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1.
Life Sci ; 234: 116779, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31430452

RESUMO

Emerging evidence has revealed that microRNAs (miRNAs) play critical roles in keloid pathogenesis. However, potential molecular mechanism of keloid formation remains unclear. In the present study, our findings showed that miR-152-3p mRNA expression level was notably up-regulated in keloid tissues and keloid fibroblasts compared with that of normal skin tissues and normal skin fibroblasts, respectively. Furthermore, miR-152-3p inhibition remarkably suppressed cell proliferation, which was increased by miR-152-3p overexpression. Cell invasion was also significantly decreased by miR-152-3p inhibition, whereas was increased by miR-152-3p overexpression. The mRNA and protein expression levels of extracellular matrix components including type I collagen, type III collagen and fibronectin were decreased by miR-152-3p inhibition, but were increased by miR-152-3p overexpression. In addition, results of dual-luciferase reporter assay indicated that FOXF1 is a direct target of miR-152-3p. FOXF1 overexpression significantly inhibits cell proliferation, invasion, and extracellular matrix in keloid fibroblasts, and the suppressive effects of miR-152-3p mimic on these functions were notably partly reversed by FOXF1 overexpression. Taken together, these findings indicated that miR-152-3p regulates cell proliferation, invasion and extracellular matrix expression through targeting FOXF1 in keloid fibroblasts, suggesting that miR-152-3p is a novel and promising molecular target for keloid treatment.


Assuntos
Matriz Extracelular/patologia , Fibroblastos/patologia , Fatores de Transcrição Forkhead/genética , Queloide/patologia , MicroRNAs/genética , Regiões 3' não Traduzidas , Adolescente , Adulto , Movimento Celular , Proliferação de Células , Células Cultivadas , Matriz Extracelular/genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Humanos , Queloide/genética , Regulação para Cima , Adulto Jovem
2.
Int J Nanomedicine ; 14: 5033-5050, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31371945

RESUMO

Background: Repairs to deep skin wounds continue to be a difficult issue in clinical practice. A promising approach is to fabricate full-thickness skin substitutes with functions closely similar to those of the natural tissue. For many years, a three-dimensional (3D) collagen hydrogel has been considered to provide a physiological 3D environment for co-cultivation of skin fibroblasts and keratinocytes. This collagen hydrogel is frequently used for fabricating tissue-engineered skin analogues with fibroblasts embedded inside the hydrogel and keratinocytes cultivated on its surface. Despite its unique biological properties, the collagen hydrogel has insufficient stiffness, with a tendency to collapse under the traction forces generated by the embedded cells. Methods: The aim of our study was to develop a two-layer skin construct consisting of a collagen hydrogel reinforced by a nanofibrous poly-L-lactide (PLLA) membrane pre-seeded with fibroblasts. The attractiveness of the membrane for dermal fibroblasts was enhanced by coating it with a thin nanofibrous fibrin mesh. Results: The fibrin mesh promoted the adhesion, proliferation and migration of the fibroblasts upwards into the collagen hydrogel. Moreover, the fibroblasts spontaneously migrating into the collagen hydrogel showed a lower tendency to contract and shrink the hydrogel by their traction forces. The surface of the collagen was seeded with human dermal keratinocytes. The keratinocytes were able to form a basal layer of highly mitotically-active cells, and a suprabasal layer. Conclusion: The two-layer skin construct based on collagen hydrogel with spontaneously immigrated fibroblasts and reinforced by a fibrin-coated nanofibrous membrane seems to be promising for the construction of full-thickness skin substitute.


