RESUMEN

Snap-through buckling instability of elastic shells can provide a variety of biological and artificial mechanical systems with an efficient strategy to generate rapid and powerful actuation. However, snapping spherical shells studied to date have typically been shallow and thus are dominantly prone to axisymmetric inversions. Here, we study diffusion-swelling stimulated snap-through inversion of bilayer shells of a wide range of depth to cover non-axisymmetric as well as axisymmetric modes. We first establish an analytical model of strain energy stored in axisymmetrically swelling shells, in order to predict the snap-through conditions based on energy minimization. Confirming that the strain energy can indicate the critical conditions for snap-through, we compare the conditions of axisymmetric and non-axisymmetric snap-through inversion using both experiments and numerical simulations. We find that differentially swelling bilayer shells snap-through with a time-lagged but increased energy release during inversion when buckled non-axisymmetrically rather than axisymmetrically.

RESUMEN

The lack of portability and high cost of multiplex real-time PCR systems limits the device to be used in POC. To overcome this issue, this paper proposes a compact and cost-effective fluorescence detection system that can be integrated to a multiplex real-time PCR equipment. An open platform camera with embedded lens was used instead of photodiodes or an industrial camera. A compact filter wheel using a sliding tape is integrated, and the excitation LEDs are fixed at a 45° angle near the PCR chip, eliminating the need of additional filter wheels. The results show precise positioning of the filter wheel with an error less than 20 µm. Fluorescence detection results using a reference dye and standard DNA amplification showed comparable performance to that of the photodiode system.

Asunto(s)

Técnicas de Amplificación de Ácido Nucleico , Análisis Costo-Beneficio , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa

RESUMEN

The polymerase chain reaction is an important technique in biological research because it tests for diseases with a small amount of DNA. However, this process is time consuming and can lead to sample contamination. Recently, real-time PCR techniques have emerged which make it possible to monitor the amplification process for each cycle in real time. Existing camera-based systems that measure fluorescence after DNA amplification simultaneously process fluorescence excitation and emission for dozens of tubes. Therefore, there is a limit to the size, cost, and assembly of the optical element. In recent years, imaging devices for high-performance, open platforms have benefitted from significant innovations. In this paper, we propose a fluorescence detector for real-time PCR devices using an open platform camera. This system can reduce the cost, and can be miniaturized. To simplify the optical system, four low-cost, compact cameras were used. In addition, the field of view of the entire tube was minimized by dividing it into quadrants. An effective image processing method was used to compensate for the reduction in the signal-to-noise ratio. Using a reference fluorescence material, it was confirmed that the proposed system enables stable fluorescence detection according to the amount of DNA.

Asunto(s)

ADN , Técnicas de Amplificación de Ácido Nucleico , Fluorescencia , Reacción en Cadena en Tiempo Real de la Polimerasa , Relación Señal-Ruido

RESUMEN

Soft-shell turtle (SST; freshwater terrapin or tortoise) is a popular and important health functional food (HFF) product in many Asian countries. HFFs containing SST must be safe, but several HFFs have been found to be contaminated with dangerous substances, such as nitrofuran metabolites (NFMs). This finding suggests that the consumption of HFFs results in the regular exposure of vulnerable individuals to hazardous substances. Importantly, nitrofuran antibiotics have been banned for use in food-producing animals since the 1990s by the European Union. Thus, in this study, we propose a reliable and quick method to reduce the time required for the detection of four NFMs in SST powder that conventional methods are unable to quantify. Our method involves the derivatization and hydrolysis of SST powder and was validated in accordance with the requirements of European Commission Decision 2002/657/EC. The method achieves an apparent mean recovery of 82.2-108.1%, repeatability of 1.5-3.8%, and reproducibility of 2.2-4.8% for 0.5-10.0 µg kg-1 of 1-aminohydantoin, semicarbazide, 3-amino-2-oxazolidinone, and 3-amino-5-morpholinomethyl-2-oxazolidinone. In addition, linearity was achieved with correlation coefficients of 0.999, and the detection capability (CCß) and decision limit (CCα) were found to be reliable, indicating that this is a fast and accurate method for the analysis of SST powder. The validated method was successfully applied to detect NFMs in SST powder in commercial HHFs.

