RESUMO
Human CD300c is expressed on various immune or cancer cells and is a novel B7 family member, functioning as an activity modulator on immune cells. To elucidate the function of CD300c, we developed CL7, a human CD300c-specific monoclonal antibody, and assessed its biological activity. The specific binding of CL7 monoclonal antibody against recombinant CD300c antigen was confirmed using enzyme-linked immunosorbent assay and surface plasmon resonance analysis. The binding affinity of CL7 was strong at the sub-nanomolar level. Furthermore, CL7 effectively bound to exogenously expressed CD300c on 293T cells. CL7 antibody differentiated monocytes to M1 macrophages, as evidenced by the upregulated expression of M1-specific cell surface markers and increased secretion of M1-specific cytokines in vitro in THP-1 cells and primary macrophages, as well as the increased population size of M1 macrophages in tumors grafted into mice. Additionally, CL7 treatment upregulated PD-L1 expression on THP-1 cells. We confirmed that the mechanism of M1 macrophage differentiation was through the mitogen-activated protein kinase and NF-κB signaling pathways. CD300c expression on various immune and cancer cells was similar to that of the well-known immune checkpoint PD-L1, suggesting the possibility of CD300c as a novel tumor biomarker. We also confirmed that the tumor size was substantially reduced by CL7 antibody treatment in the CT26 mouse model. Our study supports that CD300c is a potential therapeutic target in immuno-oncology. Overall, the CD300c-specific monoclonal antibody, CL7, is a promising immunotherapeutic agent, and it induces enhanced differentiation of M1 macrophages and/or their infiltration into the tumor microenvironment.
Assuntos
Antígeno B7-H1 , Monócitos , Humanos , Camundongos , Animais , Antígeno B7-H1/metabolismo , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/metabolismo , Macrófagos , Citocinas/metabolismoRESUMO
Liver fibrosis is an excessive wound healing process that occurs in response to liver damage depending on underlying aetiologies. Currently, there are no effective therapies and FDA-approved therapeutics for the treatment of liver fibrosis except liver transplantation. Multipotent adipose-derived stem cells (ADSCs) have received significant attention as regenerative medicine for liver fibrosis owing to their advantages over stem cells with other origins. However, intrinsic limitations of stem cell therapies, such as cellular rejection and tumor formation, have impeded clinical applications of the ADSC-based liver therapeutics. To overcome these problems, the extracellular nanovesicles (ENVs) responsible for the therapeutic effect of ADSCs (A-ENVs) have shown considerable promise as cell-free therapeutics for liver diseases. However, A-ENVs have not been used for the treatment of intractable chronic liver diseases including liver fibrosis and cirrhosis. Therefore, in this study, we investigated the in vitro and in vivo antifibrotic efficacy of A-ENVs in thioacetamide-induced liver fibrosis models. A-ENVs significantly downregulated the expression of fibrogenic markers, such as matrix metalloproteinase-2, collagen-1, and alpha-smooth muscle actin. The systemic administration of A-ENVs led to high accumulation in fibrotic liver tissue and the restoration of liver functionality in liver fibrosis models through a marked reduction in α-SMA and collagen deposition. These results demonstrate the significant potential of A-ENVs for use as extracellular nanovesicles-based therapeutics in the treatment of liver fibrosis and possibly other intractable chronic liver diseases.
