RESUMEN
EGb-761 is commonly used as a treatment for ischemic brain injury, neurodegenerative diseases and some types of tumors (Christen and Maixent, in Cell Mol Biol 48(6):601-611, 2002). However, it is unclear whether EGb-761 affects the proliferation of cells exposed to fluoride. In this study, the proliferation and apoptosis of PC-12 cells exposed to fluoride were investigated and EGb-761 was used to protect PC-12 cells against the effects of fluoride. We found that the canonical Wnt signaling pathway was involved in the anti-proliferation of PC-12 cells exposed to fluoride. Furthermore, the results also showed that EGb-761 could attenuate the anti-proliferative activity of fluoride via DDK1 in PC-12 cells. This study may provide a new method for protecting against the inhibition of cell proliferation induced by fluoride.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Exodesoxirribonucleasas/biosíntesis , Extractos Vegetales/farmacología , Fluoruro de Sodio/toxicidad , Animales , Proliferación Celular/fisiología , Relación Dosis-Respuesta a Droga , Ginkgo biloba , Células PC12 , RatasRESUMEN
A unique feature in several non-CNS-tumors is the overexpression of heat shock protein 70 (Hsp70, HSPA1A) in the cytosol, but also its unusual plasma membrane expression and release. Although in gliomas, cytosolic Hsp70 levels are not associated with histological grading, the role of membrane bound and released Hsp70 is still completely unknown. Membrane bound as well as cytosolic Hsp70 can be detected in viable tumor cells with the monoclonal antibody (mAb) cmHsp70.1. Herein, we analysed membrane bound Hsp70 levels in primary and secondary gliomas of different grades and on isolated glioma subpopulations (endothelial cells, CD133-positive cells, primary cultures) by immunohistochemistry and flow cytometry using cmHsp70.1 mAb. Extracellular Hsp70 was determined by a commercial Hsp70 sandwich ELISA (R&D) in plasma samples of glioblastoma patients and healthy volunteers. We found an overexpression of Hsp70 in primary glioblastomas compared to low-grade, anaplastic, or secondary gliomas as determined by immunohistochemistry. Especially in flow cytometry, a strong plasma membrane Hsp70 expression was only observed in primary but not secondary glioblastomas. Within the heterogeneous tumor mass, CD133-positive tumor-initiating and primary glioblastoma cells showed a high membrane Hsp70 expression density, whereas endothelial cells, isolated from glioblastoma tissues only showed a weak staining pattern. Also in plasma samples, secreted Hsp70 protein was significantly increased in patients harbouring primary glioblastomas compared to those with secondary and low grade glioblastomas. Taken together, we show for the first time that cytosolic, membrane bound and extracellular Hsp70 is uniquely overexpressed in primary glioblastomas.
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Neoplasias Encefálicas/metabolismo , Membrana Celular/metabolismo , Citosol/metabolismo , Espacio Extracelular/metabolismo , Glioblastoma/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Antígeno AC133/metabolismo , Biomarcadores/metabolismo , Encéfalo/metabolismo , Encéfalo/patología , Encéfalo/cirugía , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/terapia , Membrana Celular/patología , Estudios de Cohortes , Citosol/patología , Células Endoteliales/metabolismo , Células Endoteliales/patología , Epilepsia/metabolismo , Epilepsia/patología , Epilepsia/cirugía , Femenino , Glioblastoma/genética , Glioblastoma/patología , Glioblastoma/terapia , Humanos , Isocitrato Deshidrogenasa/genética , Isocitrato Deshidrogenasa/metabolismo , Masculino , Persona de Mediana Edad , Clasificación del Tumor , Curva ROC , Sarcoma de Células PequeñasRESUMEN
Aptamers, single stranded DNA or RNA molecules, generated by a method called SELEX (systematic evolution of ligands by exponential enrichment) have been widely used in various biomedical applications. The newly developed Cell-SELEX (cell based-SELEX) targeting whole living cells has raised great expectations for cancer biology, -therapy and regenerative medicine. Combining nanobiotechnology with aptamers, this technology opens the way to more sophisticated applications in molecular diagnosis. This paper gives a review of recent developments in SELEX technologies and new applications of aptamers.