RESUMEN

A simplified method is described for reducing the analysis time of nitrofurans (NFs) and nitrofuran metabolites (NFMs) in the aquatic animals. Most existing HPLC-MS/MS methods are intended only for NFMs and are based on their fast metabolic transformations. We optimized a method for simultaneously detecting major NFs and their metabolites, including nitrovin (NV) that imply use of an optimized buffer solution. The novel method was validated by six different aquatic animal matrices (loach, catfish, shrimp, lobster, scallop, and eel) spiked with the analytes at 0.5, 1.0, and 2.0 µg kg-1. Recovery rates and %RSDs (relative standard deviations) of 82-97% and 1-8% were observed for NFMs, respectively, with values of 70-96% and 1-8% obtained for furazolidone, furaltadone, nitrofurazone, nitrofurantoin, and NV, respectively. Linearity was observed in the 0.1-20 µg L-1 range, with correlation coefficients greater than 0.99 recorded for all compounds. The developed method is sensitive, accurate, easier to use, and faster than previous methods when applied to real samples. To the best of our knowledge, this is the first method that can simultaneously determine NFs and their metabolites, as well as NV, using a single-step extraction process.

Asunto(s)

Residuos de Medicamentos , Nitrofuranos , Animales , Cromatografía Líquida de Alta Presión , Cromatografía Liquida , Residuos de Medicamentos/análisis , Nitrofuranos/análisis , Nitrofurazona , Espectrometría de Masas en Tándem

RESUMEN

Sustained release of bioactive molecules from delivery systems is a common strategy for ensuring their prolonged bioactivity and for minimizing safety issues. However, residual toxic reagents, the use of harsh organic solvents, and complex fabrication procedures in conventional delivery systems are considered enormous impediments toward clinical use. Herein, we describe bone morphogenetic protein-2 (BMP-2)-immobilized porous polycaprolactone particles with unique leaf-stacked structures (LSS particles) prepared using clinically feasible materials and procedures. The BMP-2 immobilized in these LSS particles is continuously released up to 36 days to provide an appropriate environment for osteogenic differentiation of human periosteum-derived cells and new bone formation. Thus, the leaf-stacked structures of these LSS particles provide a simple but clinically applicable platform for effectively delivering a variety of bioactive molecules, such as growth factors, hormones, cytokines, peptides, etc.

Asunto(s)

Preparaciones de Acción Retardada , Proteína Morfogenética Ósea 2 , Regeneración Ósea , Humanos , Osteogénesis , Periostio , Porosidad , Andamios del Tejido

RESUMEN

We prepared in situ gelling alginate (ALG)/hyaluronic acid (HA) hydrogels with a controllable gelation rate using CaSO4 as a crosslinking agent and Na2HPO4 as a crosslinking retardation agent. The ALG/HA hydrogels provided sustained release of bone morphogenetic protein-2 (BMP-2) immobilized in the hydrogels over 5 weeks. The BMP-2-immobilized ALG/HA hydrogels with different ALG/HA ratios were investigated for their in vitro osteogenic differentiation behavior of human bone marrow stem cells (hBMSCs) and in vivo bone regeneration behavior using an animal model (mandibular defect model of miniature pigs). Our findings from cell culture and animal study demonstrated that the osteogenic differentiation of hBMSCs was improved with increasing HA composition in the hydrogel. The hBMSCs/BMP-2-immobilized ALG/HA hydrogel allowed greatly enhanced osteogenic differentiation of hBMSCs (in vitro) and bone regeneration (in vivo) compared with the ALG/HA hydrogel itself and single hBMSCs- or BMP-2-immobilized hydrogel groups.