Assuntos
Metaloproteinase 2 da Matriz , Células-Tronco , Tecido Adiposo , Fibrose , Humanos , Fígado/patologia , Cirrose Hepática/patologia , Cirrose Hepática/terapiaRESUMO
Ultraviolet-B (UVB) irradiation causes imbalance between dermal matrix synthesis and degradation through aberrant upregulation of matrix metalloproteinases (MMPs), which leads to overall skin photoaging. We investigated the effects of extracellular vesicles (EVs) derived from human adipose-derived stem cells (HASCs) on photo-damaged human dermal fibroblasts (HDFs). EVs were isolated from conditioned media of HASCs with tangential flow filtration and characterized using transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), western blotting, micro RNA (miRNA) arrays, cytokine arrays and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The effects of EVs on the UVB-irradiated HDFs were evaluated using scratch assay, ELISA and real-time PCR. Microarrays exhibited that EVs are rich in various miRNAs and proteins, and that these EV contents are linked to a broad range of biological functions, including fibroblast proliferation, UV protection, collagen biosynthesis, DNA repair and cell ageing. A scratch assay showed that HASC-EVs enhanced the migration ability of UVB-irradiated HDFs. Real-time RT-PCR and ELISA analyses revealed that the HASC-derived EVs significantly suppressed the overexpression of MMP-1, -2, -3 and -9 induced by UVB irradiation and enhanced the expression of collagen types I, II, III and V and elastin. In particular, tissue inhibitor of metalloproteinase (TIMP)-1 and transforming growth factor (TGF)-ß1, which are important factors involved in MMP suppression and ECM synthesis, were upregulated in EV-treated HDFs after UVB irradiation. Overall results suggest that diverse components that are enriched in HASC-derived EVs could act as a biochemical cue for recovery from skin photoaging.
RESUMO
Human adipose-derived stem cells (hADSCs) formed robust cell sheets by engineering the cells with soluble cell adhesive molecules (CAMs), which enabled unique approaches to harvest large area hADSC sheets. As a soluble CAM, fibronectin (FN) (100 pg/ml) enhanced the cell proliferation rate and control both cell-to-cell and cell-to-substrate interactions. Through this engineering of FN, a transferrable hADSC sheet was obtained as a free-stranding sheet (122.6 mm2) by a photothermal method. During the harvesting of hADSC sheets by the photothermal method, a collagen layer in-between cells and conductive polymer film (CP) was dissociated, to protect cells from direct exposure to a near infrared (NIR) source. The hADSC sheets were applied to chronic wound of genetically diabetic db/db mice in vivo, to accelerate 30% faster wound closure with a high closure effect (εwc) than that of control groups. These results indicated that the engineering of CAM and collagens allow hADSC sheet harvesting, which could be extended to engineer various stem cell sheets for efficient therapies.
Assuntos
Células-Tronco Mesenquimais/metabolismo , Engenharia de Proteínas , Cicatrização , Tecido Adiposo , Animais , Compostos Bicíclicos Heterocíclicos com Pontes/química , Fibronectinas/genética , Fibronectinas/metabolismo , Humanos , Raios Infravermelhos , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Camundongos , Polímeros/química , Dermatopatias/patologia , Dermatopatias/terapiaRESUMO
Self-assembled nanoparticles based on PEGylated human α-elastin were prepared as a potential vehicle for sustained protein delivery. The α-elastin was extracted from human adipose tissue and modified with methoxypolyethyleneglycol (mPEG) to control particle size and enhance the colloidal stability. The PEGylated human α-elastin showed sol-to-particle transition with a lower critical solution temperature (LCST) of 25°C-40°C in aqueous media. The PEGylated human α-elastin nanoparticles (PhENPs) showed a narrow size distribution with an average diameter of 330±33nm and were able to encapsulate significant amounts of insulin and bovine serum albumin (BSA) upon simple mixing at low temperature in water and subsequent heating to physiological temperature. The release profiles of insulin and BSA showed sustained release for 72h. Overall, the thermo-responsive self-assembled PhENPs provide a useful tool for a range of protein delivery and tissue engineering applications.
Assuntos
Portadores de Fármacos , Elastina/química , Nanopartículas/química , Polietilenoglicóis/química , Tecido Adiposo/química , Animais , Bovinos , Composição de Medicamentos , Liberação Controlada de Fármacos , Elastina/isolamento & purificação , Humanos , Insulina/química , Cinética , Nanopartículas/ultraestrutura , Tamanho da Partícula , Transição de Fase , Soroalbumina Bovina/química , Soluções , TemperaturaRESUMO
The local heating of poly(3,4-ethylenedioxythiophene) (PEDOT) by a photothermal effect directed by near-infrared (NIR) light induces unfolding of absorbed collagen triple helices, yielding soluble collagen single-helical structures. This dissociation of collagens allowed the harvesting of a living idiomorphic cell sheet, achieved upon irradiation with NIR light (λ=808â nm). The PEDOT layer was patterned and cells were successfully cultured on the patterned substrate. Cell sheets of various shapes mirroring the PEDOT pattern could be detached after a few minutes of irradiation with NIRâ light. The PEDOT patterns guided not only the entire shape of the cell sheets but also the spreading direction of the cells in the sheets. This photothermally induced dissociation of collagen provided a fast non-invasive harvesting method and tailor-made cell-sheet patterns.