RESUMEN
To improve cell seeding efficiency and cytocompatibility, we designed a new coating material for scaffolds. We used aptamers, highly specific cell binding nucleic acids generated by combinatorial chemistry with an in vitro selection called systematic evolution of exponential enrichment (SELEX). In this study, we functionalized Ti-alloy surfaces to enhance cell adhesion. By coating the material with a cell specific aptamer, working as a capture molecule, we could improve the attachment of cells effectively and avoid the limitations of the currently available materials. Aptamers, immobilized by partial electrochemical entrapment in oxide layers on Ti-alloy surfaces were able to capture cells out of a flowing suspension rapidly. This model proves that surface immobilized aptamers can greatly enhance the attachment of seeded cells. This technology opens new perspectives towards clinical application of stem cell and tissue engineering strategies.
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Adhesión Celular , Electroquímica/métodos , Titanio/química , Titanio/farmacología , Animales , Línea Celular , Separación Celular , Técnicas Químicas Combinatorias , ADN/química , ADN de Cadena Simple/química , Humanos , Magnetismo , Osteoblastos/metabolismo , Células Madre/citología , Propiedades de Superficie , Ingeniería de Tejidos/métodosRESUMEN
Aptamers have been introduced to analytical applications, target validation, and drug discovery processes and, recently, applied directly as therapeutic agents. Aptamers can be generated by a method called SELEX (Systematic Evolution of Ligands by Exponential Enrichment). This is quite remarkable for such a young technology, which is only created in the early 1990s. This paper reviews recent new applications of aptamers in stem cell research and tissue engineering.
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Aptámeros de Nucleótidos , Técnicas de Cultivo de Célula , Células Madre Mesenquimatosas , Ingeniería de Tejidos/métodos , Animales , Aptámeros de Nucleótidos/síntesis química , Aptámeros de Nucleótidos/química , Aptámeros de Nucleótidos/farmacología , Humanos , Investigación , Técnica SELEX de Producción de AptámerosRESUMEN
Fluoride is a common element in nature and our daily life, and excessive intake of this element can cause fluorosis and irreversible brain damage. The toxic effects of fluoride on the central nervous system may be attributed to the release of inflammatory cytokines and ROS. GSK3ß is a key protein that modulates NF-κB activity and inflammatory cytokine levels and plays an important role in the Wnt signaling pathway. In this study, we found that fluoride altered the inflammatory status and oxidative stress by inhibiting Wnt signaling pathway activity. This study thus provides a valid basis for the fluorine-induced neuroinflammation injury theory.
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Fluoruros/efectos adversos , Inflamación/inducido químicamente , Microglía/patología , Vía de Señalización Wnt/efectos de los fármacos , Línea Celular , Sistema Nervioso Central/patología , Citocinas/metabolismo , Humanos , Microglía/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismoRESUMEN
OBJECTIVE: Functioning pituitary macroadenoma and giant adenoma have large growth volumes and endocrinological abnormalities, requiring proper medical intervention. In this retrospective study, microneurosurgery and subsequent gamma knife radiosurgery (GKRS) is assessed for efficacy and safety for the treatment of functioning pituitary macroadenoma and giant adenoma. METHODS: Between January 2007 and December 2011, 59 patients with functioning pituitary macroadenoma (n=38) or giant adenoma (n=21) received microneurosurgical resection, and after three months, GKRS with average maximum radiation dose â¼42Gy (range 30-66.7Gy). The median follow-up time was 54.3 months (range 36-85 months). RESULTS: The combined treatment controlled tumor growth in 81.4% (48/59) of patients, and improved the endocrinological status in 64.4% (38/59). Complications included hypopituitarism and visual deterioration (22 and 7 patients, respectively). Large tumor size at presentation was a risk factor for tumor recurrence, but not age, gender, invasion, radiosurgical dose, pituitary hormone status or follow-up period. Better outcomes were achieved by patients with macroadenoma than giant adenoma. CONCLUSIONS: Combined microneurosurgery and GKRS are safe and effective for functioning pituitary macroadenomas or giant adenomas. Tumor control and endocrinological improvement were satisfactory, with minimal complications.
