RESUMEN
BACKGROUND: To establish a strategy for stem cell-related tissue regeneration therapy, human gingival mesenchymal stem cells (hGMSCs) were loaded with three-dimensional (3D) bioengineered Matrigel matrix scaffolds in high-cell density microtissues to promote local tissue restoration. METHODS: The biological performance and stemness of hGMSCs under 3D culture conditions were investigated by viability and multidirectional differentiation analyses. A SpragueâDawley (SD) rat full-thickness buccal mucosa wound model was established, and hGMSCs/Matrigel were injected into the submucosa of the wound. Autologous stem cell proliferation and wound repair in local tissue were assessed by histomorphometry and immunohistochemical staining. RESULTS: Three-dimensional suspension culture can provide a more natural environment for extensions and contacts between hGMSCs, and the viability and adipogenic differentiation capacity of hGMSCs were significantly enhanced. An animal study showed that hGMSCs/Matrigel significantly accelerated soft tissue repair by promoting autologous stem cell proliferation and enhancing the generation of collagen fibers in local tissue. CONCLUSION: Three-dimensional cell culture with hydrogel scaffolds, such as Matrigel, can effectively improve the biological function and maintain the stemness of stem cells. The therapeutic efficacy of hGMSCs/Matrigel was confirmed, as these cells could effectively stimulate soft tissue repair to promote the healing process by activating the host microenvironment and autologous stem cells.
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Colágeno , Combinación de Medicamentos , Laminina , Células Madre Mesenquimatosas , Proteoglicanos , Ratas Sprague-Dawley , Andamios del Tejido , Cicatrización de Heridas , Animales , Laminina/química , Proteoglicanos/química , Colágeno/química , Humanos , Ratas , Células Madre Mesenquimatosas/citología , Andamios del Tejido/química , Diferenciación Celular , Proliferación Celular , Encía/citología , Técnicas de Cultivo Tridimensional de Células/métodos , Células Cultivadas , Ingeniería de Tejidos/métodos , Masculino , Mucosa Bucal/citologíaRESUMEN
Synovial fluid (SF) is the complex biofluid that facilitates the exceptional lubrication of articular cartilage in joints. Its primary lubricating macromolecules, the linear polysaccharide hyaluronic acid (HA) and the mucin-like glycoprotein proteoglycan 4 (PRG4 or lubricin), interact synergistically to reduce boundary friction. However, the precise manner in which these molecules influence the rheological properties of SF remains unclear. This study aimed to elucidate this by employing confocal microscopy and multiscale rheometry to examine the microstructure and rheology of solutions containing recombinant human PRG4 (rhPRG4) and HA. Contrary to previous assumptions of an extensive HA-rhPRG4 network, it is discovered that rhPRG4 primarily forms stiff, gel-like aggregates. The properties of these aggregates, including their size and stiffness, are found to be influenced by the viscoelastic characteristics of the surrounding HA matrix. Consequently, the rheology of this system is not governed by a single length scale, but instead responds as a disordered, hierarchical network with solid-like rhPRG4 aggregates distributed throughout the continuous HA phase. These findings provide new insights into the biomechanical function of PRG4 in cartilage lubrication and may have implications in the development of HA-based therapies for joint diseases like osteoarthritis.
Asunto(s)
Ácido Hialurónico , Proteoglicanos , Reología , Líquido Sinovial , Líquido Sinovial/metabolismo , Líquido Sinovial/química , Humanos , Ácido Hialurónico/química , Proteoglicanos/química , Proteoglicanos/metabolismo , Lubrificación , Sustancias Macromoleculares/química , ViscosidadRESUMEN
Proteoglycans are hierarchically organized structures that play an important role in the hydration and the compression resistance of cartilage matrix. In this study, the static and dynamic properties relevant to the biomechanical function of cartilage are determined at different levels of the hierarchical structure, using complementary osmotic pressure, neutron scattering (SANS) and light scattering (DLS) measurements. In cartilage proteoglycans (PGs), two levels of bottlebrush structures can be distinguished: the aggrecan monomer, which consists of a core protein to which are tethered charged glycosaminoglycan (GAG) chains, and complexes formed of the aggrecan monomers attached around a linear hyaluronic acid backbone. The principal component of GAG, chondroitin sulfate (CS), is used as a baseline in this comparison. The osmotic modulus, measured as a function of the proteoglycan concentration, follows the order CS < aggrecan < aggrecan-HA complex. This order underlines the benefit of the increasing complexity at each level of the molecular architecture. The hierarchical bottlebrush configuration, which prevents interpenetration among the bristles of the aggrecan monomers, enhances both the mechanical properties and the osmotic resistance. The osmotic pressure of the collagen solution is notably smaller than in the proteoglycan systems. This is consistent with its known primary role to provide tensile strength to the cartilage and to confine the aggrecan-HA complexes, as opposed to load bearing. The collective diffusion coefficient D governs the rate of recovery of biological tissue after compressive load. In CS solutions the diffusion process is fast, D ≈ 3 × 10-6 cm2 s-1 at concentrations comparable with that of the GAG chains inside the aggrecan molecule. In CS solutions D is a weakly decreasing function of calcium ion concentration, while in aggrecan and its complexes with HA, the relaxation rate is insensitive to the presence of calcium.