Assuntos
Separação Celular/métodos , Colágeno/metabolismo , Colágeno/efeitos da radiação , Raios Infravermelhos , Processos Fotoquímicos , Temperatura , Compostos Bicíclicos Heterocíclicos com Pontes/química , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Humanos , Polímeros/químicaRESUMO
The human placenta, a complex organ, which facilitates exchange between the fetus and the mother, contains abundant extracellular matrix (ECM) components and well-preserved endogenous growth factors. In this study, we designed a new dermal substitute from human placentas for full-thickness wound healing. Highly porous, decellularized ECM sheets were fabricated from human placentas via homogenization, centrifugation, chemical and enzymatic treatments, molding, and freeze-drying. The physical structure and biological composition of human placenta-derived ECM sheets dramatically supported the regeneration of full-thickness wound in vivo. At the early stage, the ECM sheet efficiently absorbed wound exudates and tightly attached to the wound surface. Four weeks after implantation, the wound was completely closed, epidermic cells were well arranged and the bilayer structure of the epidermis and dermis was restored. Moreover, hair follicles and microvessels were newly formed in the ECM sheet-implanted wounds. Overall, the ECM sheet produced a dermal substitute with similar cellular organization to that of normal skin. These results suggest that human placenta-derived ECM sheets provide a microenvironment favorable to the growth and differentiation of cells, and positive modulate the healing of full-thickness wounds.
Assuntos
Matriz Extracelular/química , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Placenta/química , Pele Artificial , Pele/lesões , Pele/fisiopatologia , Cicatrização/fisiologia , Animais , Curativos Biológicos , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/química , Teste de Materiais , Gravidez , Ratos , Ratos Sprague-Dawley , Resistência à Tração , Resultado do Tratamento , Ferimentos Penetrantes/fisiopatologia , Ferimentos Penetrantes/terapiaRESUMO
Cells in tissues are surrounded by the extracellular matrix (ECM), a gel-like material of proteins and polysaccharides that are synthesized and secreted by cells. Here we propose that the ECM can be isolated from porcine adipose tissue and holds great promise as a xenogeneic biomaterial for tissue engineering and regenerative medicine. Porcine adipose tissue is easily obtained in large quantities from commonly discarded food waste. Decellularization protocols have been developed for extracting an intact ECM while effectively eliminating xenogeneic epitopes and minimally disrupting the ECM composition. Porcine adipose tissue was defatted by homogenization and centrifugation. It was then decellularized via chemical (1.5 M sodium chloride and 0.5% sodium dodecyl sulfate) and enzymatic treatments (DNase and RNase) with temperature control. After decellularization, immunogenic components such as nucleic acids and α-Gal were significantly reduced. However, abundant ECM components, such as collagen (332.9±12.1 µg/mg ECM dry weight), sulfated glycosaminoglycan (GAG, 85±0.7 µg/mg ECM dry weight), and elastin (152.6±4.5 µg/mg ECM dry weight), were well preserved in the decellularized material. The biochemical and mechanical features of a decellularized ECM supported the adhesion and growth of human cells in vitro. Moreover, the decellularized ECM exhibited biocompatibility, long-term stability, and bioinductivity in vivo. The overall results suggest that the decellularized ECM derived from porcine adipose tissue could be useful as an alternative biomaterial for xenograft tissue engineering.