Assuntos
Colágeno/farmacologia , Fibrina/farmacologia , Hidrogéis/farmacologia , Membranas Artificiais , Nanofibras/química , Poliésteres/farmacologia , Pele Artificial , Animais , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Derme/citologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Humanos , Recém-Nascido , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Ratos
3.
Cell Prolif ; 52(5): e12668, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31379046

RESUMO

OBJECTIVES: Reproducing human hair follicles in vitro is often limited by various reasons such as the lack of a systematic approach to culture distinct hair follicle cell types to reproduce their spatial relationship. Here, we reproduce hair follicle-like constructs resembling the spatial orientation of different cells in vivo, to study the role of keratinocytes in maintaining cellular compartmentalization among hair follicle-related cells. MATERIALS AND METHODS: Dermal papilla (DP) cells, HaCaT keratinocytes and human dermal fibroblast (HDF) cells were seeded sequentially into three-dimensional (3D) microwells fabricated from polyethylene glycol diacrylate hydrogels. Quantitative polymerase chain reaction was used to compare inductive gene expression of 3D and two-dimensional (2D) DP. DP and HaCaT cells were transfected with green fluorescent protein and red fluorescent protein lentivirus, respectively, to enable cell visualization using confocal microscopy. RESULTS: The 3D DP cultures showed significantly enhanced expression of essential DP genes as compared 2D cultures. Core-shell configurations containing keratinocytes forming the outer shell and DP forming the core were observed. Migratory polarization was mediated by cell-cell interaction between the keratinocytes and HDF cells, while preserving the aggregated state of the DP cells. CONCLUSIONS: Keratinocytes may play a role in maintaining compartmentalization between the DP and the surrounding HDF residing in the dermis, and therefore maintains the aggregative state of the DP cells, necessary for hair follicle development and function.


Assuntos
Técnicas de Cultura de Células/métodos , Derme/citologia , Fibroblastos/citologia , Queratinócitos/citologia , Células Cultivadas , Derme/metabolismo , Fibroblastos/metabolismo , Humanos , Hidrogéis/química , Queratinócitos/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Confocal
4.
Adv Exp Med Biol ; 1165: 305-322, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31399971

RESUMO

Renal fibrosis is a major pathological feature of chronic kidney disease, which is characterized by massive fibroblast activation and excessive production and deposition of extracellular matrix (ECM). Renal fibrosis results in progressive loss of kidney function; however, there is currently no effective therapy available clinically to treat or even reverse renal fibrosis. Although activated fibroblasts/myofibroblasts are responsible for the production and deposition of ECM, their origin has been debatable. Recent studies have provided compelling evidence that bone marrow-derived fibroblast precursors contribute significantly to the population of myofibroblasts and the development of renal fibrosis. Therefore, targeting the molecular signaling mechanisms underlying the recruitment and activation of the bone marrow-derived fibroblast precursors may serve as novel therapeutic strategy for chronic kidney disease. In this review, we appraise recent advances in our understanding of the recruitment and activation of bone marrow-derived fibroblast precursors in the kidney and the development of renal fibrosis and highlight novel molecular signaling pathways that may lead to the development of new therapies for chronic kidney disease.


Assuntos
Medula Óssea , Fibroblastos/citologia , Rim/patologia , Matriz Extracelular , Fibrose , Humanos , Miofibroblastos/citologia , Transdução de Sinais
5.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 35(6): 545-551, 2019 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-31292059

RESUMO

Objective To illuminate whether IL-17 regulates receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) expression in human periodontal ligament fibroblasts (HPDLFs) via p38MAPK signaling pathway. Methods HPDLFs were incubated in the presence of 20 ng/mL IL-17 for 0, 20, 40, 60 and 80 minutes. HPDLFs were divided randomly into 6 groups: control group, dimethyl sulfoxide (DMSO) group, p38MAPK pathway inhibitor SB203580 group, IL-17 group, IL-17 combined with DMSO group and IL-17 combined with SB203580 group. SB203580 (10 µmol/L) and IL-17 (20 ng/mL) were added to the corresponding groups. Real-time quantitative PCR was used to detect the expression of RANKL and OPG mRNAs in HPDLFs. The levels of phospho-p38MAPK (p-p38MAPK) and RANKL protein were measured using Western blot analysis. The protein level of OPG was detected by ELISA. Results After IL-17 stimulation, the expression level of p-p38MAPK protein gradually increased starting from 0 minute and reached its highest level at 60 minutes. It started to decline at 80 minutes. Stimulation with IL-17 could increase the mRNA and protein expression level of RANKL but decrease the mRNA and protein expression level of OPG. Nevertheless, unlike the IL-17 group, IL-17 combined with inhibitor SB203580 decreased the expression of RANKL mRNA and protein and increased OPG mRNA. Conclusion IL-17 can enhance the expression of RANKL in human periodontal fibroblasts and inhibit the expression of OPG mRNA through the p38MAPK signal transduction pathways.