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Agrecanos , Matriz Extracelular , Presión Osmótica , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Agrecanos/química , Agrecanos/metabolismo , Animales , Cartílago/química , Cartílago/metabolismo , Proteoglicanos/química , Proteoglicanos/metabolismo , Ácido Hialurónico/química , Ácido Hialurónico/metabolismo , Sulfatos de Condroitina/química , Sulfatos de Condroitina/metabolismo , ÓsmosisRESUMEN
Multifunctional molecules involved in tumor progression and metastasis have been identified as valuable targets for immunotherapy. Among these, chondroitin sulfate proteoglycan 4 (CSPG4), a significant tumor cell membrane-bound proteoglycan, has emerged as a promising target, especially in light of advances in chimeric antigen receptor (CAR) T-cell therapy. The profound bioactivity of CSPG4 and its role in pivotal processes such as tumor proliferation, migration, and neoangiogenesis underline its therapeutic potential. We reviewed the molecular intricacies of CSPG4, its functional attributes within tumor cells, and the latest clinical-translational advances targeting it. Strategies such as blocking monoclonal antibodies, conjugate therapies, bispecific antibodies, small-molecule inhibitors, CAR T-cell therapies, trispecific killer engagers, and ribonucleic acid vaccines against CSPG4 were assessed. CSPG4 overexpression in diverse tumors and its correlation with adverse prognostic outcomes emphasize its significance in cancer biology. These findings suggest that targeting CSPG4 offers a promising avenue for future cancer therapy, with potential synergistic effects when combined with existing treatments.
Asunto(s)
Inmunoterapia , Neoplasias , Humanos , Inmunoterapia/métodos , Neoplasias/terapia , Neoplasias/inmunología , Animales , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Proteoglicanos Tipo Condroitín Sulfato/inmunología , Proteoglicanos/metabolismo , Proteoglicanos/química , Terapia Molecular Dirigida , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Monoclonales/inmunología , Antígenos , Proteínas de la MembranaRESUMEN
Proteoglycans (PGs), consisting of glycosaminoglycans (GAGs) linked with the core protein through a tetrasaccharide linkage region, play roles in many important biological events. The chemical synthesis of PG glycopeptides is extremely challenging. In this work, the enzymes required for synthesis of chondroitin sulfate (CS) PG (CSPG) have been expressed and the suitable sequence of enzymatic reactions has been established. To expedite CSPG synthesis, the peptide acceptor was immobilized on solid phase and the glycan units were directly installed enzymatically onto the peptide. Subsequent enzymatic chain elongation and sulfation led to the successful synthesis of CSPG glycopeptides. The CS dodecasaccharide glycopeptide was the longest homogeneous CS glycopeptide synthesized to date. The enzymatic synthesis was much more efficient than the chemical synthesis of the corresponding CS glycopeptides, which could reduce the total number of synthetic steps by 80 %. The structures of the CS glycopeptides were confirmed by mass spectrometry analysis and NMR studies. In addition, the interactions between the CS glycopeptides and cathepsin G were studied. The sulfation of glycan chain was found to be important for binding with cathepsin G. This efficient chemoenzymatic strategy opens new avenues to investigate the structures and functions of PGs.