Assuntos
Tecido Adiposo/metabolismo , Materiais Biocompatíveis/farmacologia , Matriz Extracelular/química , Engenharia Tecidual/métodos , Adipogenia/efeitos dos fármacos , Adipogenia/genética , Tecido Adiposo/citologia , Tecido Adiposo/ultraestrutura , Animais , Matriz Extracelular/ultraestrutura , Feminino , Imunofluorescência , Humanos , Teste de Materiais , Camundongos , Camundongos Endogâmicos ICR , Sus scrofa , Alicerces TeciduaisRESUMO
Collagen, the most abundant protein in vertebrates, is a useful biomaterial in pharmaceutical and medical industries. So far, most collagen has been extracted from animals and cadavers. Herein, we suggest human adipose tissue, which is routinely abandoned after liposuction, as a plentiful source of human collagen. In this study, human collagen was obtained from adipose tissue through two successive major steps: (i) extraction of the extracellular matrix (ECM) by pulverization, centrifugation, alkaline, and alcohol treatment; (ii) isolation of collagen from ECM by pepsin treatment in dilute acetic acid. The purified human adipose-derived collagen was characterized by Fourier transform infrared spectroscopy, polyacrylamide gel electrophoresis, amino acid analysis, and circular dichroism spectroscopy. The extracted collagen showed a typical triple helix structure, good thermal stability due to abundant imino acids, and high solubility at acidic pH. The collagen greatly facilitated the adhesion and proliferation of human adipose-derived stem cells and normal human dermal fibroblasts on polystyrene plates. These results suggest that human adipose tissue obtained by liposuction can provide human collagen for use in cosmetics, pharmaceutics, and medicine.
Assuntos
Tecido Adiposo/química , Colágeno/isolamento & purificação , Adulto , Animais , Bovinos , Células Cultivadas , Colágeno/química , Feminino , Humanos , Mapeamento de Peptídeos , Ratos , Espectroscopia de Infravermelho com Transformada de Fourier , Adulto JovemRESUMO
Decellularized human extracellular matrices (ECMs) are an extremely appealing biomaterial for tissue engineering and regenerative medicine. In this study, we decellularized human adipose tissue, fabricated a thin ECM sheet and explored the potential of this human adipose-derived ECM sheet as a substrate to support the formation of tissues other than adipose tissue. Acellular ECM sheets were fabricated from human adipose tissue through successive physical and chemical treatments: homogenization, centrifugation, casting, freeze-drying and sodium dodecyl sulfate treatment. The ECM sheets exhibited good mechanical properties, despite their porous structure. They degraded quickly in the presence of collagenase and the degradation rate increased with the collagenase concentration in phosphate-buffered saline. Five different human cell types, covering a broad range of cells and applications (normal human dermal fibroblasts, human aortic smooth muscle cells, human chondrocytes, human umbilical vein endothelial cells and human adipose-derived stem cells), were seeded onto the ECM sheets. All the human cell types spread well, proliferated and were successfully integrated into the decellularized ECM sheet. Overall, the results suggest that recellularized ECM sheets are a promising substitute for defective or damaged human tissues.
Assuntos
Tecido Adiposo/citologia , Matriz Extracelular/metabolismo , Engenharia Tecidual/métodos , Adulto , Aorta/citologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Condrócitos/citologia , Condrócitos/efeitos dos fármacos , Derme/citologia , Matriz Extracelular/ultraestrutura , Feminino , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Indóis/metabolismo , Microscopia de Fluorescência , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Porosidade/efeitos dos fármacos , Dodecilsulfato de Sódio/farmacologia , Coloração e Rotulagem , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Estresse Mecânico , Resistência à Tração/efeitos dos fármacos , Adulto JovemRESUMO
Carrier geometry is a key parameter of drug delivery systems and has significant impact on the drug release rate and interaction with cells and tissues. Here we present a piezoelectric inkjet printing system as a simple and convenient approach for fabrication of drug-loaded polymer microparticles with well-defined and controlled shapes. The physical properties of paclitaxel (PTX)-loaded poly(lactic-co-glycolic acid) (PLGA) inks, such as volatility, viscosity and surface tension, were optimized for piezoelectric inkjet printing, and PTX-loaded PLGA microparticles were fabricated with various geometries, such as circles, grids, honeycombs, and rings. The resulting microparticles with 10% (w/w) PTX exhibited a fairly homogeneous shape and size. The microparticle fabrication by piezoelectric inkjet printing was precise, reproducible, and highly favorable for mass production. The microparticles exhibited a biphasic release profile with an initial burst due to diffusion and a subsequent, slow second phase due to degradation of PLGA. The release rate was dependent on the geometry, mainly the surface area, with a descending rate order of honeycomb>grid, ring>circle. The PTX-loaded microparticles showed a comparable activity in inhibiting the growth of HeLa cells. Our results demonstrate that a piezoelectric inkjet printing system would provide a new approach for large-scale manufacturing of drug carriers with a desired geometry.
