RESUMO
BACKGROUND: Bovine tuberculosis (TB) is caused by Mycobacterium bovis, a well-known cause of zoonotic tuberculosis in cattle and deer, and has been investigated in many physiological and molecular studies. However, detailed genome-level studies of M. bovis have not been performed in Korea. OBJECTIVES: To survey whole genome-wide single-nucleotide polymorphism (SNP) variants in Korean M. bovis field isolates and to define M. bovis groups in Korea by comparing SNP typing with spoligotyping and variable number tandem repeat typing. METHODS: A total of 46 M. bovis field isolates, isolated from laryngopharyngeal lymph nodes and lungs of Korean cattle, wild boar, and Korean water deer, were used to identify SNPs by performing whole-genome sequencing. SNP sites were confirmed via polymerase chain reaction using 87 primer pairs. RESULTS: We identified 34 SNP sites with different frequencies across M. bovis isolates, and performed SNP typing and epidemiological analysis, which divided the 46 field isolates into 16 subtypes. CONCLUSIONS: Through SNP analysis, detailed differences in samples with identical spoligotypes could be detected. SNP analysis is, therefore, a useful epidemiological tracing tool that could enable better management of bovine TB, thus preventing further outbreaks and reducing the impact of this disease.
Assuntos
Cervos , Técnicas de Genotipagem/veterinária , Mycobacterium bovis/genética , Polimorfismo de Nucleotídeo Único , Sus scrofa , Tuberculose Bovina/microbiologia , Animais , Bovinos , Técnicas de Genotipagem/métodos , Mycobacterium bovis/isolamento & purificação , República da CoreiaRESUMO
Bovine tuberculosis is a chronic disease impacting both public health and the livestock industry. The interferon (IFN)-γ assay has been introduced as an ancillary test for diagnosing bovine tuberculosis to overcome limitations of the skin test. The objective of this study was to assess the IFN-γ assay in terms of diagnostics and as a nationwide surveillance program in South Korea. From 2012 to 2013, cattle (n = 120) with bovine tuberculosis and cattle (n = 426) from bovine tuberculosis free herds were subjected to the IFN-γ assay to evaluate the sensitivity and specificity of the assay, respectively, depending on various cut-offs (0-3.5). When optical density of the cut-off was 0.1, the sensitivity and specificity were found to be 81.7% (74.7-88.6) and 99.5% (98.9-100.0), respectively. After introducing the IFN-γ assay as part of the national control program, the IFN-γ assay and single caudal fold skin test data were collected from 47 regional veterinary services to compare the results of these two tests. Overall, the agreement between the IFN-γ assay and the single caudal fold skin test (n = 492,068) was 98.2%, and Cohen's kappa value for the two methods was 0.47. Serial and parallel use of the IFN-γ assay and skin test for the bovine tuberculosis control program were compared using samples (n = 91) from cattle confirmed as bovine tuberculosis positive in laboratories from 2014 to 2016. Parallel screening for bTB showed much higher sensitivity (86/91, 94.5%) than the following screening approaches: serial (47.2%, 43/91), single screening using CFT (63.7%, 58/91), or the IFN-γ assay (78.0%, 71/91). These results indicate that the IFN-γ assay and single caudal fold skin test are complementary to each other; therefore, parallel use of these two tests is considered a useful approach to reduce the prevalence of bovine tuberculosis in South Korea.
RESUMO
Mesenchymal stem cells (MSC) secrete a great variety of cytokines that have beneficial paracrine actions. Hepatocyte growth factor (HGF) promotes proliferation in several cell types. The aim of the present study was to investigate the protective effect of HGF gene-transfected MSC (HGF-MSC) in acetaminophen (AAP)-treated hepatocytes. We transfected the HGF gene into MSCs and confirmed HGF expression by RT-PCR and western blot. The concentration of HGF in HGF-MSC conditioned media (HGFCM) was upregulated compared with that in control MSCCM samples. Cell viability was increased in HGFCM-treated hepatocytes. Expression of Mcl-1, an anti-apoptosis protein, was increased and expression of pro-apoptosis proteins (Bad, Bik and Bid) was decreased in HGFCM-treated hepatocytes. HGF-MSC had protective effects on AAP-induced hepatocyte damage by enhancing proliferation. These results suggest that HGF-expressing MSCs may provide regenerative potential for liver cell damage.