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Sulfatos de Condroitina , Glicopéptidos , Glicopéptidos/química , Glicopéptidos/síntesis química , Glicopéptidos/metabolismo , Sulfatos de Condroitina/química , Sulfatos de Condroitina/síntesis química , Técnicas de Síntesis en Fase Sólida , Proteoglicanos/químicaRESUMEN
Proteoglycans contain glycosaminoglycans (GAGs) which are negatively charged linear polymers made of repeating disaccharide units of uronic acid and hexosamine units. They play vital roles in numerous physiological and pathological processes, particularly in governing cellular communication and attachment. Depending on their sulfonation state, acetylation, and glycosidic linkages, GAGs belong to different families. The high molecular weight, heterogeneity, and flexibility of GAGs hamper their characterization at atomic resolution, but this may be circumvented via coarse-grained (CG) approaches. In this work, we report a CG model for a library of common GAG types in their isolated or proteoglycan-linked states compatible with version 2.2 (v2.2) of the widely popular CG Martini force field. The model reproduces conformational and thermodynamic properties for a wide variety of GAGs, as well as matching structural and binding data for selected proteoglycan test systems. The parameters developed here may thus be employed to study a range of GAG-containing biomolecular systems, thereby benefiting from the efficiency and broad applicability of the Martini framework.
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Glicosaminoglicanos , Simulación de Dinámica Molecular , Termodinámica , Glicosaminoglicanos/química , Proteoglicanos/químicaRESUMEN
The evaluation of anti-tumor drugs is critical for their development and clinical guidance. Tumor organoid models are gaining increased attention due to their ability to better mimic real tumor tissues, as well as lower time and economic costs, which makes up for the shortcomings of cell lines and xenograft models. However, current tumor organoid cultures based on the Matrigel have limitations in matching with high-throughput engineering methods due to slow gelation and low mechanical strength. Here, we present a novel composite bioink for culturing colorectal cancer organoids that provides an environment close to real tissue growth conditions and exhibits excellent photocrosslinking properties for rapid gel formation. Most importantly, the tumor organoids viability in the composite bioink after printing was as high as 97%, which also kept multicellular polar structures consistent with traditional culture methods in the Matrigel. Using 3D bioprinting with this composite bioink loaded with organoids, we demonstrated the feasibility of this drug evaluation model by validating it with clinically used colorectal cancer treatment drugs. Our results suggested that the composite bioink could effectively cultivate tumor organoids using 3D bioprinting, which had the potential to replace less reliable manual operations in promoting the application of tumor organoids in drug development and clinical guidance.
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Bioimpresión , Organoides , Impresión Tridimensional , Organoides/citología , Organoides/efectos de los fármacos , Humanos , Neoplasias Colorrectales/patología , Antineoplásicos/farmacología , Antineoplásicos/química , Línea Celular Tumoral , Laminina/química , Laminina/farmacología , Proteoglicanos/química , Proteoglicanos/farmacología , Colágeno , Combinación de MedicamentosRESUMEN
The extracellular matrix (ECM) provides dynamic structural and molecular signals that affect the form and function of developing tissues. In order to parse how the individual features of the ECM impact cell- and tissue-level behavior during development, engineered culture models should reproduce key structural and molecular features of native ECM. Here, we describe a protocol for bioprinting epithelial cell aggregates embedded within a collagen-Matrigel ink in order to study the dynamic interplay between epithelial tissues and aligned networks of type I collagen fibers. Collagen fiber alignment and geometry can be spatially controlled by modulating the printing speed, nozzle geometry, surface chemistry, and degree of molecular crowding in the printing ink. We provide detailed procedures for generating epithelial cell aggregates, microextrusion printing collagen-Matrigel bioinks, culturing the three-dimensional (3D)-printed tissues, and imaging 3D-printed collagen-Matrigel constructs.