Assuntos
Fibroblastos/citologia , Interleucina-17/farmacologia , Sistema de Sinalização das MAP Quinases , Osteoprotegerina/metabolismo , Ligamento Periodontal/citologia , Ligante RANK/metabolismo , Células Cultivadas , Dimetil Sulfóxido/farmacologia , Humanos , Imidazóis/farmacologia , Piridinas/farmacologia , Distribuição Aleatória , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
6.
J Photochem Photobiol B ; 197: 111539, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31301638

RESUMO

Treatment of burn injury is clinically challenging one, therefore several steps and noteworthy approaches have been taken to improve wound mechanisms. Citrus pectin plays a stabilizing agent to synthesis of ZnO nanoparticles (ZnO NPs). The present study is focused on ZnO loaded collagen/chitosan nanofibrous were synthesized by electrospinning method using ZnO NPs. The chemical structure, phase purity and morphological observation were investigated under spectroscopic and mircoscopic techniques and demonstrated their suitable properties as a wound healing material. In addition, that prepared nanoparticles loaded biopolymeric fibrous nanomaterial showed suitable antibacterial activity against S. aureus and E. coli bacterial pathogens and also in vitro studies was confirmed the enhanced proliferation, cell viability and biocompatibility. In vitro evaluations have been exhibited acceptable cell proliferation is observed throughout the ZnO loaded Coll/CS nanofibrous within 3 days, which was comparable to the control material. In vivo wound healing ability was monitored on the rat wound experimental model. From the in vivo observations, revealed that the loaded of ZnO NPs with Coll/CS nanofibrous can effectively quicken wound healing mechanism, expressed in the initial stage healing process. These results suggest that ZnO loaded collagen/chitosan nanofibrous is a potential candidate for wound healing applications with enhanced biological properties.


Assuntos
Queimaduras/patologia , Quitosana/química , Colágeno/química , Nanopartículas Metálicas/química , Nanofibras/química , Animais , Antibacterianos/química , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Bacteriemia/prevenção & controle , Queimaduras/veterinária , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Nanofibras/uso terapêutico , Nanofibras/toxicidade , Ratos , Pele/efeitos dos fármacos , Pele/patologia , Staphylococcus aureus/efeitos dos fármacos , Cicatrização/efeitos dos fármacos , Óxido de Zinco/química
7.
Aquat Toxicol ; 213: 105229, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31255889

RESUMO

Although the global use of the 1,1,1-trichloro-2,2-bis (4-chlorophenyl) ethane (p,p'-DDT) has been prohibited, its persistence in the environment has caused long-lasting exposure on marine mammals. Our previous studies revealed exceedingly high residue levels of DDTs in Indo-Pacific humpback dolphins (Sousa chinensis) from the Pearl River Estuary region, China. However, the molecular mechanisms of p,p'-DDT toxicity on the dolphin are largely unknown. This study conducted the first cytotoxicity effect exploration of p,p'-DDT on the dolphin skin fibroblasts (ScSFs) to enhance the understanding of the cellular and molecular regulation impacts. ScSF cells were exposed to p,p'-DDT (28∼168 µM) for 24, 48 and 72 h. The exposure remarkably decreased viability of ScSF cells, possibly due to the synergetic effects of cell cycle arrest and apoptosis via DNA damage and mitochondria dysfunction. The DNA damage and mitochondria dysfunction were likely triggered by an increase of cellular reactive oxygen species (ROS), alteration in mitochondrial membrane potential, reduction in the cellular ATP levels, decreased expression of the genes CDK1, CDK4, cyclin B1, cyclin D1 and apoptosis regulator Bcl-2, release of cytochrome c, and activation of caspase-3, caspase-8 and caspase-9. Moreover, caspase inhibitor displayed protective activity against p,p'-DDT-induced apoptosis, indicating that caspases played a central role in p,p'-DDT-triggered apoptosis in the ScSF cells. We hypothesize apoptosis likely plays a minor role in cytocidal effects induced by p,p'-DDT exposure, but the mechanisms remain unclear. Overall, this research provides new evidence of the cytotoxic mechanisms underlying p,p'-DDT exposure on humpback dolphin skin cells, and suggests that p,p'-DDT contamination is one of key health concern issues for the protection of this marine mammal.