Assuntos
Acetaminofen/efeitos adversos , Analgésicos não Narcóticos/efeitos adversos , Meios de Cultivo Condicionados/farmacologia , Fator de Crescimento de Hepatócito/metabolismo , Hepatócitos/metabolismo , Células-Tronco Mesenquimais/metabolismo , Acetaminofen/farmacologia , Analgésicos não Narcóticos/farmacologia , Animais , Proteínas Reguladoras de Apoptose/biossíntese , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/biossíntese , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Fragmentação do DNA/efeitos dos fármacos , Expressão Gênica , Fator de Crescimento de Hepatócito/biossíntese , Fator de Crescimento de Hepatócito/genética , Humanos , L-Lactato Desidrogenase/metabolismo , Fígado/lesões , Proteínas de Membrana/biossíntese , Camundongos , Proteínas Mitocondriais , Proteína de Sequência 1 de Leucemia de Células Mieloides/biossíntese , Transfecção , Proteína de Morte Celular Associada a bcl/biossínteseRESUMO
From the whole blood of Korean native cattle, Hanwoo (Bos taurus coreanae), a previously undescribed, rapidly growing, scotochromogenic isolate of the genus Mycobacterium is reported. Its 16S rRNA gene sequence, and the sequences of three other genes (hsp65, recA and rpoB) were unique and phylogenetic analysis based on 16S rRNA gene sequence (1420 bp) placed the organism into the rapidly growing Mycobacterium group close to Mycobacterium smegmatis (98.5% sequence similarity). However, phylogenetic analyses based on three different gene sequences (hsp65, recA and rpoB) revealed its location to be distinct from the branch of rapidly growing species. Culture and biochemical characteristics were generally similar to those of Mycobacterium fortuitum. Unique matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS profiles of lipids, unique fatty acid profile, unique mycolic acids profiles and a low DNA-DNA relatedness to M. fortuitum (23.6%) and M. smegmatis (39.7%) strongly supported the taxonomic status of this strain as a representative of a novel species of rapidly growing mycobacteria named Mycobacterium anyangense. The type strain is strain QIA-38(T) ( = JCM 30275(T) = KCTC 29443(T)).
Assuntos
Doenças dos Bovinos/microbiologia , Bovinos/microbiologia , Infecções por Mycobacterium/veterinária , Micobactérias não Tuberculosas/classificação , Filogenia , Animais , DNA Bacteriano/genética , Ácido Diaminopimélico/química , Ácidos Graxos/química , Genes Bacterianos , Dados de Sequência Molecular , Infecções por Mycobacterium/microbiologia , Ácidos Micólicos/química , Micobactérias não Tuberculosas/genética , Micobactérias não Tuberculosas/isolamento & purificação , Hibridização de Ácido Nucleico , Peptidoglicano/química , Pigmentação , RNA Ribossômico 16S/genética , República da Coreia , Análise de Sequência de DNARESUMO
OBJECTIVES: Cell-based therapy has been reported to repair or restore damaged salivary gland (SG) tissue after irradiation. This study was aimed at determining whether systemic administration of human adipose-derived mesenchymal stem cells (hAdMSCs) can ameliorate radiation-induced SG damage. METHODS: hAdMSCs (1 × 10(6)) were administered through a tail vein of C3H mice immediately after local irradiation, and then this infusion was repeated once a week for 3 consecutive weeks. At 12 weeks after irradiation, functional evaluations were conducted by measuring salivary flow rates (SFRs) and salivation lag times, and histopathologic and immunofluorescence histochemistry studies were performed to assay microstructural changes, apoptosis, and proliferation indices. The engraftment and in vivo differentiation of infused hAdMSCs were also investigated, and the transdifferentiation of hAdMSCs into amylase-producing SG epithelial cells (SGCs) was observed in vitro using a co-culture system. RESULTS: The systemic administration of hAdMSCs exhibited improved SFRs at 12 weeks after irradiation. hAdMSC-transplanted SGs showed fewer damaged and atrophied acinar cells and higher mucin and amylase production levels than untreated irradiated SGs. Immunofluorescence TUNEL assays revealed fewer apoptotic cells in the hAdMSC group than in the untreated group. Infused hAdMSCs were detected in transplanted SGs at 4 weeks after irradiation and some cells were found to have differentiated into SGCs. In vitro, a low number of co-cultured hAdMSCs (13%-18%) were observed to transdifferentiate into SGCs. CONCLUSION: The findings of this study indicate that hAdMSCs have the potential to protect against irradiation-induced cell loss and to transdifferentiate into SGCs, and suggest that hAdMSC administration should be viewed as a candidate therapy for the treatment of radiation-induced SG damage.