Assuntos
Sistemas de Liberação de Medicamentos , Nanopartículas , Antineoplásicos Fitogênicos/administração & dosagem , Química Farmacêutica , Cromatografia Líquida de Alta Pressão , Colorimetria , Excipientes , Células HeLa , Humanos , Tinta , Cinética , Ácido Láctico , Microscopia Eletrônica de Varredura , Paclitaxel/administração & dosagem , Tamanho da Partícula , Ácido Poliglicólico , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Solubilidade , Espectrofotometria Ultravioleta , Sais de Tetrazólio , ViscosidadeRESUMO
Extracellular matrix (ECM) secreted from the resident cell of tissue is an ideal biomaterial evolved by nature. Cartilage is also built from well-organized ECM components in a gel-like structure with a high collagen and proteoglycan content. Here, we explored cartilage tissue engineering using ECM scaffolds seeded with stem cells. Both scaffolds and stem cells were isolated from human adipose tissue, which is abundant and easily harvested in the human body. The human ECM scaffolds contained various endogenous bioactive factors, including transforming growth factor-beta1 (TGF-ß1, 8782±4989 pg/g, dry ECM), insulin growth factor-1 (13319±1388 pg/g, dry ECM), basic fibroblast growth factor (82373±9572 pg/g, dry ECM), and vascular endothelial growth factor (25647±2749 pg/g, dry ECM). A composite of ECM and stem cells was prepared and cultured in chondrogenic medium (with 10 ng/mL TGF-ß1 or not) for 45 days. The volumes and weights of the composites increased during culture and the surface gradually became smooth. Cell viability remained high throughout the 45 days of in vitro culture. Composites showed the formation of cartilage-like tissue with the synthesis of cartilage-specific proteins such as collagen and glycosaminoglycan. Important chondrogenic markers were expressed including Sox-9, aggrecan, and collagen type II and XI. These results demonstrate that a cell/ECM composite containing endogenous bioactive factors could provide biochemical cues for the promotion of cartilage formation.
Assuntos
Tecido Adiposo/citologia , Cartilagem/fisiologia , Matriz Extracelular/metabolismo , Células-Tronco/citologia , Engenharia Tecidual/métodos , Alicerces Teciduais/química , Adulto , Biomarcadores/metabolismo , Células Cultivadas , Condrogênese/genética , Colágeno/metabolismo , DNA/metabolismo , Feminino , Citometria de Fluxo , Imunofluorescência , Géis , Regulação da Expressão Gênica , Glicosaminoglicanos/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Microscopia Eletrônica de Varredura , Microscopia de Fluorescência , Células-Tronco/metabolismo , Células-Tronco/ultraestrutura , Adulto JovemRESUMO
Stem cell therapy requires large numbers of stem cells to replace damaged tissues, but only limited numbers of stem cells can be harvested from a single patient. To obtain large quantities of stem cells with differentiation potential, we explored a spinner culture system using human extracellular matrix (hECM) powders. The hECM was extracted from adipose tissue and fabricated into powders. Human adipose-derived stem cells (hASCs) were isolated, seeded on hECM powders, and cultivated in a spinner flask. The 3-D culture system, using hECM powders, was highly effective for promoting cell proliferation. The number of hASCs in the 3-D culture system significantly increased for 10 days, resulting in an approximately 10-fold expansion, whereas a traditional 2-D culture system showed just a 2.8-fold expansion. Surface markers, transcriptional factors, and differentiation potential of hASCs were assayed to identify the characteristics of proliferated cells in 3-D culture system. The hASCs expressed the pluripotency markers, Oct-4 and Sox-2 during 3-D culture and retained their capacity to differentiate into adipogenic, osteogenic, and chondrogenic lineages. These findings demonstrate that the 3-D culture systems using hECM powders provide an efficient in vitro environment for stem cell proliferation, and could act as stem cell delivery carriers for autologous tissue engineering and cell therapy.
