RESUMEN
OBJECTIVE: The mesenchymal stem cells (MSCs) have been widely studied for their anti-tumor property, due to the characteristic of homing towards tumor sites and immunosuppression. Nevertheless, the underlying molecular mechanisms that link MSCs to the targeted tumor cells, such as glioma, are not clear. MATERIALS AND METHODS: Here, we examined the inhibitory properties and new molecular mechanisms of the human umbilical cord (hUC-MSCs) derived exosomes on the human glioma U87 cells using a co-culture system in vitro. The cell counting kit-8 (CCK-8) assay was performed to measure the anti-tumor activity of hUC-MSCs derived exosomes. The cell apoptosis was assessed by flow cytometry and the immunoblotting assay was applied in order to assess the associated proteins level. The data revealed that hUC-MSCs derived exosomes could repress cell proliferation and induce cell apoptosis. RESULTS: Mechanistically, we identified that lncRNA PTENP1 could be packaged into exosome from hUC-MSCs, transferred to U87 cells, and then stabilized PTEN by binding miR-10a-5p competitively. CONCLUSIONS: Therefore, our data suggested that the exosomes from hUC-MSCs possess a higher anti-tumor capacity, at least partially, via regulating miR-10a-5p/PTEN signaling, which thereby may represent a possible target for early diagnosis and treatment of glioma clinically.
Asunto(s)
Neoplasias Encefálicas/genética , Exosomas/genética , Glioma/genética , MicroARNs/genética , Fosfohidrolasa PTEN/genética , ARN Largo no Codificante/genética , Cordón Umbilical/citología , Apoptosis , Neoplasias Encefálicas/metabolismo , Diferenciación Celular , Línea Celular Tumoral , Proliferación Celular , Técnicas de Cocultivo , Progresión de la Enfermedad , Exosomas/metabolismo , Femenino , Glioma/metabolismo , Humanos , Células Madre Mesenquimatosas/química , Células Madre Mesenquimatosas/citología , Embarazo , Transducción de Señal , Cordón Umbilical/químicaRESUMEN
BACKGROUND: Apoptosis plays an important role in renal ischemia/reperfusion (IR) injury. Evidence has shown that erythropoietin (EPO) has an antiapoptotic effect. Therefore, this study aimed to explore the effect and potential mechanism of EPO in renal IR injury. METHODS: Kidney IR injury in rats was established by clamping the left renal artery for 30 minutes followed by 24 hours of reperfusion, along with contralateral nephrectomy. Renal function, renal histology, and expression of EPOR, p-EPOR, ERK, p-ERK, p-p53, p53, Bcl-2, Bcl-xl, Bad, and Bax were examined. RESULTS: Pretreatment with EPO significantly reduced renal dysfunction, pathologic change, and expression of Bad and Bax. Furthermore, EPO treatment enhanced the expression of p-ERK, p-p53, Bcl-2, and Bcl-xl with no influence on the expression of EPOR, ERK, and p53. CONCLUSIONS: These findings demonstrated that EPO pretreatment can attenuate renal IR injury by inhibiting apoptosis by promoting activation of the ERK/p53 signaling.
Asunto(s)
Apoptosis/efectos de los fármacos , Eritropoyetina/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sustancias Protectoras/farmacología , Daño por Reperfusión/fisiopatología , Proteína p53 Supresora de Tumor/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/metabolismo , Isquemia/fisiopatología , Riñón/irrigación sanguínea , Riñón/lesiones , Riñón/fisiopatología , Sistema de Señalización de MAP Quinasas/fisiología , Masculino , Ratas , Ratas Sprague-Dawley , Receptores de Eritropoyetina/metabolismoRESUMEN
The process of thymocyte development culminates in the maturation of helper (CD4+) and cytotoxic (CD8+) T cells from their common precursors, the CD4+CD8+ double-positive cells. A crucial step during lineage specification is the termination of expression of either the CD4 or the CD8 coreceptor. A silencer element within the first intron of the CD4 gene is sufficient for CD4 transcriptional repression in cells of the cytotoxic lineage, as well as in thymocytes at earlier stages of differentiation. Here we show that the function of the CD4 silencer is required only at distinct stages of development. Its deletion before the initiation of lineage specification resulted in CD4 derepression throughout thymocyte differentiation. By contrast, once cells committed to the cytotoxic CD8+ lineage, the CD4 locus remained silent through subsequent mitoses, even when the silencer element was excised. The epigenetic inheritance of the silenced CD4 locus was not affected by the inhibition of DNA methylation or histone deacetylation, and may thus involve other mechanisms that ensure a stable state of gene expression.