Assuntos
Tecido Adiposo/fisiologia , Sobrevivência de Enxerto/fisiologia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/fisiologia , Lesões Experimentais por Radiação/terapia , Glândulas Salivares/efeitos da radiação , Tecido Adiposo/citologia , Animais , Diferenciação Celular , Transdiferenciação Celular , Técnicas de Cocultura , Células Epiteliais/citologia , Células Epiteliais/fisiologia , Feminino , Humanos , Injeções Intravenosas , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Endogâmicos C3H , Lesões Experimentais por Radiação/patologia , Regeneração , Glândulas Salivares/patologia , Salivação/fisiologia , Transplante Heterólogo , Raios X/efeitos adversosRESUMO
OBJECTIVES: We investigated whether mouse bone marrow-derived clonal mesenchymal stem cells (BM-cMSCs) could promote vocal fold (VF) wound healing by using a xenograft animal model. METHODS: Homogeneous BM-cMSCs isolated by a subfractionation culturing method from the bone marrow aspirates of green fluorescent protein transgenic mice were injected into the VFs of rabbits immediately after direct mechanical injury. Macroscopic, biomechanical (rheometric), histologic, immunohistochemical, and transcriptional evaluations were performed on the scarred VFs 1 to 3 months after injury. Engraftment of the implanted BM-cMSCs was determined by detection of green fluorescent protein cells in the recipient VF by confocal microscopy. RESULTS: The BM-cMSC-treated VFs showed improved morphological properties and viscoelasticity as compared to control VFs injected with phosphate-buffered saline solution. Histologic and immunohistochemical evaluations showed less excessive collagen deposition and increased density of glycosaminoglycans in the BM-cMSC-treated VFs as compared to the control VFs at 3 months after injury (p = 0.003 and p = 0.037, respectively). BM-cMSC transplantation led to a significant attenuation of fibronectin (p = 0.036) and transforming growth factor beta1 (p = 0.042) messenger RNA expression at 1 month after injury. Green fluorescent protein-expressing BM-cMSCs engrafted in recipient VFs were found at 1 month after implantation. CONCLUSIONS: BM-cMSCs appeared to survive in the injured xenogeneic VFs after transplantation for up to 1 month and favorably enhanced the wound healing of VFs after injury. We conclude that BM-cMSCs are a possible source of cell therapy for vocal fold regeneration.
Assuntos
Células da Medula Óssea/citologia , Terapia Baseada em Transplante de Células e Tecidos/métodos , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/citologia , Prega Vocal/cirurgia , Cicatrização , Ferimentos e Lesões/cirurgia , Animais , Células Cultivadas , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos C57BL , Coelhos , Transplante Heterólogo , Prega Vocal/lesões , Prega Vocal/patologia , Ferimentos e Lesões/patologiaRESUMO
OBJECTIVES: Vocal fold (VF) scarring remains a therapeutic challenge. Granulocyte-macrophage colony-stimulating factor (GM-CSF) facilitates epithelial wound healing, and recently, growth factor therapy has been applied to promote tissue repair. This study was undertaken to investigate the effect of GM-CSF on VF wound healing in vivo and in vitro. METHODS: VF scarring was induced in New Zealand white rabbits by direct injury. Immediately thereafter, either GM-CSF or PBS was injected into the VFs of rabbits. Endoscopic, histopathological, immunohistochemical, and biomechanical evaluations of VFs were performed at 3 months post-injury. Human vocal fold fibroblasts (hVFFs) were cultured with GM-CSF. Production of type I and III collagen was examined immunocytochemically, and the synthesis of elastin and hyaluronic acids was evaluated by ELISA. The mRNA levels of genes related to ECM components and ECM production-related growth factors, such as HGF and TGF-ß1, were examined by real time RT-PCR. RESULTS: The GM-CSF-treated VFs showed reduced collagen deposition in comparison to the PBS-injected controls (P<0.05). Immunohistochemical staining revealed lower amounts of type I collagen and fibronectin in the GM-CSF-treated VFs (P<0.05 and P<0.01, respectively). Viscous and elastic shear moduli of VF samples were significantly lower in the GM-CSF group than in the PBS-injected group (P<0.001 and P<0.01, respectively). Mucosal waves in the GM-CSF group showed significant improvement when compared to the PBS group (Pâ=â0.0446). GM-CSF inhibited TGF-ß1-induced collagen synthesis by hVFFs (P<0.05) and the production of hyaluronic acids increased at 72 hours post-treatment (P<0.05). The expressions of HAS-2, tropoelastin, MMP-1, HGF, and c-Met mRNA were significantly increased by GM-CSF, although at different time points (P<0.05). CONCLUSION: The present study shows that GM-CSF offers therapeutic potential for the remodeling of VF wounds and the promotion of VF regeneration.