Asunto(s)
Bioimpresión , Colágeno , Células Epiteliales , Matriz Extracelular , Hidrogeles , Impresión Tridimensional , Ingeniería de Tejidos , Bioimpresión/métodos , Hidrogeles/química , Colágeno/química , Colágeno/metabolismo , Ingeniería de Tejidos/métodos , Células Epiteliales/citología , Células Epiteliales/metabolismo , Matriz Extracelular/metabolismo , Matriz Extracelular/química , Animales , Morfogénesis , Humanos , Proteoglicanos/química , Proteoglicanos/metabolismo , Andamios del Tejido/química , Laminina/química , Combinación de Medicamentos , Perros , Epitelio/metabolismo , Epitelio/crecimiento & desarrolloRESUMEN
This study proposed an optimized histogel construction method for histological analysis by applying lung cancer patient-derived organoids (PDOs) to the developed histo-pillar strip. Previously, there is the cultured PDOs damage problem during the histogel construction due to forced detachment of the Matrigel spots from the 96-well plate bottom. To address this issue, we cultured PDO on the proposed Histo-pillar strips and then immersed them in 4% paraformaldehyde fixation solution to self-isolate PDO without damage. The 4µl patient-derived cell (PDC)/Matrigel mixtures were dispensed on the surface of a U-shaped histo-pillar strip, and the PDCs were aggregated by gravity and cultured into PDOs. Cultured PDOs were self-detached by simply immersing them in a paraformaldehyde fixing solution without physical processing, showing about two times higher cell recovery rate than conventional method. In addition, we proposed a method for embedding PDOs under conditions where the histogel temperature was maintained such that the histogel did not harden, thereby improving the problem of damaging the histogel block in the conventional sandwich histogel construction method. We performed histological and genotyping analyses using tumor tissues and PDOs from two patients with lung adenocarcinoma. Therefore, the PDO culture and improved histogel block construction method using the histo-pillar strip proposed in this study can be employed as useful tools for the histological analysis of a limited number of PDCs.
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Neoplasias Pulmonares , Organoides , Humanos , Organoides/metabolismo , Organoides/efectos de los fármacos , Organoides/patología , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/metabolismo , Biomarcadores de Tumor/metabolismo , Laminina/química , Geles/química , Colágeno/química , Colágeno/metabolismo , Combinación de Medicamentos , Proteoglicanos/químicaRESUMEN
Sustained inflammation can halt or delay wound healing, and macrophages play a central role in wound healing. Inflammatory macrophages are responsible for the removal of pathogens, debris, and neutrophils, while anti-inflammatory macrophages stimulate various regenerative processes. Recombinant human Proteoglycan 4 (rhPRG4) is shown to modulate macrophage polarization and to prevent fibrosis and scarring in ear wound healing. Here, dissolvable microneedle arrays (MNAs) carrying rhPRG4 are engineered for the treatment of skin wounds. The in vitro experiments suggest that rhPRG4 modulates the inflammatory function of bone marrow-derived macrophages. Degradable and detachable microneedles are developed from gelatin methacryloyl (GelMA) attach to a dissolvable gelatin backing. The developed MNAs are able to deliver a high dose of rhPRG4 through the dissolution of the gelatin backing post-injury, while the GelMA microneedles sustain rhPRG4 bioavailability over the course of treatment. In vivo results in a murine model of full-thickness wounds with impaired healing confirm a decrease in inflammatory biomarkers such as TNF-α and IL-6, and an increase in angiogenesis and collagen deposition. Collectively, these results demonstrate rhPRG4-incorporating MNA is a promising platform in skin wound healing applications.
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Gelatina , Agujas , Piel , Cicatrización de Heridas , Animales , Cicatrización de Heridas/efectos de los fármacos , Humanos , Piel/lesiones , Piel/efectos de los fármacos , Ratones , Gelatina/química , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Macrófagos/inmunología , Proteoglicanos/química , Proteoglicanos/farmacología , Ratones Endogámicos C57BL , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/farmacología , MetacrilatosRESUMEN
Poly-l-lysine (PLL) and Matrigel, both classical coating materials for culture substrates in neural stem cell (NSC) research, present distinct interfaces whose effect on NSC behavior at cellular and molecular levels remains ambiguous. Our investigation reveals intriguing disparities: although both PLL and Matrigel interfaces are hydrophilic and feature amine functional groups, Matrigel stands out with lower stiffness and higher roughness. Based on this diversity, Matrigel surpasses PLL, driving NSC adhesion, migration, and proliferation. Intriguingly, PLL promotes NSC differentiation into astrocytes, whereas Matrigel favors neural differentiation and the physiological maturation of neurons. At the molecular level, Matrigel showcases a wider upregulation of genes linked to NSC behavior. Specifically, it enhances ECM-receptor interaction, activates the YAP transcription factor, and heightens glycerophospholipid metabolism, steering NSC proliferation and neural differentiation. Conversely, PLL upregulates genes associated with glial cell differentiation and amino acid metabolism and elevates various amino acid levels, potentially linked to its support for astrocyte differentiation. These distinct transcriptional and metabolic activities jointly shape the divergent NSC behavior on these substrates. This study significantly advances our understanding of substrate regulation on NSC behavior, offering novel insights into optimizing and targeting the application of these surface coating materials in NSC research.