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Adenoma/radioterapia , Adenoma/cirugía , Procedimientos Neuroquirúrgicos/métodos , Neoplasias Hipofisarias/radioterapia , Neoplasias Hipofisarias/cirugía , Adolescente , Adulto , Anciano , Femenino , Estudios de Seguimiento , Humanos , Masculino , Microcirugia/efectos adversos , Microcirugia/métodos , Persona de Mediana Edad , Procedimientos Neuroquirúrgicos/efectos adversos , Radiocirugia/efectos adversos , Radiocirugia/métodos , Resultado del Tratamiento , Adulto JovenRESUMEN
Oligonucleotides (ODN) with hexameric motifs containing central unmethylated CpG dinucleotides are immunostimulatory. Also ODN with continuous guanosines (polyG motif) show a wide range of immunological activity. Depending on the position, the chemical property of the ODN backbone and the cell type, polyG motifs have either an enhancing or a suppressing effect on the immunostimulatory activity of the CpG-ODN. Microglial cells are central components of the innate immune system of the brain and are activated by CpG-ODN in vitro and in vivo. Here we present the analysis of the immunomodulatory effects of CpG-ODN carrying a polyG motif on the microglial cell line N9. Our data show that N9 cells express Toll-like receptor 9 (TLR9) and are activated by CpG-ODN, which leads to expression of interleukin-12p40 (IL12p40), tumor necrosis factor-alpha (TNF-alpha) and inducible nitric oxide synthase (iNOS). A 3'-end polyG motif inhibits phosphothioate (PS) CpG-ODN immunostimulatory activity but enhances the immunostimulatory activity of phosphodiester (PE) CpG-ODN. Correspondingly, a 3'-end polyG motif improves the cellular uptake of PE CpG-ODN but does not change their cellular distribution pattern. Furthermore, PE CpG-ODN with a 3'-end polyG motif interact with a much higher number of cellular proteins than PE CpG-ODN. These data indicate that the 3'-end polyG motif could enhance the immunostimulatory activity of PE CpG-ODN in microglial N9 cells through increasing interaction with cellular proteins. Therefore PE CpG-ODN containing a 3'-end polyG motif resulting in increased immunostimulatory activity might be promising alternate analogues for studies in the central nervous system.
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Regulación de la Expresión Génica/efectos de los fármacos , Microglía/efectos de los fármacos , Oligodesoxirribonucleótidos/farmacología , Animales , Línea Celular , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Relación Dosis-Respuesta Inmunológica , Interacciones Farmacológicas , Regulación de la Expresión Génica/fisiología , Indoles , Interleucina-12/genética , Interleucina-12/metabolismo , Subunidad p40 de la Interleucina-12 , Ratones , Microglía/inmunología , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo II , Oligodesoxirribonucleótidos/inmunología , Poli G/química , Poli G/inmunología , Poli G/metabolismo , Poli G/farmacología , Polisacáridos/farmacología , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , ARN Mensajero/biosíntesis , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Receptor Toll-Like 9 , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
OBJECTIVE: Wnt signalling pathways regulate proliferation, motility and survival in a variety of human cell types. Dickkopf 1 (DKK1) gene codes for a secreted Wnt inhibitory factor. It functions as tumour suppressor gene in breast cancer and as a pro-apoptotic factor in glioma cells. In this study, we aimed to demonstrate whether the different expression of DKK1 in human glioma-derived cells is dependent on microenvironmental factors like hypoxia and regulated by the intercellular crosstalk with bone-marrow-derived mesenchymal stem cells (bmMSCs). METHODS: Glioma cell line U87-MG, three cell lines from human glioblastoma grade IV (glioma-derived mesenchymal stem cells) and three bmMSCs were selected for the experiment. The expression of DKK1 in cell lines under normoxic/hypoxic environment or co-culture condition was measured using real-time PCR and enzyme-linked immunoadsorbent assay. The effect of DKK1 on cell migration and proliferation was evaluated by in vitro wound healing assays and sulphorhodamine assays, respectively. RESULTS: Glioma-derived cells U87-MG displayed lower DKK1 expression compared with bmMSCs. Hypoxia led to an overexpression of DKK1 in bmMSCs and U87-MG when compared to normoxic environment, whereas co-culture of U87-MG with bmMSCs induced the expression of DKK1 in both cell lines. Exogenous recombinant DKK1 inhibited cell migration on all cell lines, but did not have a significant effect on cell proliferation of bmMSCs and glioma cell lines. CONCLUSION: In this study, we showed for the first time that the expression of DKK1 was hypoxia dependent in human malignant glioma cell lines. The induction of DKK1 by intracellular crosstalk or hypoxia stimuli sheds light on the intense adaption of glial tumour cells to environmental alterations.