Assuntos
Fibroblastos/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Prega Vocal/efeitos dos fármacos , Prega Vocal/lesões , Cicatrização/efeitos dos fármacos , Animais , Células Cultivadas , Colágeno Tipo I/antagonistas & inibidores , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Colágeno Tipo III/genética , Colágeno Tipo III/metabolismo , Elastina/genética , Elastina/metabolismo , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibronectinas/antagonistas & inibidores , Fibronectinas/genética , Fibronectinas/metabolismo , Expressão Gênica/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Fator de Crescimento de Hepatócito/genética , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Ácido Hialurônico/biossíntese , Injeções Intralesionais , Coelhos , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo , Prega Vocal/metabolismo , Cicatrização/fisiologiaRESUMO
OBJECTIVES: External irradiation in head and neck cancers may induce irreversible hyposalivation and consequent xerostomia, stemming from radiation damage to salivary glands (SGs). As cell-based therapy has been reported to be able to repair or restore damaged SG tissues, we attempted to determine whether bone marrow-derived clonal mesenchymal stem cells (BM-cMSCs) can ameliorate irradiation-induced salivary gland damage via a murine model. METHODS: External irradiation at a dose of 15Gy was delivered to the neck fields of C57BL/6 mice. We directly administered either homologous mouse BM-cMSCs labeled with PKH26 (treatment group) or PBS (control group) into SGs 24h after irradiation. Salivary flow rate (SFR) and lag time of salivation were measured at 12weeks after transplantation. At 4 and 12weeks post-transplantation, we performed morphological, histological, and immunofluorescent examinations. Transdifferentiation of administered BM-cMSCs into salivary epithelial cells was observed by confocal microscopy. RESULTS: SFR was significantly increased in BM-cMSCs-transplanted mice compared with PBS-injected mice at 12weeks after transplantation. Administration of BM-cMSCs preserved the microscopic morphologies of SGs, with more functional acini in BM-cMSC-transplanted SGs than in PBS-injected SGs. Immunofluorescent staining revealed less apoptotic cells and increased microvessel density in BM-cMSC-transplanted SGs compared with PBS-injected SGs. PKH-26 labeled BM-cMSCs were detected in transplanted SGs at 4weeks after transplantation and in vivo transdifferentiation of BM-cMSCs into acinar cells was also observed. CONCLUSION: This study suggests that BM-cMSCs can ameliorate salivary damage following irradiation and can be used as a source of cell-based therapy for restoration of irradiation-induced salivary hypofunction.
Assuntos
Células-Tronco Mesenquimais/citologia , Glândulas Salivares/efeitos da radiação , Transplante de Células-Tronco , Animais , Peso Corporal , Diferenciação Celular , Sobrevivência Celular , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Glândulas Salivares/patologia , Glândulas Salivares/fisiopatologiaRESUMO
This paper describes a temperature-controllable bead affinity chromatography (BAC) in a microsystem for biomarker detection, and preparing samples for matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) analysis. Cancer marker proteins were captured in the microsystem by BAC with RNA aptamer-immobilized microbeads. The captured proteins were then denatured and released from the microbeads by controlling temperature. The microsystem consists of a microreactor for trapping microbeads and a temperature control unit for thermal treatment of the trapped beads. We used polymethylsilxoane or single crystalline silicon in fabricating two different types of reaction chamber to compare the differences in performance originated from the materials. Carcinoembryonic antigen was concentrated and purified from human serum using the microsystem and detected by MALDI-TOF MS to demonstrate the usefulness of the microsystem. The microsystem simplifies a sample preparation process required for protein analysis and cancer biomarker detection, which will accelerate the process of cancer research.