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Diferenciación Celular , Proliferación Celular , Colágeno , Combinación de Medicamentos , Laminina , Células-Madre Neurales , Polilisina , Proteoglicanos , Polilisina/química , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Células-Madre Neurales/efectos de los fármacos , Laminina/química , Laminina/farmacología , Colágeno/química , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Proteoglicanos/química , Proteoglicanos/farmacología , Animales , Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , RatonesRESUMEN
There is no curative treatment for chronic auto-inflammatory diseases including rheumatoid arthritis, and current treatments can induce off-target side effects due to systemic immune suppression. This work has previously shown that dexamethasone-pulsed tolerogenic dendritic cells loaded with the arthritis-specific antigen human proteoglycan can suppress arthritis development in a proteoglycan-induced arthritis mouse model. To circumvent ex vivo dendritic cell culture, and enhance antigen-specific effects, drug delivery vehicles, such as liposomes, provide an interesting approach. Here, this work uses anionic 1,2-distearoyl-sn-glycero-3-phosphoglycerol liposomes with enhanced loading of human proteoglycan-dexamethasone conjugates by cationic lysine tetramer addition. Antigen-pulsed tolerogenic dendritic cells induced by liposomal dexamethasone in vitro enhanced antigen-specific regulatory T cells to a similar extent as dexamethasone-induced tolerogenic dendritic cells. In an inflammatory adoptive transfer model, mice injected with antigen-dexamethasone liposomes have significantly higher antigen-specific type 1 regulatory T cells than mice injected with antigen only. The liposomes significantly inhibit the progression of arthritis compared to controls in preventative and therapeutic proteoglycan-induced arthritis mouse models. This coincides with systemic tolerance induction and an increase in IL10 expression in the paws of mice. In conclusion, a single administration of autoantigen and dexamethasone-loaded liposomes seems to be a promising antigen-specific treatment strategy for arthritis in mice.
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Autoantígenos , Células Dendríticas , Dexametasona , Liposomas , Animales , Liposomas/química , Dexametasona/química , Dexametasona/farmacología , Ratones , Autoantígenos/inmunología , Autoantígenos/química , Células Dendríticas/inmunología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/metabolismo , Humanos , Artritis Experimental/inmunología , Artritis Experimental/tratamiento farmacológico , Artritis Experimental/terapia , Proteoglicanos/química , Proteoglicanos/farmacología , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/efectos de los fármacos , Artritis Reumatoide/inmunología , Artritis Reumatoide/tratamiento farmacológico , Artritis Reumatoide/terapia , Artritis Reumatoide/inducido químicamenteRESUMEN
The intricate electrophysiological functions and anatomical structures of spinal cord tissue render the establishment of in vitro models for spinal cord-related diseases highly challenging. Currently, both in vivo and in vitro models for spinal cord-related diseases are still underdeveloped, complicating the exploration and development of effective therapeutic drugs or strategies. Organoids cultured from human induced pluripotent stem cells (hiPSCs) hold promise as suitable in vitro models for spinal cord-related diseases. However, the cultivation of spinal cord organoids predominantly relies on Matrigel, a matrix derived from murine sarcoma tissue. Tissue-specific extracellular matrices are key drivers of complex organ development, thus underscoring the urgent need to research safer and more physiologically relevant organoid culture materials. Herein, we have prepared a rat decellularized brain extracellular matrix hydrogel (DBECMH), which supports the formation of hiPSC-derived spinal cord organoids. Compared with Matrigel, organoids cultured in DBECMH exhibited higher expression levels of markers from multiple compartments of the natural spinal cord, facilitating the development and maturation of spinal cord organoid tissues. Our study suggests that DBECMH holds potential to replace Matrigel as the standard culture medium for human spinal cord organoids, thereby advancing the development of spinal cord organoid culture protocols and their application in in vitro modeling of spinal cord-related diseases.