Assuntos
Tecido Adiposo/citologia , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Matriz Extracelular/metabolismo , Células-Tronco/citologia , Adulto , Adesão Celular , Diferenciação Celular/genética , Linhagem da Célula/genética , Proliferação de Células , Matriz Extracelular/ultraestrutura , Citometria de Fluxo , Regulação da Expressão Gênica , Humanos , Imuno-Histoquímica , Células-Tronco Pluripotentes/citologia , Pós , Células-Tronco/ultraestrutura , Adulto JovemRESUMO
Decellularized tissues composed of extracellular matrix (ECM) have been clinically used to support the regeneration of various human tissues and organs. Most decellularized tissues so far have been derived from animals or cadavers. Therefore, despite the many advantages of decellularized tissue, there are concerns about the potential for immunogenicity and the possible presence of infectious agents. Herein, we present a biomaterial composed of ECM derived from human adipose tissue, the most prevalent, expendable, and safely harvested tissue in the human body. The ECM was extracted by successive physical, chemical, and enzymatic treatments of human adipose tissue isolated by liposuction. Cellular components including nucleic acids were effectively removed without significant disruption of the morphology or structure of the ECM. Major ECM components were quantified, including acid/pepsin-soluble collagen, sulfated glycosaminoglycan (GAG), and soluble elastin. In an in vivo experiment using mice, the decellularized ECM graft exhibited good compatibility to surrounding tissues. Overall results suggest that the decellularized ECM containing biological and chemical cues of native human ECM could be an ideal scaffold material not only for autologous but also for allograft tissue engineering.
Assuntos
Tecido Adiposo/química , Materiais Biocompatíveis/química , Matriz Extracelular/química , Engenharia Tecidual/métodos , Adulto , Animais , Feminino , Humanos , Camundongos , Camundongos Endogâmicos ICR , Transplante Homólogo , Adulto JovemRESUMO
Although hyaluronic acid (HA) has been widely used in clinics as an injectable biomaterial, it may not be appropriate as an injectable stem cell carrier because highly hydrophilic HA hydrogels provide an unfavorable environment in which the encapsulated stem cells are likely to be constrained to a round shape, thereby losing their native morphology. Herein, we hypothesized that dextran microspheres (DMs) can improve stem cell viability in HA hydrogels because they can act as substrates for stem cell adhesion, spreading and proliferation. DMs with a mean diameter of 80 µm were mixed with HA hydrogels. Human adipose-derived stem cells (hASCs) were isolated from human adipose tissue and seeded into the DM-incorporated HA hydrogels. When compared with the hydrogels alone, the number of viable cells was significantly increased in the presence of the DMs. Initially, hASCs appeared to be round in the HA hydrogels. At 12 h after seeding, the hASCs apparently attached onto the DMs and became slightly flattened. One day after seeding, the hASCs seemed to spread onto the surface of the DMs. Fluorescence micrography of live and dead cells confirmed that the cell viability was significantly improved by use of the DMs in HA hydrogels. Overall results demonstrated that the microsphere/hydrogel composite supported stem cell survival and spreading. These characteristics show the potential for use of the composite in cell-delivery and tissue-engineering applications.
Assuntos
Dextranos/química , Dextranos/farmacologia , Ácido Hialurônico/farmacologia , Hidrogéis/farmacologia , Microesferas , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Tecido Adiposo/citologia , Adulto , Sobrevivência Celular/efeitos dos fármacos , Colorimetria , Feminino , Humanos , Microscopia de Fluorescência , Pele/citologia , Adulto JovemRESUMO
Adipose tissue is found over the whole body and easily obtained in large quantities with minimal risk by a common surgical operation, liposuction. Although liposuction was originally intended for the removal of undesired adipose tissue, it may provide an ideal material for tissue engineering scaffolds. Here we present novel, porous scaffolds prepared from human adipose tissues. The scaffolds were fabricated in a variety of macroscopic shapes such as round dishes, squares, hollow tubes, and beads. The microscopic inner porous structure was controlled by the freezing temperature, with a decrease in pore size as the freezing temperature decreased. The scaffold prepared from human adipose tissue contains extracellular matrix components including collagen. Preliminary in vitro studies showed that human adipose-derived stem cells attached to a human extracellular matrix scaffold and proliferated. This scaffold based on human adipose tissue holds great promise for many clinical applications in regenerative medicine, particularly in patients requiring soft-tissue regeneration.