Assuntos
Biomarcadores/sangue , Cromatografia de Afinidade/instrumentação , Neoplasias/diagnóstico , Temperatura , Sequência de Aminoácidos/genética , Humanos , Dados de Sequência Molecular , Compostos de Organossilício/química , Peptídeos/química , Peptídeos/genética , Polietilenoglicóis/química , Silanos/química , Propriedades de SuperfícieRESUMO
OBJECTIVES: To compare the rheological characteristics of structurally different hyaluronic acid (HA)-based biomaterials that are presently used for phonosurgery and to investigate their influence on the viscoelastic properties of vocal folds after implantation in an in vivo rabbit model. STUDY DESIGN: In vitro and in vivo rheometric investigation. SETTING: Experimental laboratory, Inha and Seoul National Universities. METHODS: Viscoelastic shear properties of 3 HA-based biomaterials (Rofilan, Restylane, and Reviderm) were measured with a strain-controlled rheometer. These biomaterials were injected into the deep layers of rabbit vocal folds, and viscoelastic moduli of the injected vocal folds were determined 2 months after the injection. The vocal fold specimens were observed using a light microscope and a transmission electron microscope. RESULTS: All HA-based biomaterials showed similar levels of shear viscosity, which were slightly higher than that of human vocal folds reported in previous studies. Compared with noninjected control vocal folds, there were no significant differences in the magnitudes of both elastic shear modulus (G') and viscous modulus (G") of injected vocal folds among all of the materials. Light microscopic images showed that all materials were observed in the deep layers of vocal folds and electron scanning images revealed that injected HA particles were homogeneously distributed in regions of collagenous fibers. CONCLUSION: HA-based biomaterials could preserve the viscoelastic properties of the vocal folds, when they were injected into vocal folds in an in vivo rabbit model. However, further studies on the influence of the biomaterials on the viscoelasticity of human vocal folds in ECM surroundings are still needed.
Assuntos
Materiais Biocompatíveis , Ácido Hialurônico/análogos & derivados , Próteses e Implantes , Prega Vocal/fisiopatologia , Prega Vocal/cirurgia , Animais , Elasticidade , Injeções , Microscopia Eletrônica de Transmissão , Coelhos , Reologia , Resistência ao Cisalhamento , Viscosidade , Prega Vocal/patologiaRESUMO
A simple technique is presented for controlling the shapes of micro- and nanodrops by patterning surfaces with special hydrophilic regions surrounded by hydrophobic boundaries. Finite element method simulations link the shape of the hydrophilic regions to that of the droplets. Shaped droplets are used to controllably pattern planar surfaces and microwell arrays with microparticles and cells at the micro- and macroscales. Droplets containing suspended sedimenting particles, initially at uniform concentration, deposit more particles under deeper regions than under shallow regions. The resulting surface concentration is thus proportional to the local fluid depth and agrees well with the measured and simulated droplet profiles. A second application is also highlighted in which shaped droplets of prepolymer solution are crosslinked to synthesize microgels with tailored 3D geometry.
Assuntos
Géis/síntese química , Interações Hidrofóbicas e Hidrofílicas , Nanotecnologia/métodos , Animais , Camundongos , Microscopia Eletrônica de Varredura , Células NIH 3T3 , Propriedades de SuperfícieRESUMO
High-throughput preparation of multi-component solutions is an integral process in biology, chemistry and materials science for screening, diagnostics and analysis. Compact microfluidic systems enable such processing with low reagent volumes and rapid testing. Here we present a microfluidic device that incorporates two gradient generators, a tree-like generator and a new microfluidic active injection system, interfaced by intermediate solution reservoirs to generate diluted combinations of input solutions within an 8 × 8 or 10 × 10 array of isolated test chambers. Three input solutions were fed into the device, two to the tree-like gradient generator and one to pre-fill the test chamber array. The relative concentrations of these three input solutions in the test chambers completely characterized device behaviour and were controlled by the number of injection cycles and the flow rate. Device behaviour was modelled by computational fluid dynamics simulations and an approximate analytic formula. The device may be used for two-dimensional (2D) combinatorial dilution by adding two solutions in different relative concentrations to each of its three inputs. By appropriate choice of the two-component input solutions, test chamber concentrations that span any triangle in 2D concentration space may be obtained. In particular, explicit inputs are given for a coarse screening of a large region in concentration space followed by a more refined screening of a smaller region, including alternate inputs that span the same concentration region but with different distributions. The ability to probe arbitrary subspaces of concentration space and to control the distribution of discrete test points within those subspaces makes the device of potential benefit for high-throughput cell biology studies and drug screening.