Asunto(s)
Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/metabolismo , Linaje de la Célula/genética , Citotoxicidad Inmunológica , Silenciador del Gen , Linfocitos T Citotóxicos/citología , Linfocitos T Citotóxicos/metabolismo , Animales , Linfocitos T CD4-Positivos/inmunología , Diferenciación Celular/genética , Metilación de ADN , Citometría de Flujo , Regulación de la Expresión Génica , Ratones , Ratones Transgénicos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T Citotóxicos/inmunología , Transcripción GenéticaRESUMEN
Antibody-secreting plasma cells are nonrecirculatory and lodge in splenic red pulp, lymph node medullary cords, and bone marrow. The factors that regulate plasma cell localization are poorly defined. Here we demonstrate that, compared with their B cell precursors, plasma cells exhibit increased chemotactic sensitivity to the CXCR4 ligand CXCL12. At the same time, they downregulate CXCR5 and CCR7 and have reduced responsiveness to the B and T zone chemokines CXCL13, CCL19, and CCL21. We demonstrate that CXCL12 is expressed within splenic red pulp and lymph node medullary cords as well as in bone marrow. In chimeric mice reconstituted with CXCR4-deficient fetal liver cells, plasma cells are mislocalized in the spleen, found in elevated numbers in blood, and fail to accumulate normally in the bone marrow. Our findings indicate that as B cells differentiate into plasma cells they undergo a coordinated change in chemokine responsiveness that regulates their movements in secondary lymphoid organs and promotes lodgment within the bone marrow.
Asunto(s)
Quimiocinas CXC/metabolismo , Quimiocinas/metabolismo , Plasma/citología , Plasma/metabolismo , Receptores CXCR4/metabolismo , Animales , Médula Ósea/metabolismo , Movimiento Celular , Quimiocina CCL19 , Quimiocina CCL21 , Quimiocina CXCL12 , Quimiocina CXCL13 , Quimiocinas CC/metabolismo , Quimiocinas CXC/genética , Femenino , Ganglios Linfáticos/fisiología , Masculino , Ratones , Ratones Endogámicos , Ratones Mutantes , Receptores CCR7 , Receptores CXCR4/genética , Receptores CXCR5 , Receptores de Quimiocina/metabolismo , Receptores de Citocinas/metabolismo , Bazo/fisiologíaRESUMEN
Most developing thymocytes undergo apoptosis because they cannot interact productively with molecules encoded by the major histocompatibility complex. Here, we show that mice lacking the orphan nuclear hormone receptor RORgamma lose thymic expression of the anti-apoptotic factor Bcl-xL. RORgamma thus regulates the survival of CD4+8+ thymocytes and may control the temporal window during which thymocytes can undergo positive selection. RORgamma was also required for development of lymph nodes and Peyer's patches, but not splenic follicles. In its absence, there was loss of a population of CD3-CD4+CD45+ cells that normally express RORgamma and that are likely early progenitors of lymphoid organs. Hence, RORgamma has critical functions in T cell repertoire selection and lymphoid organogenesis.
Asunto(s)
Quinasas CDC2-CDC28 , Tejido Linfoide/crecimiento & desarrollo , Receptores Citoplasmáticos y Nucleares/fisiología , Receptores de Ácido Retinoico , Receptores de Hormona Tiroidea , Proteínas Represoras , Subgrupos de Linfocitos T/citología , Timo/citología , Factores de Transcripción , Animales , Apoptosis , Recuento de Células , Ciclo Celular , Supervivencia Celular , Cruzamientos Genéticos , Quinasa 2 Dependiente de la Ciclina , Quinasas Ciclina-Dependientes/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Femenino , Marcación de Gen , Proteína 2 Inhibidora de la Diferenciación , Tejido Linfoide/citología , Tejido Linfoide/embriología , Masculino , Ratones , Ratones Endogámicos C57BL , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Proteína bcl-XAsunto(s)
Proteínas Nucleares , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Ácido Retinoico , Receptores de Hormona Tiroidea , Linfocitos T/citología , Linfocitos T/metabolismo , Animales , Apoptosis/efectos de los fármacos , Ciclo Celular , Supervivencia Celular , Proteínas de Unión al ADN/metabolismo , Dexametasona/farmacología , Humanos , Tejido Linfoide/crecimiento & desarrollo , Ratones , Ratones Noqueados , Modelos Biológicos , Factores de Transcripción NFATC , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Transducción de Señal , Linfocitos T/inmunología , Factores de Transcripción/metabolismo , Transcripción Genética , Proteína bcl-XRESUMEN
Chemokines and their receptors are important in cell migration during inflammation, in the establishment of functional lymphoid microenvironments, and in organogenesis. The chemokine receptor CXCR4 is broadly expressed in cells of both the immune and the central nervous systems and can mediate migration of resting leukocytes and haematopoietic progenitors in response to its ligand, SDF-1. CXCR4 is also a major receptor for strains of human immunodeficiency virus-1 (HIV-1) that arise during progression to immunodeficiency and AIDS dementia. Here we show that mice lacking CXCR4 exhibit haematopoietic and cardiac defects identical to those of SDF-1-deficient mice, indicating that CXCR4 may be the only receptor for SDF-1. Furthermore, fetal cerebellar development in mutant animals is markedly different from that in wild-type animals, with many proliferating granule cells invading the cerebellar anlage. This is, to our knowledge, the first demonstration of the involvement of a G-protein-coupled chemokine receptor in neuronal cell migration and patterning in the central nervous system. These results may be important for designing strategies to block HIV entry into cells and for understanding mechanisms of pathogenesis in AIDS dementia.