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Encéfalo , Hidrogeles , Células Madre Pluripotentes Inducidas , Organoides , Médula Espinal , Organoides/efectos de los fármacos , Organoides/citología , Organoides/metabolismo , Humanos , Animales , Médula Espinal/citología , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Hidrogeles/química , Hidrogeles/farmacología , Encéfalo/metabolismo , Ratas , Matriz Extracelular Descelularizada/química , Matriz Extracelular Descelularizada/farmacología , Matriz Extracelular/metabolismo , Matriz Extracelular/química , Laminina/farmacología , Laminina/química , Proteoglicanos/química , Ratas Sprague-Dawley , Combinación de Medicamentos , ColágenoRESUMEN
Three-dimensional (3D) organoid models have been instrumental in understanding molecular mechanisms responsible for many cellular processes and diseases. However, established organic biomaterial scaffolds used for 3D hydrogel cultures, such as Matrigel, are biochemically complex and display significant batch variability, limiting reproducibility in experiments. Recently, there has been significant progress in the development of synthetic hydrogels for in vitro cell culture that are reproducible, mechanically tuneable, and biocompatible. Self-assembling peptide hydrogels (SAPHs) are synthetic biomaterials that can be engineered to be compatible with 3D cell culture. Here we investigate the ability of PeptiGel® SAPHs to model the mammary epithelial cell (MEC) microenvironment in vitro. The positively charged PeptiGel®Alpha4 supported MEC viability, but did not promote formation of polarised acini. Modifying the stiffness of PeptiGel® Alpha4 stimulated changes in MEC viability and changes in protein expression associated with altered MEC function, but did not fully recapitulate the morphologies of MECs grown in Matrigel. To supply the appropriate biochemical signals for MEC organoids, we supplemented PeptiGels® with laminin. Laminin was found to require negatively charged PeptiGel® Alpha7 for functionality, but was then able to provide appropriate signals for correct MEC polarisation and expression of characteristic proteins. Thus, optimisation of SAPH composition and mechanics allows tuning to support tissue-specific organoids.
Asunto(s)
Técnicas de Cultivo Tridimensional de Células , Colágeno , Combinación de Medicamentos , Células Epiteliales , Hidrogeles , Laminina , Péptidos , Proteoglicanos , Laminina/farmacología , Laminina/química , Hidrogeles/química , Hidrogeles/farmacología , Proteoglicanos/farmacología , Proteoglicanos/química , Colágeno/química , Colágeno/farmacología , Péptidos/farmacología , Péptidos/química , Células Epiteliales/efectos de los fármacos , Células Epiteliales/citología , Humanos , Femenino , Técnicas de Cultivo Tridimensional de Células/métodos , Supervivencia Celular/efectos de los fármacos , Materiales Biocompatibles/química , Materiales Biocompatibles/farmacología , Glándulas Mamarias Humanas/citología , Organoides/efectos de los fármacos , Organoides/citología , Técnicas de Cultivo de Célula/métodosRESUMEN
SUMMARY: Matrigel is a basement membrane matrix extracted from the EHS mouse tumor containing extracellular matrix protein, its main components are laminin, type IV collagen, nestin, heparin sulfate, growth factor and matrix metalloproteinase.At room temperature, Matrigel polymerized to form a three dimensional matrix with biological activity. It can simulate the structure, composition, physical properties and functions of the cell basement membrane in vivo, which is beneficial to the culture and differentiation of the cells in vitro, and can be used for the study of cell morphology, biochemical function, migration, infection and gene expression. In this study, Matrigel three-dimensional culture model of bone marrow mesenchymal stem cells(BMSCs) was established, and its morphology, proliferation and survival were observed. BMSCs were isolated and cultured with whole bone marrow adherence method. The Second generation BMSCs with good growth condition were selected and mixed with Matrigel to form cell gel complexes. The morphology and proliferation of mesenchymal stem cells were observed by phase contrast microscope and HE staining,Live/Dead staining was used to evaluate the cell activity.Phase contrast microscopy showed that BMSCs were reticulated in Matrigel and proliferated well, After 7 days, the matrix gel gradually became soft and collapsed, a few cell reticular crosslinking growth was seen at 14 days; HE staining showed that the cytoplasm of the cells was larger on the fourth day and the cells were elongated and cross-linked on the seventh day; Live/dead staining showed that most cells showed green fluorescence with the prolongation of culture time, on the first, 4 and 7 days, the activity of bone marrow mesenchymal stem cells in Matrigel gradually increased, and the percentages were 92.57 %, 95.54 % and 97.37 %, respectively. Matrigel three-dimensional culture system can maintain the morphology, function and proliferation ability of bone marrow mesenchymal stem cells.