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Comunicación Celular , Regulación Neoplásica de la Expresión Génica , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Vía de Señalización Wnt , Hipoxia de la Célula , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Técnicas de Cocultivo , Expresión Génica , Glioma , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Células Madre Mesenquimatosas/metabolismo , Células Madre Neoplásicas/metabolismoRESUMEN
Ginkgo biloba extract EGb761 is widely used to treat patients with learning and memory impairment in Alzheimer's disease and Parkinson's disease in China. However, it is not yet clear whether the analog of EGb761 (EGb) has a protective effect on the learning and memory damage induced by chronic fluorosis. In this study, 30 Wistar rats were randomly divided into three groups: a control group, a sodium fluoride (NaF) + EGb group, and a NaF group. The rats were administered 0.5 ml water containing NaF (100 mg/l) and EGb (120 mg/kg) per day via gavage. After 3 months, the rats' capacity for learning and memory was tested using a Y-maze. Damage to hippocampal neurons was evaluated by histological examination of the CA3 area. Superoxide dismutase (SOD) activity and the levels of glutathione peroxidase (GSH-Px) and malondialdehyde (MDA) were measured. Furthermore, the expression levels of Bcl-2 and Bax and the levels of cleaved Caspase3 in the hippocampus were evaluated by RT-PCR and Western blotting. The results showed that EGb could improve learning and memory abilities, enhance the activities of SOD and GSH-Px, attenuate the level of MDA, upregulate the ratio of Bcl-2/Bax, and downregulate the level of cleaved Caspase3.
Asunto(s)
Trastornos del Conocimiento/tratamiento farmacológico , Fluorosis Dental/complicaciones , Ginkgo biloba/química , Extractos Vegetales/uso terapéutico , Animales , Secuencia de Bases , Western Blotting , Región CA3 Hipocampal/efectos de los fármacos , Región CA3 Hipocampal/enzimología , Región CA3 Hipocampal/metabolismo , Enfermedad Crónica , Trastornos del Conocimiento/etiología , Cartilla de ADN , Masculino , Malondialdehído/metabolismo , Aprendizaje por Laberinto , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND: Malignant gliomas are highly-vascularised tumours. Neoangiogenesis is a crucial factor in the malignant behaviour of tumour and prognosis of patients. Several mechanisms are suspected to lead to neoangiogenesis, one of them is the recruitment of multipotent progenitor cells towards the tumour. Factors such as Vascular endothelial growth factor-A (VEGF-A) were described to recruit bone marrow-derived endothelial progenitor cells (EPCs) to the glioma stroma and vasculature. Little is known about isolating EPCs from normal or malignant tissues. MATERIALS AND METHODS: In this study, we addressed the topic of characterization of tumour-isolated EPCs and re-defined the clonal relationship between EPCs and hematopoietic stem cells (HSCs) in gliomas. We first checked public gene expression data of glioma for putative marker expression, pointing towards a prevalence of EPCs and HSCs in glioma. Immunohistochemical staining of glioma tissue confirmed the higher expression of these progenitor markers in glioma tissue. EPCs and HSCs were consequently isolated and characterized at the phenotypic and functional levels. We applied a new isolation method, for the first time, to specimen from patients with high grade glioma including seven grade IV glioblastoma, five-grade III astrocytoma, and three grade III oligoastrocytoma. RESULTS: In all samples, we were able to isolate the tumour-derived EPCs, which were positive for characteristic markers: CD31, CD34 and VEGFR2. The EPCs formed capillary networks in vitro and had the ability to take up acetylated low-density lipoprotein. Glioma-derived HSCs were positive for CD34 and CD45, but they were unable to form a capillary network in vitro. These findings on tumour-derived EPCs/HSCs were in concordance with the results, derived from peripheral blood of healthy volunteers. CONCLUSION: In our study, we established a new method for EPC/HSC isolation from human gliomas, defined the contribution of EPCs and HSCs to the tumour tissue, and highlighted the intense in vivo tumour host interaction.