Assuntos
Tecido Adiposo , Matriz Extracelular , Tecido Adiposo/citologia , Tecido Adiposo/metabolismo , Adulto , Sequência de Bases , Materiais Biocompatíveis , Células Cultivadas , Colágeno/metabolismo , Primers do DNA , Feminino , Humanos , Microscopia Eletrônica de Varredura , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células-Tronco/citologia , Células-Tronco/metabolismo , Engenharia TecidualRESUMO
Here, we present extracellular matrix (ECM) powders derived from human adipose tissue as injectable cell delivery carriers for adipose tissue engineering. We postulate that human adipose tissue may provide an ideal biomaterial because it contains large amounts of ECM components including collagen. Fresh human adipose tissue was obtained by a simple surgical operation (liposuction). After removing blood and oil components, the tissue was homogenized, centrifuged, freeze-dried, and ground to powders by milling. In an in vitro study, the human ECM powders were highly effective for promotion of cell attachment and proliferation for three-dimensional (3D) cell culture. In in vivo studies, suspensions of human ECM powders containing human adipose-derived stem cells (hASCs) were subcutaneously injected into nude mice. At eight weeks post-injection, numerous blood vessels were observed and the newly formed tissue exhibited adipogenesis with accumulated intracellular small lipid droplets. Overall, the grafts showed well-organized adipose tissue constructs without any signs of tissue necrosis, cystic spaces, or fibrosis. We believe that human ECM powders could act as efficient injectable biomaterials for tissue engineering and have great potential for meeting clinical challenges in regenerative medicine, particularly in relation to adipose tissue engineering.
Assuntos
Adipócitos/fisiologia , Tecido Adiposo/química , Matriz Extracelular/química , Células-Tronco/fisiologia , Adulto , Animais , Adesão Celular , Proliferação de Células , Separação Celular , Portadores de Fármacos , Sistemas de Liberação de Medicamentos , Humanos , Camundongos , Microscopia Eletrônica de Varredura , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Engenharia Tecidual , Adulto JovemRESUMO
Mass mortality occurred among Penaeus vannamei shrimp cultured in Korea in 2004. In an earlier study, we reported white spot syndrome virus (WSSV) as a causative agent of mass mortality of P. monodon shrimp in Korea (Moon et al. 2003; Dis Aquat Org 53:11-13). However, in the present study, we detected Taura syndrome virus (TSV) from the moribund 2004 P. vannamei shrimp by reverse transcription polymerase chain reaction (RT-PCR). In addition, during our regular screening for the TSV in stocks of P. vannamei imported from Hawaii, USA, we also detected TSV by RT-PCR. The nucleotide sequences of the partial capsid protein VP1 of 2 Korean isolates were 99% identical to each other and 96 to 99% identical to those of TSVs isolated from the Americas, Taiwan, and Thailand. Phylogenetic analysis revealed that the 2 Korean isolates were closely related to TSV types from Thailand. This is the first report on the detection of TSV during an epizootic among cultured P. vannamei in Korea, and our results suggests the possibility that TSV has been introduced via the imported stock of P. vannamei.