RESUMEN: Matrigel es una matriz de membrana basal extraída del tumor de ratón EHS que contiene proteína de matriz extracelular. Los componentes principales son laminina, el colágeno tipo IV, nestina, sulfato de heparina, factor de crecimiento y metaloproteinasa de matriz. A temperatura ambiente, Matrigel se polimerizó para formar una matriz tridimensional. Es posible simular la estructura, la composición, las propiedades físicas y las funciones de la membrana basal celular in vivo, lo que es beneficioso para el cultivo y la diferenciación de las células in vitro, y se puede utilizar para el estudio de la morfología celular, la función bioquímica, la migración, infección y expresión génica. En este estudio, se estableció el modelo de cultivo tridimensional Matrigel de células madre mesenquimales de médula ósea (BMSC), y se observó su morfología, proliferación y supervivencia. Las BMSC fueron aisladas y cultivadas con el método de adherencia de la médula ósea completa. Se seleccionaron las BMSC de segunda generación con buenas condiciones de crecimiento y se mezclaron con Matrigel para formar complejos de gel de células. La morfología y la proliferación de las células madre mesenquimales se observaron con microscopio de contraste de fase y se tiñó con Hematoxilina-Eosina (HE); para evaluar la actividad celular se usó la tinción Live/Dead. La microscopía de contraste mostró que las BMSC se reticularon en Matrigel y proliferaron bien. Después de 7 días, se observó que el gel de matriz gradualmente se volvió blando y colapsó, y se visualizó un cruce transversal de algunas células reticulares a los 14 días. La tinción mostró que la mayoría de las células mostraron una fluorescencia verde con la prolongación del tiempo de cultivo; en los primeros 4 y 7 días, la actividad de las células madre mesenquimales de la médula ósea en Matrigel aumentó gradualmente y los porcentajes fueron de 92,57 %, 95,54 % y 97,37 %, respectivamente. El sistema de cultivo tridimensional de Matrigel puede mantener la morfología, la función y la capacidad de proliferación de las células madre mesenquimales de la médula ósea.
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Animales , Perros , Proteoglicanos/química , Colágeno/química , Laminina/química , Técnicas de Cultivo de Célula/métodos , Células Madre Mesenquimatosas/citología , Ingeniería de Tejidos , Combinación de MedicamentosRESUMEN
The syndecans, heparan sulfate proteoglycans, are abundant molecules associated with the cell surface and extracellular matrix and consist of a protein core to which heparan sulfate chains are covalently attached. Each of the syndecan core proteins has a short cytoplasmic domain that binds cytosolic regulatory factors. The syndecans also contain highly conserved transmembrane domains and extracellular domains for which important activities are becoming known. These protein domains locate the syndecan on cell surface sites during development and tumor formation where they interact with other receptors to regulate signaling and cytoskeletal organization. The functions of cell surface heparan sulfate proteoglycan have been centered on the role of heparan sulfate chains, located on the outer side of the cell surface, in the binding of a wide array of ligands, including extracellular matrix proteins and soluble growth factors. More recently, the core proteins of the syndecan family transmembrane proteoglycans have also been shown to be involved in cell signaling through interaction with integrins and tyrosine kinase receptors.