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Neoplasias Encefálicas/patología , Separación Celular/métodos , Células Endoteliales/citología , Glioma/patología , Células Madre Hematopoyéticas/citología , Células de la Médula Ósea/citología , Humanos , Inmunohistoquímica , Neovascularización Patológica/patología , Células Madre/citologíaRESUMEN
Gas chromatography-mass spectrometry (GC-MS) profiles were generated from U87 glioma cells and human mesenchymal stem cells (hMSC). 37 metabolites representing glycolysis intermediates, TCA cycle metabolites, amino acids and lipids were selected for a detailed analysis. The concentrations of these metabolites were compared and Pearson correlation coefficients were used to calculate the relationship between pairs of metabolites. Metabolite profiles and correlation patterns differ significantly between the two cell lines. These profiles can be considered as a signature of the underlying biochemical system and provide snap-shots of the metabolism in mesenchymal stem cells and tumor cells.
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Aminoácidos/metabolismo , Glioma/metabolismo , Metabolismo de los Lípidos , Células Madre Mesenquimatosas/metabolismo , Redes y Vías Metabólicas , Línea Celular , Biología Computacional , Cromatografía de Gases y Espectrometría de Masas/métodos , HumanosRESUMEN
One class of oligonucleotides with a high potential for use in medical applications is short nucleic acids, widely known as aptamers. Although several aptamers are already being used clinically, there are very few studies dealing with the impact aptamers have on the hemostatic system. In this study, we have performed a comprehensive evaluation of the hemostatic system including coagulation, platelets, complement, and inflammatory activation by using different aptamer concentrations and fresh human whole blood in a well-established flow model. We found that single-stranded aptamers did not have a negative influence on platelets, complement, or inflammation but were able to activate factor XII, kallikrein, and prothrombin in a concentration-dependent manner. Consequently, the influence of aptamers on the coagulation system should be taken into consideration before the use of any aptamer-based drugs in patients.
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Aptámeros de Nucleótidos/farmacología , Coagulación Sanguínea/efectos de los fármacos , Aptámeros de Nucleótidos/toxicidad , Biomarcadores , Proteínas del Sistema Complemento/efectos de los fármacos , ADN de Cadena Simple/farmacología , ADN de Cadena Simple/toxicidad , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Factor XII/metabolismo , Hemorreología , Heparina/farmacología , Hirudinas/farmacología , Humanos , Técnicas In Vitro , Inflamación , Calicreínas/metabolismo , Leucocitos/efectos de los fármacos , Activación Plaquetaria/efectos de los fármacos , Protrombina/metabolismo , Técnica SELEX de Producción de AptámerosRESUMEN
Isolation of huge quantities of primary cells from whole organs like the heart becomes increasingly important, especially for the emerging research field of tissue engineering and regenerative medicine. This study deals with the isolation of pig cardiomyocytes, in contrast to the standard mouse or rat models, because we aimed to draw attention to the species, which are genetically more closely related to the human organism. The bigger operative and veterinary expenditure of the pig-heart model can only be justified by a technique that supplies a big amount of qualitative high-grade cardiomyocytes. In our model, the quality is guaranteed by protection of the heart, already in situ, by a cardioplegia and a careful application of collagenase to soften the tissue. The construction of a new apparatus which includes enormous costs was not necessary, since the perfusion equipment was realized from two commercially available HLMsets, which were modified and connected to each other. Our model makes it possible to rinse the whole myocardium, which leads to a better output than models that only prepare a part of the myocardium around a coronary artery. The careful harvesting of high-grade cardiomyocytes is an important source of successful cell cultures to be used for numerous experimental applications, may reduce animal experiments and, additionally, represents a chance for perfusionists to become an important partner in interdisciplinary research projects.