Asunto(s)

Trasplante Óseo/métodos , Ácido Hialurónico/química , Hidrogeles/química , Andamios del Tejido/química , Alginatos/química , Animales , Proteína Morfogenética Ósea 2/administración & dosificación , Proteína Morfogenética Ósea 2/farmacología , Humanos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/efectos de los fármacos , Osteogénesis , Porcinos , Porcinos Enanos

RESUMEN

Easy isolation, lack of ethical issues, high proliferation, multi-lineage differentiation potential and immunomodulatory properties of umbilical cord (UC)-derived mesenchymal stem cells (MSCs) make them a valuable tool in stem cell research. Recently, Wharton's jelly (WJ) was proven as the best MSC source among various compartments of UC. However, it is still unclear whether or not Wharton's jelly-derived MSCs (WJMSCs) from different parts of the whole cord exhibit the same characteristics. There may be varied MSCs present in different parts of WJ throughout the length of the UC. For this purpose, using an explant attachment method, WJMSCs were isolated from three different parts of the UC, mainly present towards the placenta (mother part), the center of the whole cord (central part) and the part attached to the fetus (baby part). WJMSCs from all three parts were maintained in normal growth conditions (10% ADMEM) and analyzed for mesenchymal markers, pluripotent genes, proliferation rate and tri-lineage differentiation potential. All WJMSCs were highly proliferative, positively expressed CD90, CD105, CD73 and vimentin, while not expressing CD34, CD45, CD14, CD19 or HLA-DR, differentiated into adipocytes, osteocytes and chondrocytes and expressed pluripotency markers OCT-4, SOX-2 and NANOG at gene and protein levels. Furthermore, MSCs derived from all the parts were shown to have potency towards hepatocyte-like cell differentiation. Human bone marrow-derived MSCs were used as a positive control. Finally, we conclude that WJMSCs derived from all the parts are valuable sources and can be efficiently used in various fields of regenerative medicine.

Asunto(s)

Células Madre Mesenquimatosas/citología , Cordón Umbilical/citología , Gelatina de Wharton/citología , Antígenos CD/metabolismo , Diferenciación Celular/genética , Linaje de la Célula , Proliferación Celular , Separación Celular , Femenino , Regulación de la Expresión Génica , Hepatocitos/citología , Humanos

RESUMEN

Although oxygen concentrations affect the growth and function of mesenchymal stem cells (MSCs), the impact of hypoxia on osteoblastic differentiation is not understood. Likewise, the effect of hypoxia-induced epigenetic changes on osteoblastic differentiation of MSCs is unknown. The aim of this study was to examine the in vitro hypoxic response of human periosteum-derived cells (hPDCs). Hypoxia resulted in greater proliferation of hPDCs as compared with those cultured in normoxia. Further, hypoxic conditions yielded decreased expression of apoptosis- and senescence-associated genes by hPDCs. Osteoblast phenotypes of hPDCS were suppressed by hypoxia, as suggested by alkaline phosphatase activity, alizarin red-S-positive mineralization, and mRNA expression of osteoblast-related genes. Chromatin immunoprecipitation assays showed an increased presence of H3K27me3, trimethylation of lysine 27 on histone H3, on the promoter region of bone morphogenetic protein-2. In addition, mRNA expression of histone lysine demethylase 6B (KDM6B) by hPDCs was significantly decreased in hypoxic conditions. Our results suggest that an increased level of H3K27me3 on the promoter region of bone morphogenetic protein-2, in combination with downregulation of KDM6B activity, is involved in the suppression of osteogenic phenotypes of hPDCs cultured in hypoxic conditions. Although oxygen tension plays an important role in the viability and maintenance of MSCs in an undifferentiated state, the effect of hypoxia on osteoblastic differentiation of MSCs remains controversial. In addition, evidence regarding the importance of epigenetics in regulating MSCs has been limited. This study was to examine the role hypoxia on osteoblastic differentiation of hPDCs, and we examined whether histone methylation is involved in the observed effect of hypoxia on osteogenic differentiation of hPDCs.