Asunto(s)
Animales , Humanos , Adhesión Celular/fisiología , Proteoglicanos de Heparán Sulfato/fisiología , Glicoproteínas de Membrana/fisiología , Proteoglicanos/fisiología , Transducción de Señal/fisiología , Proteínas de la Matriz Extracelular/fisiología , Proteoglicanos de Heparán Sulfato/química , Glicoproteínas de Membrana/química , Unión Proteica/fisiología , Proteoglicanos/química , Receptores de Superficie Celular/fisiología , SindecanosRESUMEN
The structure of the large proteoglycan present in the bullfrog epiphyseal cartilage was studied by immunochemical and biochemical methods. The isolated monomer showed a polydisperse behavior on Sepharose CL2B, with a peak at Kav = 0.14. Chondroitin sulfate chains were identified by HPLC analysis of the products formed by chondroitinase digestion and mercuric acetate treatment. These chains have approximately 38 disaccharides, a Di45:Di68 ratio of 1.6 and GalNAc4S + GalNAc4,6S are the main non-reducing terminals. Keratan sulfate was identified by the use of two monoclonal antibodies in Western blots after chondroitinase ABC treatment. A keratan sulfate-rich region (~110 kDa) was isolated by sequential treatment with chondroitinase ABC and proteases. We also employed antibodies in Western blotting experiments and showed that the full length deglycosylated core protein is about 300 kDa after SDS-PAGE. Domain-specific antibodies revealed the presence of immunoreactive sites corresponding to G1/G2 and G3 globular domains and the characterization of this large proteoglycan as aggrecan. The results indicate the high conservation of the aggrecan domain structure in this lower vertebrate
Asunto(s)
Animales , Anfibios/fisiología , Placa de Crecimiento/química , Sulfato de Queratano/química , Proteoglicanos/química , Western Blotting , Sulfatos de Condroitina/aislamiento & purificación , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Placa de Crecimiento/fisiología , Rana catesbeianaRESUMEN
Leukemia represents the clonal expansion of an individual cell lineage of the hematopoietic system at a specific point of its maturation and development. This dysregulated expansion of cells in often accompanied by altered adherence to the bone marrow microenvironment and abnormalities in endogenous cytokine production by neoplastic cells. Proteoglycans (PGs) synthesized by neoplastic cells may interact with extracellular matrix (ECM) molecules and/or locally produced cytokines. It is believed that these events may be mediated by the glycosaminoglycan (GAG) moiety of PGs such as heparan or chondroitin sulfate, and depends on its charge. The strength of GAG-cytokine binding may be determined by the extent to sulfation of the GAG chains. The synthesis, metabolism and biological role of PGs in hematopoietic malignancies have not been clearly defined. In order to study how alterations of GAGs in leukemic cells may alter cellular behavior, we treated the murine myeloid leukemic cell line WeHi-3B with sodium chlorate. This drug reduces the sulfation of GAGs, since chlorate is a potent inhibitor of sulfate adenylyltransferase. The undersulfated GAGs produced by WeHi-3B cells were not efficient in controlling the mitotic rat of the cells, since a decrease in cell proliferation was observed in vitro. These data suggest that the complexes formed by GAGs with ECM components and/or cytokines may have an important role in the induction of leukemic cell proliferation. It is possible that the stimulatory activity elicited by this binding may be dependent upon the organization of these complexes.
Asunto(s)
Humanos , Línea Celular/química , Glicosaminoglicanos/química , Técnicas In Vitro , Leucemia Experimental , Proteoglicanos/química , Matriz Extracelular/químicaRESUMEN
The sequence of the disacharide units of eight heparan sulfate proteoglycans of different origins is described. All heparan sulfates contain 5 variable regions made of oligosaccharide blocks of disaccharides, namely GlcUA(1-4) GlcNAc, GlcUA(1-4)GlcNS, IdoUA (104)GlcNS) and monosaccharides (GlcNS, and GlcNS,65) at the non-reducing terminal. The N-acetylated region of the heparan sulfates is linked to the serine of the protein core through a trisaccharide of Xyl-Gal-Gal. Heparan sulfates differ from one another in terms of the number of disaccharides that compose each block
Asunto(s)
Bovinos , Perros , Conejos , Animales , Heparitina Sulfato/química , Oligosacáridos/química , Proteoglicanos/química , Acetilación , Secuencia de Carbohidratos , Fraccionamiento Químico , Disacáridos/química , Datos de Secuencia Molecular , Polisacárido Liasas/análisis , Análisis de SecuenciaRESUMEN
1. Two proteoglycans, PG1 and PG2, have been isolated from shark cartilage. Both are highly polydisperse and large (molecular mass: 1-10 x 10**6 Daltons) and contain chondroitin sulfate and keratan sulfate side chains, but PG2 is somewhat smaller tham PG1 and contains less keratan sulfate. 2. Monoclonal antibodies were raised against PG1. Many antibodies were obtained and one of them, MST1, was subcloned and furter characterized. This monoclonal antibody reacts with PG1 and PG2 from shark cartilage and also with aggrecan from bovine trachea cartilage. Chondroitinase AC-treated proteglycans react MST1, indicating that the antibody does not reconize chondroitin sulfate. MST1 also recognizes aggrecan from human cartilage and a proteoglycan from bovine brain (neurocan) but it does reconize proteoglycans from rat Walker tumor, fetal calf muscle and decorin from human myoma. 3. Using MST1 we were able to demonstrate that both PG1 aggregate with hyaluronic acid