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Separación Celular/métodos , Miocitos Cardíacos/citología , Perfusión/instrumentación , Perfusión/métodos , Ingeniería de Tejidos/métodos , Anestesia , Animales , Aorta , Células Cultivadas , Colagenasas , Diseño de Equipo , Paro Cardíaco Inducido , Máquina Corazón-Pulmón , Especificidad de la Especie , Sus scrofaRESUMEN
Small interfering RNA (siRNA), double-stranded RNA (dsRNA) 21-23 nucleotides (nt) long with two nt 3' overhangs, has been shown to mediate powerful sequence-specific gene silence in mammalian cells through RNA interference (RNAi). Due to its high efficiency and high specificity siRNA has been used as a powerful post genomic tool and a potent therapeutic candidate. However, there is still a lot to learn about the mobility of siRNA inside cells and the cellular factors that might interfere with the specificity and activity of siRNA. Microglia are the brain's effector cells of the innate immune system and suitable targets in the development of novel therapeutic strategies. Here, we show the cellular uptake and intracellular distribution of siRNA in murine microglial N9 cells. siRNA was internalized by microglial N9 cells without transfection reagent and mainly localized to the endosomes However, no significant gene silencing effects were observed. Its cellular uptake and cellular distribution pattern were similar with that of a same length single stranded DNA (ssDNA). Further, cellular binding proteins of siRNA were purified and identified by mass spectrometry. Negative control siRNA and siRNA targeted to beta-actin were used in this part of experiment. Most of the siRNA binding proteins for negative control siRNA and siRNA targeted to beta-actin were dsRNA-binding proteins, such as dsRNA-dependent protein kinase R (PKR). Furthermore, both control siRNA and siRNA targeted to beta-actin activated PKR in N9 cells, which suggest that siRNA might cause off-target effects through activation of PKR.
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Proteínas Portadoras/metabolismo , Ligandos , Microglía/metabolismo , ARN Interferente Pequeño/metabolismo , eIF-2 Quinasa/metabolismo , Animales , Células Cultivadas , ADN de Cadena Simple/metabolismo , Células Endoteliales/metabolismo , Silenciador del Gen , Humanos , Ratones , Microscopía Fluorescente , ARN Interferente Pequeño/aislamiento & purificación , ARN Interferente Pequeño/farmacologíaRESUMEN
As ATP-gated ion channels, P2X4 receptors (P2X4R) of microglial cells play a crucial role in central nervous system (CNS) inflammation. In this study, we used rat microglial cell cultures to examine P2X4R expression in response to stimulation by combination of toll-like receptors (TLRs) and nucleotide-binding oligomerization domain 2 (NOD2) receptors. Various TLR1-9 ligands and NOD2 agonist muramyldipeptide (MDP) were investigated. Our results showed that certain combination of ligands had additive effects on upregulating microglial P2X4R at both mRNA and protein levels, and induced nitric oxide increase and tumor necrosis factor-alpha production. Thus TLRs and NOD2 combinations are contributors to the signaling cascades resulting in purinergic microglial activation.
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Péptidos y Proteínas de Señalización Intracelular/metabolismo , Microglía/metabolismo , Receptores Purinérgicos P2/metabolismo , Receptores Toll-Like/metabolismo , Animales , Células Cultivadas , Dipéptidos/farmacología , Citometría de Flujo , Péptidos y Proteínas de Señalización Intracelular/agonistas , Ligandos , Microglía/efectos de los fármacos , Óxido Nítrico/metabolismo , Proteína Adaptadora de Señalización NOD2 , ARN Mensajero/genética , Ratas , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2X4 , Factor de Necrosis Tumoral alfa/biosíntesis , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Adult mesenchymal stem cells (aMSCs) are a stem cell population present in bone marrow, which can be isolated and expanded in culture and characterized. Due to the lack of specific surface markers, it is difficult to separate the MSCs from bone marrow directly. Here, we present a novel method using high-specific nucleic acids called aptamers. Porcine MSCs were used as a target to generate aptamers by combinatorial chemistry out of a huge random library with in vitro technology called systematic evolution of ligands by exponential enrichment (SELEX). After cloning and sequencing, the binding affinity was detected using a cell-sorting assay with streptavidin-coated magnetic microbeads. We also used 12-well plates immobilized with aptamers to fish out MSCs from the cell solution and a fluorescein isothiocyanate-labeled aptamer to sort MSCs from bone marrow using high-speed fluorescence-activated cell sorting. The cells showed high potency to differentiate into osteogenic, as well as into adipogenic, lineages with typical morphological characteristics. Surface marker staining showed that the attached cells were CD29(+), CD44(+), CD45(-), CD90(+), SLA class I(+), SLA DQ(-), and SLA DR(-). Compared with existing methods, this study established a novel, rapid, and efficient method for direct isolation of aMSCs from porcine bone marrow by using an aptamer as a probe to fish out the aMSCs. This new application of aptamers can facilitate aMSC isolation and enrichment greatly, thereby enhancing the rate of aMSC-derived cells after in vitro differentiation for various applications in the emerging field of tissue engineering and regenerative medicine.