Asunto(s)

Hipoxia de la Célula , Histonas/metabolismo , Osteoblastos/metabolismo , Periostio/citología , Apoptosis , Proteína Morfogenética Ósea 2/genética , Proteína Morfogenética Ósea 2/metabolismo , Diferenciación Celular , Proliferación Celular , Supervivencia Celular , Células Cultivadas , Senescencia Celular , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Histona Demetilasas con Dominio de Jumonji/genética , Histona Demetilasas con Dominio de Jumonji/metabolismo , Metilación , Osteoblastos/citología , Osteogénesis , ARN Mensajero/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo

RESUMEN

Polycaprolactone (PCL) is a biodegradable polyester that is bioresorbable and biocompatible, and is widely used in medical fields. This study examines in vitro and in vivo osteogenic activities of cultured human periosteum-derived osteoblasts (POs) seeded into growth factor (bone morphogenic protein 2 [BMP-2] or vascular endothelial growth factor [VEGF])-releasing scaffolds of PCL beads coated with Pluronic F127. Each growth factor immobilized in the PCL/F127 is cumulatively released from the beads for more than 40 days (up to 3.04 ± 0.08 ng mg-1 BMP-2 and 3.41 ± 0.18 ng mg-1 VEGF in 42 days). Long-term (∼2 years) experimental results obtained in a miniature pig model suggest that POs seeded into BMP-2 + VEGF-releasing PCL/P-F127 beads are the most effective for bone repair. In in vitro assays, osteogenic activities were higher in POs seeded into BMP-2-releasing PCL/Pluronic F127 beads at earlier time points and in POs seeded into BMP-2 + VEGF-releasing PCL/Pluronic F127 beads at later time points. These results suggest that the combination of BMP-2 and VEGF more sufficiently stimulates (in particular at late time points) osteoblast differentiation of POs seeded in the PCL/F127 in vitro and in vivo, and thus allows effective bone regeneration. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 363-376, 2017.

Asunto(s)

Proteína Morfogenética Ósea 2/química , Osteoblastos/metabolismo , Osteogénesis , Periostio/metabolismo , Poloxámero/química , Poliésteres/química , Andamios del Tejido/química , Factor A de Crecimiento Endotelial Vascular/química , Animales , Femenino , Humanos , Masculino , Osteoblastos/citología , Periostio/citología , Porcinos , Porcinos Enanos

RESUMEN

To study gene expression and to determine distinctive characteristics of embryos produced by different methods, normalisation of the gene(s) of interest against reference gene(s) has commonly been employed. Therefore, the present study aimed to assess which reference genes tend to express more stably in single porcine blastocysts produced in vivo (IVO) or by parthenogenetic activation (PA), in vitro fertilisation (IVF) and somatic cell nuclear transfer (SCNT) using different analysis programs, namely geNorm, Normfinder and Bestkeeper. Commonly used reference genes including 18S rRNA (18S), H2A histone family, member Z (H2A), hypoxanthine phosphoribosyltransferase1 (HPRT1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein 4 (RPL4), peptidylprolyl isomerase A (PPIA), beta actin (ACTB), succinate dehydrogenase complex, subunit A (SDHA) and hydroxymethylbilane synthase (HMBS2) were analysed; most of them resulted in significantly (P<0.05) different cycle threshold (CT) values in porcine embryos except for SDHA and H2A. In evaluation of stable reference genes across in vivo and in vitro porcine blastocysts, three kinds of programs showed slightly different results; however, there were similar patterns about the rankings of more or less stability overall. In conclusion, SDHA and H2A were determined as the most appropriate reference genes for reliable normalisation in order to find the comparative gene expression in porcine blastocysts produced by different methods, whereas 18S was regarded as a less-stable reference gene. The present study has evaluated the stability of commonly used reference genes for accurate normalisation in porcine embryos to obtain reliable results.