Asunto(s)
Antígenos CD , Cerebelo/embriología , Hematopoyesis/fisiología , Receptores CXCR4/fisiología , Animales , Linfocitos B/citología , Quimiocina CXCL12 , Quimiocinas CXC/fisiología , Quimiotaxis , Desarrollo Embrionario y Fetal/fisiología , Muerte Fetal , Defectos de los Tabiques Cardíacos/etiología , Antígenos Comunes de Leucocito/biosíntesis , Leucosialina , Hígado/citología , Hígado/embriología , Ratones , Mutación , Neuronas/fisiología , ARN Mensajero/metabolismo , Receptores CXCR4/deficiencia , Receptores CXCR4/genética , Sialoglicoproteínas/biosíntesis , Transducción de Señal , Linfocitos T/citologíaRESUMEN
BACKGROUND: The bacteriophage-derived Cre-loxP recombination system operates efficiently in mammalian cells. This system is particularly useful in gene-targeting experiments in the mouse, and has already been used to generate 'clean' deletions of target genes in the germ line, as well as to inactivate target genes in a conditional manner (based on regulated expression of the Cre recombinase). In principle, Cre-loxP-mediated recombination should also allow gene replacement, and thus the introduction of virtually any kind of mutation into the genome. RESULTS: We used the Cre-loxP system, in mouse embryonic stem cells, to replace the mouse gene C gamma 1, which encodes the constant region of the heavy chain of IgG1 antibodies, with its human counterpart. The mutation was transmitted through the mouse germ line, and the resulting mutant mice were crossed with mice expressing kappa light chains with a human, instead of a mouse, constant region. Mice homozygous for both mutations produce humanized, kappa-chain-bearing IgG1 antibodies at the same level and efficiency as wild-type mice produce murine IgG1 antibodies. These animals should enable the ex vivo production of humanized, chimeric monoclonal antibodies specific for any antigen to which the mouse can respond. CONCLUSIONS: Cre-loxP-mediated gene replacement is a simple and efficient general method of targeted mutagenesis in the mouse.
Asunto(s)
Marcación de Gen/métodos , Inmunoglobulina G/genética , Integrasas , Proteínas Virales , Animales , Secuencia de Bases , Células Cultivadas , ADN Nucleotidiltransferasas/genética , Cartilla de ADN , Expresión Génica , Humanos , Regiones Constantes de Inmunoglobulina/genética , Regiones Constantes de Inmunoglobulina/metabolismo , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/metabolismo , Cadenas kappa de Inmunoglobulina/genética , Cadenas kappa de Inmunoglobulina/metabolismo , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Células MadreAsunto(s)
Antígenos CD , Linfocitos B/fisiología , Animales , Diferenciación Celular , Regulación de la Expresión Génica , Reordenamiento Génico/genética , Técnicas de Transferencia de Gen , Genes de Inmunoglobulinas/genética , Leucosialina , Ratones , Mutación , Sialoglicoproteínas/genética , Sialoglicoproteínas/inmunología , Células MadreRESUMEN
Humanized antibodies are highly efficient as immunotherapeutic reagents and have many advantages over rodent antibodies. A mouse strain was generated by gene targeting to replace the mouse kappa light chain constant (C) region gene with the human C kappa gene. Mice homozygous for the replacement mutation (C kappa R) produced normal concentrations of serum antibodies, most of which carry chimeric kappa light chains, and mounted normal immune responses to hapten-protein conjugates. This technology provides a feasible option for the generation of high-affinity humanized antibodies by means of the powerful somatic hypermutation-selection mechanism.
