inferior gluteal arteries existed simultaneously on both sides, despite the general assumption that the inferior gluteal artery is a remnant of sciatic artery regression.