Asunto(s)

Embrión de Mamíferos/metabolismo , Perfilación de la Expresión Génica/métodos , Genes Esenciales , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Animales , Porcinos

RESUMEN

Mesenchymal stem cells (MSCs) have been established after isolation from various tissue sources, including bone marrow and synovial fluid. Recently, synovial-fluid-derived MSCs were reported to have multi-lineage differentiation potential and immunomodulatory features, which indicates that these cells can be used for tissue engineering and systemic treatments. This study presents a protocol for simple and non-invasive isolation of MSCs derived from the bone marrow and synovial fluid of minipigs to analyze cell surface markers for cell phenotyping and in vitro culturing. Using sexually mature six-month-old minipigs, bone marrow was extracted from the iliac crest bone using a bone marrow extractor, and the synovial fluid was aspirated from the femorotibial joint. Procedures for the collection of samples from both sources were non-invasive. The protocols for effective isolation of MSCs from harvested cell sources and for creating in vitro culture conditions to expand stable MSCs from minipigs and the application of systemic autologous treatments are provided. For cell phenotyping, the cell surface markers of both cells were analyzed using flow cytometry. In the results, the MSCs were isolated from the synovial fluid of the minipigs and showed that synovial-fluid-derived MSCs have a similar morphology and cell phenotype to bone-marrow-derived MSCs. Therefore, non-invasively obtained synovial fluid is a valuable source of MSCs.

Asunto(s)

Células Madre Mesenquimatosas , Animales , Médula Ósea , Células de la Médula Ósea , Diferenciación Celular , Porcinos , Porcinos Enanos , Líquido Sinovial

RESUMEN

AIMS: The aim of this study was to find out a mesenchymal stem cells (MSCs) source from human dental tissues of the same donor (follicle, papilla and pulp), which exhibits higher neurogenic differentiation potential in vitro. MAIN METHODS: MSCs were isolated from dental tissues (follicle, papilla and pulp) by digestion method. All MSCs were analyzed for pluripotent makers by western blot, cell surface markers by flow cytometry, adipo- and osteocytes markers by RT-qPCR. The neuronal differentiated MSCs were characterized for neuronal specific markers by RT-qPCR and immunofluorescence. Functional neuronal properties were analyzed by electrophysiology and synaptic markers expression. KEY FINDINGS: All MSCs expressed pluripotent markers (Oct4, Sox2 and Nanog) and were found positive for mesenymal markers (CD44, CD90, CD105) while negative for hematopoietic markers (CD34 and CD45). Furthermore, MSCs were successfully differentiated into adipocytes, osteocytes and trans-differentiated into neuronal cells. Among them, dental pulp derived MSCs exhibits higher neurogenic differentiation potential, in term of expression of neuronal specific markers at both gene and protein level, and having higher Na(+) and K(+) current with the expression of synaptic markers. SIGNIFICANCE: The three types of dental MSCs from a single donor broadly possessed similar cellular properties and can differentiate into neuronal cells; however, pulp derived MSCs showed higher neurogenic potential than the follicle and papilla, suggesting their use in future stem cells therapy for the treatment of neurodegenerative disorders.

Asunto(s)

Diferenciación Celular , Pulpa Dental/citología , Células Madre Mesenquimatosas/citología , Neuronas/citología , Adolescente , Células Cultivadas , Humanos , Masculino