Assuntos
Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , DDT/toxicidade , Golfinhos/metabolismo , Exposição Ambiental , Fibroblastos/citologia , Mitocôndrias/metabolismo , Pele/citologia , Animais , Caspases/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Poluentes Químicos da Água/toxicidade
8.
Soft Matter ; 15(27): 5455-5463, 2019 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-31231747

RESUMO

Development of fast force volume (FFV), PeakForce Tapping (PFT), and related AFM techniques allow fast acquisition and mapping of a sample's mechanical properties. The methods are well-suited for studying soft biological samples like living cells in a liquid environment. However, the question remains how the measured mechanical properties are related to those acquired with the classical force volume (FV) technique conducted at low indentation rates. The difference is coming mostly from the pronounced viscoelastic behavior of cells, making apparent elastic parameters depending on the probing rate. Here, the viscoelastic analysis was applied directly to the force curves acquired with force volume or PeakForce Tapping by their post-processing based on the Ting's model. Maps from classical force volume, FFV and PFT obtained using special PFT cantilevers and cantilevers modified with microspheres were compared here. With the correct viscoelastic model, which was found to be the power-law rheology model, all the techniques have provided self-consistent results. The techniques were further modified for the mapping of the viscoelastic model-independent complex Young's modulus.


Assuntos
Fibroblastos/citologia , Animais , Linhagem Celular , Simulação por Computador , Módulo de Elasticidade , Camundongos , Microscopia de Força Atômica , Ratos , Reologia , Propriedades de Superfície , Viscosidade
9.
Histochem Cell Biol ; 152(2): 133-143, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31154480

RESUMO

Actin fulfills important cytoplasmic but also nuclear functions in eukaryotic cells. In the nucleus, actin modulates gene expression and chromatin remodeling. Monomeric (G-actin) and polymerized actin (F-actin) have been analyzed by fluorescence microscopy in the nucleus; however, the resolution at the ultrastructural level has not been investigated in great detail. We provide a first documentation of nuclear actin in mouse fibroblasts by electron microscopy (EM). For this, we employed correlative light and electron microscopy on the same section using actin-directed nanobodies recognizing endogenous monomeric and polymeric actin proteins (so-called nuclear Actin-chromobody-GFP; nAC-GFP). Indeed, using this strategy, we could identify actin proteins present in the nucleus. Here, immunogold-labeled actin proteins were spread throughout the entire nucleoplasm. Of note, nuclear actin was complementarily localized to DAPI-positive areas, the latter marking preferentially transcriptionally inactive heterochromatin. Since actin aggregates in rod structures upon cell stress including neurodegeneration, we analyzed nuclear actin at the ultrastructural level after DMSO or UV-mediated cell damage. In those cells the ratio between cytoplasmic and nuclear gold-labeled actin proteins was altered compared to untreated control cells. In summary, this EM analysis (i) confirmed the presence of endogenous nuclear actin at ultrastructural resolution, (ii) revealed the actin abundance in less chromatin-dense regions potentially reflecting more transcriptionally active euchromatin rather than transcriptionally inactive heterochromatin and (iii) showed an altered abundance of actin-associated gold particles upon cell stress.