Assuntos
Penaeidae/virologia , Picornaviridae/classificação , Picornaviridae/genética , Animais , Aquicultura , Primers do DNA/química , DNA Viral/química , Coreia (Geográfico) , Dados de Sequência Molecular , Filogenia , Picornaviridae/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodosRESUMO
OBJECTIVE: Zinc inhibits the growth of several carcinoma cells through induction of cell cycle arrest and apoptosis. The intracellular concentration of zinc and its dynamic changes are critically important in cell biology. We investigated the effects of zinc-citrate compound (CIZAR) on normal human ovarian epithelial cells (NOSE) and human epithelial ovarian cancer cell line, OVCAR-3. METHODS: To investigate the potential effect of CIZAR on cell growth and survival, cells were treated with different doses and exposed to different times. Intracellular concentration of zinc was measured by colorimetric assay. Mitochondrial aconitase activity was determined in cell extracts using aconitase assay. The flow cytometric assay, DNA laddering, and morphological analysis were done to investigate cytotoxic effects of CIZAR. Molecular mechanism of cell death was investigated by p53, Bcl-xL, Bcl-2, Bax protein, activity of caspase-3 and -12, and activity of telomerase. RESULTS: CIZAR-induced zinc accumulation in OVCAR-3 cells was higher than that in NOSE cells. CIZAR(R) treatment resulted in a time- and dose-dependent decrease in cell number in OVCAR-3 cells in comparison with NOSE cells. M-aconitase activity was significantly decreased in OVCAR-3 cells within 4 h exposure to CIZAR but relatively constant in NOSE cells. The flow cytometric assay, DNA laddering, and morphological analysis indicated apoptosis in OVCAR-3 cells but not in NOSE cells. CIZAR increased the expression of p21(waf1) which is a part of p53-independent pathway and induced reduction of telomerase activity. CIZAR reduced expression of Bcl-2 and Bcl-xL proteins but induced expression of Bax protein. CIZAR induced apoptosis of OVCAR-3 cells by activation of caspase-12 and caspase-3 pathway. CONCLUSIONS: Exposure to CIZAR induces apoptosis in OVCAR-3 cells which accumulate high intracellular levels of zinc, but not in NOSE cells, which do not accumulate high levels of zinc. CIZAR(R) prevents the proliferation of OVCAR-3 cells by inactivation of m-aconitase activity and induces apoptosis by induction of proapoptotic gene (Bax), repression of antiapoptotic genes (Bcl-2, Bcl-xL), and consequently activation of caspase-3. CIZAR also induced activation of caspase-12. The CIZAR will offer new window in prevention and treatment of epithelial ovarian cancer.
Assuntos
Adenocarcinoma/tratamento farmacológico , Cloretos/farmacologia , Ácido Cítrico/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Compostos de Zinco/farmacologia , Aconitato Hidratase/metabolismo , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Apoptose/efeitos dos fármacos , Western Blotting , Caspase 12 , Caspase 3 , Caspases/metabolismo , Ciclo Celular/efeitos dos fármacos , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Citometria de Fluxo , Humanos , Mitocôndrias/enzimologia , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Telomerase/metabolismo , Zinco/metabolismoRESUMO
OBJECTIVE: From the knowledge of risk factors of epithelial ovarian cancer, we deduced a hypothesis that human seminal plasma (HSP) has a preventive role in the development of epithelial ovarian cancer. To examine whether HSP directly influences the growth of ovarian cancer, we have investigated the in vitro and in vivo effect of HSP on ovarian adenocarcinoma cell lines (SK-OV-3 and OVCAR-3) in comparison with its effects on normal ovarian surface epithelial cells (NOSE). METHODS: Cell viability was determined by MTT assay. Cytotoxic effect was evaluated by flow cytometry analysis, by DNA laddering, and by morphological analysis. In vivo therapeutic effect of HSP was evaluated by the subcutaneous inoculation of SK-OV-3 cells in nude mice (BALB-c) model. RESULTS: HSP at a final concentration of 1:50 induced a time- and dose-dependent inhibition of SK-OV-3 and OVCAR-3 growth, whereas NOSE was not affected. Flow cytometric analysis, DNA laddering, and morphological analysis indicated that HSP induced necrosis, rather than apoptosis, of both ovarian carcinoma cell lines. In in vivo experiment that used the nude mice (Balb-C) with tumor inoculation of SK-OV-3 cells, HSP induced necrosis of tumor with no detectable toxic effects on the major organs. CONCLUSION: These results show that HSP inhibits the growth and induces the necrosis of epithelial ovarian cancer cells and suggests that one or more components of HSP may provide a scientific basis for preventing epithelial ovarian cancer.