RESUMEN

It is commonly accepted that the sustained release of bone morphogenetic protein-2 (BMP-2) can enhance bone regeneration and minimize its safety issues. However, little is known regarding the appropriate duration of BMP-2 stimulation for sufficient osteogenic differentiation and new bone formation because of the short half-life of BMP-2 in the physiological environment and the lack of a well-defined delivery matrix that can regulate the release period of BMP-2. In this study, we prepared porous poly(lactic-co-glycolic acid) (PLGA) beads with different surface pore sizes that can regulate the release period of BMP-2 (i.e., 7, 17, and 30 days) while providing the BMP-2 concentration required for bone regeneration. Our findings in both in vitro cell culture and in vivo animal studies using these BMP-2-loaded beads demonstrate that release of BMP-2 within 7 days affects only the initial differentiation of human periosteum-derived cells (hPDCs) and does not significantly enhance their subsequent differentiation into mature functional cells. However, extending the duration of BMP-2 stimulation over 17 days can provide a suitable environment for osteogenic differentiation of hPDCs and new bone formation.

Asunto(s)

Proteína Morfogenética Ósea 2/metabolismo , Regeneración Ósea/fisiología , Diferenciación Celular , Ácido Láctico/química , Periostio/citología , Ácido Poliglicólico/química , Animales , Células Cultivadas , Semivida , Humanos , Técnicas In Vitro , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Porosidad , Porcinos , Factores de Tiempo

RESUMEN

The facile nature of mesenchymal stem cell (MSC) acquisition in relatively large numbers has made Wharton's jelly (WJ) tissue an alternative source of MSCs for regenerative medicine. However, freezing of such tissue using dimethyl sulfoxide (DMSO) for future use impedes its clinical utility. In this study, we compared the effect of two different cryoprotectants (DMSO and cocktail solution) on post-thaw cell behavior upon freezing of WJ tissue following two different freezing protocols (Conventional [-1°C/min] and programmed). The programmed method showed higher cell survival rate compared to conventional method of freezing. Further, cocktail solution showed better cryoprotection than DMSO. Post-thaw growth characteristics and stem cell behavior of Wharton's jelly mesenchymal stem cells (WJMSCs) from WJ tissue cryopreserved with a cocktail solution in conjunction with programmed method (Prog-Cock) were comparable with WJMSCs from fresh WJ tissue. They preserved their expression of surface markers, pluripotent factors, and successfully differentiated in vitro into osteocytes, adipocytes, chondrocytes, and hepatocytes. They also produced lesser annexin-V-positive cells compared to cells from WJ tissue stored using cocktail solution in conjunction with the conventional method (Conv-Cock). Real-time PCR and Western blot analysis of post-thaw WJMSCs from Conv-Cock group showed significantly increased expression of pro-apoptotic factors (BAX, p53, and p21) and reduced expression of anti-apoptotic factor (BCL2) compared to WJMSCs from the fresh and Prog-Cock group. Therefore, we conclude that freezing of fresh WJ tissue using cocktail solution in conjunction with programmed freezing method allows for an efficient WJ tissue banking for future MSC-based regenerative therapies. J. Cell. Biochem. 117: 2397-2412, 2016. © 2016 Wiley Periodicals, Inc.

Asunto(s)

Apoptosis/efectos de los fármacos , Criopreservación/métodos , Crioprotectores/farmacología , Dimetilsulfóxido/farmacología , Células Madre Mesenquimatosas/efectos de los fármacos , Cordón Umbilical/efectos de los fármacos , Gelatina de Wharton/efectos de los fármacos , Western Blotting , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Femenino , Humanos , Masculino , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Cordón Umbilical/citología , Cordón Umbilical/metabolismo , Gelatina de Wharton/citología , Gelatina de Wharton/metabolismo