Assuntos
Actinas/análise , Núcleo Celular/química , Microscopia Eletrônica/métodos , Microscopia de Fluorescência/métodos , Actinas/metabolismo , Animais , Núcleo Celular/metabolismo , Células Cultivadas , Fibroblastos/química , Fibroblastos/citologia , Camundongos , Células NIH 3T3 , Tamanho da Partícula , Conformação Proteica
10.
Cell Mol Life Sci ; 76(20): 3953-3967, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31250034

RESUMO

The brain tissue has only a limited capacity for generating new neurons. Therefore, to treat neurological diseases, there is a need of other cell sources for brain repair. Different sources of cells have been subject of intense research over the years, including cells from primary tissue, stem cell-derived cells and reprogrammed cells. As an alternative, direct reprogramming of resident brain cells into neurons is a recent approach that could provide an attractive method for treating brain injuries or diseases as it uses the patient's own cells for generating novel neurons inside the brain. In vivo reprogramming is still in its early stages but holds great promise as an option for cell therapy. To date, both inhibitory and excitatory neurons have been obtained via in vivo reprogramming, but the precise phenotype or functionality of these cells has not been analysed in detail in most of the studies. Recent data shows that in vivo reprogrammed neurons are able to functionally mature and integrate into the existing brain circuitry, and compose interneuron phenotypes that seem to correlate to their endogenous counterparts. Interneurons are of particular importance as they are essential in physiological brain function and when disturbed lead to several neurological disorders. In this review, we describe a comprehensive overview of the existing studies involving brain repair, including in vivo reprogramming, with a focus on interneurons, along with an overview on current efforts to generate interneurons for cell therapy for a number of neurological diseases.


Assuntos
Lesões Encefálicas/terapia , Terapia Baseada em Transplante de Células e Tecidos/métodos , Células-Tronco Pluripotentes Induzidas/citologia , Interneurônios/citologia , Doenças Neurodegenerativas/terapia , Regeneração/fisiologia , Animais , Biomarcadores/metabolismo , Encéfalo/citologia , Encéfalo/metabolismo , Lesões Encefálicas/genética , Lesões Encefálicas/metabolismo , Lesões Encefálicas/patologia , Transdiferenciação Celular , Reprogramação Celular , Fibroblastos/citologia , Fibroblastos/metabolismo , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Injeções Intraventriculares , Interneurônios/metabolismo , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Neurogênese/genética , Transplante de Células-Tronco/métodos
11.
Bioelectrochemistry ; 129: 106-115, 2019 Oct.
Artigo em Espanhol | MEDLINE | ID: mdl-31153125

RESUMO

Due to their desirable elastic modulus and density that are similar to natural bone, non-toxic element containing magnesium alloys are regarded as promising bio-degradable materials. A biodegradable HA-particle-reinforced magnesium-matrix composite Mg-3Zn-0.2Ca-1HA (wt%) was fabricated for biomedical application by a combination of high shear solidification (HSS) and hot extrusion technology. The microstructure, mechanical properties, corrosion resistance and cell biocompatibility of the composite were subsequently investigated. In comparison with the matrix alloy, the as-cast Mg-3Zn-0.2Ca-1HA composite obtained by HSS technology exhibited a uniform and fine grained structure, further refined after a hot extrusion ratio of 36:1. The yield strength (0.2%YS), ultimate tensile strength and elongation of the extruded composite were 322 MPa, 341 MPa and 7.6%, respectively. The corrosion rate of the as-extruded Mg-3Zn-0.2Ca-1HA composite was measured to be 1.52 mm/y. Electrochemical and immersion tests showed that the corrosion resistance of the composite is slightly improved comparing to that of the matrix alloy.