Assuntos
Técnicas Analíticas Microfluídicas/instrumentação , Algoritmos , Simulação por Computador , Desenho de EquipamentoRESUMO
The anti-cancer activity of sulforaphane (SFN) has recently been investigated in several cancer cell lines, including human hepatic cancers. However, the mechanism of SFN-induced cell death in human hepatic cancer cells is still not well understood. The aim of the present work is to explore the possible mechanisms of SFN-induced apoptosis in hepatocellular carcinoma cells using proteomic analysis. A two-dimensional electrophoresis (2-DE)-based-proteomic analysis was employed for identification of possible target-related proteins of SFN-induced apoptosis. Among eleven proteins identified as regulated, we focused on the down-regulation of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase4 (PFKFB4) protein, which has been known as a key modulator of glycolysis. We also showed that SFN down-regulated the expression of the transcriptional factor, hypoxia inducible factor-1α (HIF-1α), which strongly regulates PFKFB4 expression. In order to obtain a broad understanding of the correlation of HIF-1α and SFN, we observed the inhibition of the activity of mitogen-activated protein kinases, regulators of HIF-1α activity. Our findings suggest that SFN is a potent inducer of apoptosis in hepatocellular carcinoma cells via PFKFB4-inhibition pathways. HIF-1 pathway inhibition may be mediated by the inhibition of mitogen-activated protein kinases.
Assuntos
Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/patologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Neoplasias Hepáticas/patologia , Fosfofrutoquinase-2/antagonistas & inibidores , Tiocianatos/farmacologia , Anticarcinógenos/farmacologia , Apoptose/genética , Carcinoma Hepatocelular/enzimologia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/fisiologia , Técnicas de Silenciamento de Genes , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Isotiocianatos , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Modelos Biológicos , Fosfofrutoquinase-2/genética , Fosfofrutoquinase-2/metabolismo , RNA Interferente Pequeno/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Sulfóxidos , Células Tumorais CultivadasRESUMO
The organization of cells within a well-defined microenvironment is important in generating the resulting tissue function. However, the cellular organization within biodegradable scaffolds often does not resemble those of native tissues. In this study, we present directed assembly of microgels to organize cells for building porous 3D tissue constructs. Cell-laden microgels were generated by molding photocrosslinkable polyethylene glycol diacrylate within a poly(dimethyl siloxane) stencil. The resulting microgels were subsequently packed as individual layers (1 mm in height) on a glass substrate by removing the excess prepolymer solution around the microgels. These clusters were crosslinked and stacked on one another to fabricate thick 3D constructs that were greater than 1 cm in width and 3 mm in thickness. To generate pores within the engineered structures, sodium alginate microgels were integrated in the engineered constructs and used as a sacrificial template. These pores may be potentially useful for fabricating a vascular network to supply oxygen and nutrients to the engineered tissue constructs. This simple and versatile building approach may be a useful tool for various 3D tissue culture and engineering applications.
Assuntos
Fibroblastos/citologia , Géis/farmacologia , Alicerces Teciduais/química , Alginatos , Animais , Sobrevivência Celular/efeitos dos fármacos , Reagentes de Ligações Cruzadas , Fibroblastos/efeitos dos fármacos , Ácido Glucurônico , Ácidos Hexurônicos , Luz , Camundongos , Células NIH 3T3 , Polietilenoglicóis/farmacologia , Porosidade , Engenharia TecidualRESUMO
We describe a microfluidic device for generating nonlinear (exponential and sigmoidal) concentration gradients, coupled with a microwell array for cell storage and analysis. The device has two inputs for coflowing multiple aqueous solutions, a main coflow channel and an asymmetrical grid of fluidic channels that allows the two solutions to combine at intersection points without fully mixing. Due to this asymmetry and diffusion of the two species in the coflow channel, varying amounts of the two solutions enter each fluidic path. This induces exponential and sigmoidal concentration gradients at low and high flow rates, respectively, making the microfluidic device versatile. A key feature of this design is that it is space-saving, as it does not require multiplexing or a separate array of mixing channels. Furthermore, the gradient structure can be utilized in concert with cell experiments, to expose cells captured in microwells to various concentrations of soluble factors. We demonstrate the utility of this design to assess the viability of fibroblast cells in response to a range of hydrogen peroxide (H(2)O(2)) concentrations.