Asunto(s)
Linfocitos B/inmunología , Genes de Inmunoglobulinas , Regiones Constantes de Inmunoglobulina/biosíntesis , Cadenas kappa de Inmunoglobulina/biosíntesis , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Reordenamiento Génico , Humanos , Regiones Constantes de Inmunoglobulina/genética , Isotipos de Inmunoglobulinas/biosíntesis , Cadenas kappa de Inmunoglobulina/genética , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Mutación , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes de Fusión/biosíntesis , Células Madre , TransfecciónRESUMEN
We have employed a method based on the Cre-loxP recombination system of bacteriophage P1 to generate a mouse strain in which the JH segments and the intron enhancer in the IgH locus are deleted. By analysis of immunoglobulin isotype switch recombination in heterozygous mutant B cells activated by lipopolysaccharide plus interleukin-4, we show that, on the mutant chromosome, switch recombination at the mu gene switch region is strongly suppressed, whereas the switch region of the gamma 1 gene is efficiently rearranged. These data demonstrate an independent control of switch recombination at individual switch regions and suggest that, in the process of switch recombination, the alignment of the recombining strands occurs independently of and probably after the introduction of double-strand breaks into the switch regions involved.
Asunto(s)
Reordenamiento Génico de Cadena Pesada de Linfocito B , Genes de Cambio , Animales , Secuencia de Bases , Femenino , Genes de Inmunoglobulinas , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Datos de Secuencia Molecular , Recombinación Genética , Células Tumorales CultivadasRESUMEN
Immunoglobulins (Ig) secreted from a plasma cell contain either kappa or lambda light chains, but not both. This phenomenon is termed isotypic kappa-lambda exclusion. While kappa-producing cells have their lambda chain genes in germline configuration, in most lambda-producing cells the kappa chain genes are either non-productively rearranged or deleted. To investigate the molecular mechanism for isotypic kappa-lambda exclusion, in particular the role of the Ig kappa intron enhancer, we replaced this enhancer by a neomycin resistance (neoR) gene in embryonic stem (ES) cells. B cells heterozygous for the mutation undergo V kappa-J kappa recombination exclusively in the intact Ig kappa locus but not in the mutated Ig kappa locus. Homozygous mutant mice exhibited no rearrangements in their Ig kappa loci. However, splenic B cell numbers were only slightly reduced as compared with the wild-type, and all B cells expressed lambda chain bearing surface Ig. These findings demonstrate that rearrangement in the Ig kappa locus is not essential for lambda gene rearrangement. We also generated homozygous mutant mice in which the neoR gene was inserted at the 3' end of the Ig kappa intron enhancer. Unexpectedly, mere insertion of the neoR gene showed some suppressive effect on V kappa-J kappa recombination. However, the much more pronounced inhibition of V kappa-J kappa recombination by the replacement of the Ig kappa intron enhancer suggests that this enhancer is essential for V kappa-J kappa recombination.
Asunto(s)
Elementos de Facilitación Genéticos , Reordenamiento Génico de Cadena Ligera de Linfocito B , Cadenas kappa de Inmunoglobulina/genética , Cadenas lambda de Inmunoglobulina/genética , Intrones , Eliminación de Secuencia , Animales , Secuencia de Bases , Quimera , ADN Recombinante , Femenino , Heterocigoto , Homocigoto , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Mutación , Reacción en Cadena de la PolimerasaRESUMEN
The production of lambda chain-expressing B cells was studied in mice in which either the gene encoding the constant region of the kappa chain (C kappa) or the intron enhancer in the Ig kappa locus was inactivated by insertion of a neomycin resistance gene. The two mutants have similar phenotypes: in heterozygous mutant mice the fraction of lambda chain-bearing B cells is twice that in the wildtype. Homozygous mutants produce approximately 7 times more lambda-expressing B cells (and about 2.3 times fewer total B cells) in the bone marrow than their normal counterparts, suggesting that B cell progenitors can differentiate into either kappa- or lambda-producing cells and do the latter in the mutants. Whereas gene rearrangements in the Ig kappa locus are blocked in the case of enhancer inactivation, they still occur in that of the C kappa mutant, although in this mutant RS rearrangement is lower than in the wildtype. This indicates that gene rearrangements in the Ig lambda locus can occur in the absence of a putative positive signal resulting from gene rearrangements in Ig kappa, including RS recombination. Complementing these results, we also present data indicating that in normal B cell development kappa chain rearrangement can be preceded by lambda chain rearrangement and that the frequency of kappa/lambda double producers is small and insufficient to explain the massive production of lambda chain-expressing B cells in the mutants.