RESUMEN

The biological properties of mesenchymal stem cells (MSCs) are influenced by donor age, gender and/or tissue sources. The present study investigated the cellular and molecular properties of porcine mesenchymal stromal/stem cells (MSCs) isolated from different tissues (adipose & dermal skin) and sex at different ages (1 week & 8 months after birth) with similar genetic and environmental backgrounds. MSCs were analyzed for alkaline phosphatase (AP) activity, CD90 and Oct3/4 expression, in vitro differentiation ability, senescence-associated ß-galactosidase (SA-ß-Gal) activity, telomeric properties, cell cycle status and expression of senescence (IL6, c-myc, TGFß, p53 and p21)- and apoptosis (Bak and Bcl2)-related proteins. An age-dependent decline in AP activity and adipogenesis was observed in all MSCs, except for male A-MSCs. CD90 expression did not change, but SA-ß-Gal activity increased with advancement in age, except in A-MSCs. Telomeric properties were similar in all MSCs, whereas expression levels of Oct3/4 protein declined with the advancement in age. p21 expression was increased with increase in donor age. Male derived cells have shown higher IL6 expression. The expression of p53 was slightly lower in MSCs of dermal tissue than in adipose tissue. Bak was expressed in all MSCs regardless of age, but up regulation of Bcl2 was observed in DS-MSCs derived at 1 week after birth. In conclusion, adipose tissue-derived MSCs from young female individuals were found to be more resistant to senescence under in vitro culture conditions.

Asunto(s)

Células Madre Mesenquimatosas/fisiología , Porcinos/anatomía & histología , Tejido Adiposo/citología , Factores de Edad , Fosfatasa Alcalina/metabolismo , Animales , Femenino , Masculino , Células Madre Mesenquimatosas/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Fenotipo , Factores Sexuales , Piel/citología , Antígenos Thy-1/metabolismo , beta-Galactosidasa/metabolismo

RESUMEN

Pharmacologic blockade of the activation of signal transducer and activator of transcription 3 (STAT3) in tyrosine kinase inhibitor (TKI)-resistant chronic myeloid leukemia (CML) cell lines characterized by kinase-independent resistance was shown to re-sensitize CML cells to TKI therapy, suggesting that STAT3 inhibitors in combination with TKIs are an effective combinatorial therapeutic for the treatment of CML. Benzoic acid- and hydroxamic acid-based STAT3 inhibitors SH-4-054 and SH-5-007, developed previously in our laboratory, demonstrated promising activity against these resistant CML cell lines. However, pharmacokinetic studies in murine models (CD-1 mice) revealed that both SH-4-054 and SH-5-007 are susceptible to glutathione conjugation at the para position of the pentafluorophenyl group via nucleophilic aromatic substitution (SN Ar). To determine whether the electrophilicity of the pentafluorophenyl sulfonamide could be tempered, an in-depth structure-activity relationship (SAR) study of the SH-4-054 scaffold was conducted. These studies revealed that AM-1-124, possessing a 2,3,5,6-tetrafluorophenylsulfonamide group, retained STAT3 protein affinity (Ki =15 µm), as well as selectivity over STAT1 (Ki >250 µm). Moreover, in both hepatocytes and in in vivo pharmacokinetic studies (CD-1 mice), AM-1-124 was found to be dramatically more stable than SH-4-054 (t1/2 =1.42 h cf. 10 min, respectively). AM-1-124 is a promising STAT3-targeting inhibitor with demonstrated bioavailability, suitable for evaluation in preclinical cancer models.

Asunto(s)

Antineoplásicos/farmacología , Antineoplásicos/farmacocinética , Resistencia a Antineoplásicos/efectos de los fármacos , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Factor de Transcripción STAT3/antagonistas & inhibidores , Sulfonamidas/farmacología , para-Aminobenzoatos/farmacología , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Disponibilidad Biológica , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Ratones , Estructura Molecular , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/metabolismo , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacocinética , Factor de Transcripción STAT3/metabolismo , Relación Estructura-Actividad , Sulfonamidas/síntesis química , Sulfonamidas/química , para-Aminobenzoatos/síntesis química , para-Aminobenzoatos/química