Assuntos
Técnicas Citológicas/instrumentação , Técnicas Analíticas Microfluídicas , Dinâmica não Linear , Animais , Sobrevivência Celular/efeitos dos fármacos , Citotoxinas/toxicidade , Difusão , Relação Dose-Resposta a Droga , Camundongos , Células NIH 3T3RESUMO
In this paper, we describe a microfluidic mechanism that combines microfluidic valves and deep wells for cell localization and storage. Cells are first introduced into the device via externally controlled flow. Activating on-chip valves was used to interrupt the flow and to sediment the cells floating above the wells. Thus, valves could be used to localize the cells in the desired locations. We quantified the effect of valves in the cell storage process by comparing the total number of cells stored with and without valve activation. We hypothesized that in deep wells external flows generate low shear stress regions that enable stable, long-term docking of cells. To assess this hypothesis we conducted numerical calculations to understand the influence of well depth on the forces acting on cells. We verified those predictions experimentally by comparing the fraction of stored cells as a function of the well depth and input flow rate upon activation of the valves. As expected, upon reintroduction of the flow the cells in the deep wells were not moved whereas those in shallow wells were washed away. Taken together, our paper demonstrates that deep wells and valves can be combined to enable a broad range of cell studies.
Assuntos
Células/citologia , Microfluídica/métodos , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , HumanosRESUMO
Mesenchymal stem cells (MSCs) are multipotent progenitor cells with the ability to secrete growth factors. Because wound healing is associated with fibroblast cells and extracellular matrix (ECM) in the dermis and epidermis, we used fibroblast cells to resolve the question of whether or not MSCs regulate wound healing in vitro via a regenerative function. Using a cell proliferation assay, we demonstrated that conditioned media (CM) obtained from MSCs significantly enhanced the cell survival ability of fibroblast cells. Moreover, by measurement of mRNA and protein, we observed that CM also promoted the production or secretion of collagen, elastin, and fibronectin. To better understand the effects of ECM-related wound healing, we measured the level of collagen-degradative enzyme (matrix metalloprotease-1), and observed that CM suppressed matrix metalloprotease-1 expression. For the determination of oxidative stress, which has an influence on wound healing, we performed the superoxide dismutase and glutathione peroxidase assays; our results suggested that CM inhibited the oxidative stress of fibroblast cells. In order to widely investigate the wound-healing effects of MSCs, we performed in vivo experiments, and observed that MSCs stimulated wound healing. In summary, the results of this study suggest that MSCs inhibit the loss of fibroblast cells and ECM, and accumulation of oxidative stress. We found that MSCs stimulate wound healing in vitro and in vivo, suggesting that MSCs have the potential to enhance wound healing.
Assuntos
Fibroblastos/metabolismo , Células-Tronco Mesenquimais/fisiologia , Pele/citologia , Cicatrização , Animais , Movimento Celular , Proliferação de Células , Colágeno/metabolismo , Meios de Cultivo Condicionados , Elastina/metabolismo , Fibronectinas/metabolismo , Glutationa Peroxidase/metabolismo , Técnicas In Vitro , Masculino , Metaloproteinase 1 da Matriz/metabolismo , Estresse Oxidativo , Ratos , Ratos Sprague-Dawley , Pele/lesões , Superóxido Dismutase/metabolismoRESUMO
Luteolin has been shown to exhibit anti-cancer activity against several forms of cancers, including human hepatic cancers. Many in vitro studies have reported anti-oxidant effects of luteolin. Here, we demonstrate using ROS (reactive oxygen species) detection in the human hepatocellular carcinoma cell line, Huh-7, that anti-cancer action of luteolin are mediated through an increasing in intracellular ROS levels. To identify proteins potentially involved in this mechanism, a two-dimensional electrophoresis (2-DE)-based-proteomic approach was employed. Proteomic analysis revealed that several proteins were associated with the anti-cancer effects of luteolin. Interestingly, these proteins included peroxiredoxin 6 (PRDX6) and prohibitin (PHB), which are implicated in ROS metabolism and apoptosis. Western blot analyses confirmed the expression of these proteins in Huh-7 cells following luteolin application. On the basis of these results, we suggest that PRDX6 and PHB are key targets of luteolin that the mechanism of luteolin-induced apoptosis in Huh-7 cells is mediated through effects involving intracellular ROS.