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Aptámeros de Nucleótidos/metabolismo , Células de la Médula Ósea/citología , Médula Ósea/metabolismo , Células Madre Mesenquimatosas/citología , Animales , Células de la Médula Ósea/metabolismo , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular , Citometría de Flujo , Células Madre Mesenquimatosas/metabolismo , Reproducibilidad de los Resultados , PorcinosRESUMEN
The improvement of the cytocompatibility of medical implants is a major goal in biomaterials research. During the last years many researchers worked on the fascinating approach to seed the respective cell types on various artificial substrates before implantation. For instance, cell-seeded implants are supposed to be better candidates for transplantable bone substitutes than conventional artificial bone grafts. To improve cell seeding efficiency and cytocompatibility, we designed a new coating material for medical implants. We used aptamers, highly specific cell binding nucleic acids generated by combinatorial chemistry with an in vitro selection called systematic evolution of exponential enrichment (SELEX). Aptamers do have high binding affinity and selectivity to their target. In our study, human osteoblasts from osteosarcoma tissue were used as a target to create the aptamer. Single aptamer mediated cell sorting assays showed the binding affinity with osteoblasts. Additionally, the aptamers immobilized on tissue culture plates could capture osteoblasts directly and rapidly from the cell solution. This model proves that aptamer coated artificial surfaces can greatly enhance cell adhesion. We assume that this strategy is capable to improve the clinical application of tissue engineered implants.
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Adhesión Celular/fisiología , Osteoblastos/fisiología , Secuencia de Bases , Línea Celular , ADN/química , ADN/ultraestructura , Cartilla de ADN , Citometría de Flujo , Humanos , Modelos Moleculares , Conformación de Ácido Nucleico , Osteoblastos/citología , Prótesis e ImplantesRESUMEN
Human tumor necrosis factor alpha (hTNF-alpha) is one of the most important inflammatory cytokines that acts as a mediator in inflammatory and immune response and plays a key role in host defense against infection. The over expression of hTNF-alpha is associated with serious consequences, such as shock, hypotension, thrombus, septicemia and even death. It has been implicated in many autoimmune and inflammatory diseases, such as rheumatoid arthritis, Crohn's disease, chronic heart failure and septic shock. Inhibiting the bio-activity of hTNF-alpha is one of the strategy for the treatment of these diseases. Compared with traditional recombinant protein drugs, small molecule drugs have many advantages, such as high affinity, low immunogenecity and low cost. Systematic evolution of ligands by exponential enrichment (SELEX) is a powerful method for the selection of oligonucleotides that bind with high affinity and specificity to target proteins. Such oligonucleotides are called aptamers, and are potential therapeutics for blocking the activity of pathologically relevant proteins. To obtain oligonucleotide aptamers specifically binding to TNF, a 40nt random DNA combinatorial library flanked by 31nt fixed sequences was chemically synthesized. The random library was amplified with PCR and subjected to selection by SELEX protocol against hTNFalpha. After incubation of the library with hTNFalpha, the mixture was blotted onto Immobilon-NC transfer membrane. The no-specific binding was washed away and the hTNFa binding aptamers were eluted and detached from the target protein. The eluted oligo nucleotides were amplified with PCR and served as the DNA library for the next round selection. After 12 rounds of such selection, the selected aptamers were cloned to pGEM-T vector. Positive clones were identified by restriction enzyme digestion and DNA sequencing. Oligo DNA were synthesized according to the sequence data and tested for their activities. Binding activity of the aptamers to hTNFalpha were detected by ELISA and dot blot with biotin-streptavidin-horseradish peroxidase system. Mouse L929 cells were used to test the anti-hTNFa activity of the DNA aptamers. The aptamers were incubated with hTNFalpha and added to the L929 cells. The results were read under microscope and with MTT staining. It was shown that these DNA aptamers bound to hTNFalpha with high affinity, and can inhibit the cytotoxicity of hTNFalpha on cell culture. The affinity of these aptamers are different and may related to their structure. These ssDNA aptamers are potential for the treatment and diagnosis of hTNFalpha related diseases.