RESUMEN

The identification of stable reference genes is a prerequisite for ensuring accurate validation of gene expression, yet too little is known about stable reference genes of porcine MSCs. The present study was, therefore, conducted to assess the stability of reference genes in porcine MSCs derived from bone marrow (BMSCs), adipose (AMSCs), and skin (SMSCs) with their in vitro differentiated cells into mesenchymal lineages such as adipocytes, osteocytes, and chondrocytes. Twelve commonly used reference genes were investigated for their threshold cycle (Ct) values by qRT-PCR. The Ct values of candidate reference genes were analyzed by geNorm software to clarify stable expression regardless of experimental conditions. Thus, Pearson's correlation was applied to determine correlation between the three most stable reference genes (NF3) and optimal number of reference genes (NFopt). In assessment of stability of reference gene across experimental conditions by geNorm analysis, undifferentiated MSCs and each differentiated status into mesenchymal lineages showed slightly different results but similar patterns about more or less stable rankings. Furthermore, Pearson's correlation revealed high correlation (r > 0.9) between NF3 and NFopt. Overall, the present study showed that HMBS, YWHAZ, SDHA, and TBP are suitable reference genes for qRT-PCR in porcine MSCs.

RESUMEN

The in vitro differentiation and immunosuppressive capacity of mesenchymal stem cells (MSCs) derived from synovial fluid (SF-MSCs) and bone marrow extract (BM-MSCs) in an isogenic background of minipigs were comparatively analyzed in a collagen-induced arthritis (CIA) mouse model of rheumatoid arthritis (RA). The proliferation capacity and expression of pluripotent transcription factors (Oct3/4 and Sox2) were significantly (P<0.05) higher in SF-MSCs than in BM-MSCs. The differentiation capacity of SF-MSCs into adipocytes, osteocytes and neurocytes was significantly (P<0.05) lower than that of BM-MSCs, and the differentiation capacity of SF-MSCs into chondrocytes was significantly (P<0.05) higher than that of BM-MSCs. Systemic injection of BM- and SF-MSCs significantly (P<0.05) ameliorated the clinical symptoms of CIA mice, with SF-MSCs having significantly (P<0.05) higher clinical and histopathological recovery scores than BM-MSCs. Furthermore, the immunosuppressive properties of SF-MSCs in CIA mice were associated with increased levels of the anti-inflammatory cytokine interleukin (IL)-10, and decreased levels of the pro-inflammatory cytokine IL-1ß and osteoclast-related sRANKL. In conclusion, SF-MSCs exhibited eminent pluripotency and differentiation capacity into chondrocytes, addition to substantial in vivo immunosuppressive capacity by elevating IL-10 and reducing IL-1ß levels in CIA mice.

Asunto(s)

Artritis Reumatoide/terapia , Células Madre Mesenquimatosas/fisiología , Animales , Artritis Reumatoide/inmunología , Médula Ósea/inmunología , Células de la Médula Ósea/fisiología , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Citocinas/metabolismo , Terapia de Inmunosupresión , Inmunoterapia , Masculino , Trasplante de Células Madre Mesenquimatosas , Ratones Endogámicos DBA , Porcinos , Porcinos Enanos , Líquido Sinovial/citología

RESUMEN

OBJECTIVE: This article reviews the imaging and histopathologic findings of various axillary diseases and suggests management guidelines for radiologists based on imaging findings with clinical correlation. CONCLUSION: Although axillary diseases may reveal nonspecific imaging findings, a knowledge of the characteristic radiologic manifestations of specific diseases according to anatomic origin (nodal, accessory breast, adipocytic, fibrous, nerve, vascular, stromal, and dermal) and postsurgical lesions aids in establishing an appropriate differential diagnosis and determining whether intervention is necessary.

Asunto(s)

Axila/patología , Diagnóstico por Imagen , Enfermedades de la Mama/diagnóstico , Implantes de Mama/efectos adversos , Diagnóstico Diferencial , Femenino , Humanos , Enfermedades Linfáticas/diagnóstico , Masculino , Neoplasias de la Vaina del Nervio/diagnóstico , Guías de Práctica Clínica como Asunto , Sarcoidosis/diagnóstico , Neoplasias Vasculares/diagnóstico