Assuntos
Ligas/química , Materiais Biocompatíveis/química , Durapatita/química , Magnésio/química , Zinco/química , Animais , Linhagem Celular , Corrosão , Fibroblastos/citologia , Teste de Materiais , Camundongos , Resistência à Tração
12.
Mol Med Rep ; 19(6): 5039-5045, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31059063

RESUMO

Wound healing is a dynamic process that involves highly coordinated cellular events, including proliferation and migration. Oral gingival fibroblasts serve a central role in maintaining oral mucosa homeostasis, and their functions include the coordination of physiological tissue repair. Recently, surface pre­reacted glass­ionomer (S­PRG) fillers have been widely applied in the field of dental materials for the prevention of dental caries, due to an excellent ability to release fluoride (F). In addition to F, S­PRG fillers are known to release several types of ions, including aluminum (Al), boron (B), sodium (Na), silicon (Si) and strontium (Sr). However, the influence of these ions on gingival fibroblasts remains unknown. The aim of the present study was to examine the effect of various concentrations of an S­PRG filler eluate on the growth and migration of gingival fibroblasts. The human gingival fibroblast cell line HGF­1 was treated with various dilutions of an eluent solution of S­PRG, which contained 32.0 ppm Al, 1,488.6 ppm B, 505.0 ppm Na, 12.9 ppm Si, 156.5 ppm Sr and 136.5 ppm F. Treatment with eluate at a dilution of 1:10,000 was observed to significantly promote the migration of HGF­1 cells. In addition, the current study evaluated the mechanism underlying the mediated cell migration by the S­PRG solution and revealed that it activated the phosphorylation of extracellular signal­regulated kinase 1/2 (ERK1/2), but not of p38. Furthermore, treatment with a MEK inhibitor blocked the cell migration induced by the solution. Taken together, these results suggest that S­PRG fillers can stimulate HGF­1 cell migration via the ERK1/2 signaling pathway, indicating that a dental material containing this type of filler is useful for oral mucosa homeostasis and wound healing.


Assuntos
Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Alumínio/química , Boro/química , Linhagem Celular , Movimento Celular , Proliferação de Células/efeitos dos fármacos , Fibroblastos/citologia , Fibroblastos/metabolismo , Gengiva/citologia , Humanos , Íons/química , Íons/farmacologia , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Silício/química , Sódio/química , Estrôncio/química , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
13.
Genes Cells ; 24(7): 473-484, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31099158

RESUMO

Induced pluripotent stem (iPS) cells hold great promise for regenerative medicine and the treatment of various diseases. Before proceeding to clinical trials, it is important to test the efficacy and safety of iPS cell-based treatments using experimental animals. The common marmoset is a new world monkey widely used in biomedical studies. However, efficient methods that could generate iPS cells from a variety of cells have not been established. Here, we report that marmoset cells are efficiently reprogrammed into iPS cells by combining RNA transfection and chemical compounds. Using this novel combination, we generate transgene integration-free marmoset iPS cells from a variety of cells that are difficult to reprogram using conventional RNA transfection method. Furthermore, we show this is similarly effective for human and cynomolgus monkey iPS cell generation. Thus, the addition of chemical compounds during RNA transfection greatly facilitates reprogramming and efficient generation of completely integration-free safe iPS cells in primates, particularly from difficult-to-reprogram cells.


Assuntos
Reprogramação Celular , Fibroblastos/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Preparações Farmacêuticas/administração & dosagem , RNA/administração & dosagem , Transfecção/métodos , Idoso , Animais , Diferenciação Celular , Células Cultivadas , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/fisiologia , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/fisiologia , Platirrinos
14.
Nanomedicine (Lond) ; 14(10): 1267-1289, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31124760

RESUMO

Aim: Magnetic hyperthermia is limited by the low selective susceptibility of neoplastic cells interspersed within healthy tissues, which we aim to improve on. Materials & methods: Two superparamagnetic calcium phosphates nanocomposites, that is, iron-doped hydroxyapatite and iron oxide (Mag) nanoparticles coated with amorphous calcium phosphate (Mag@CaP), were synthesized and tested for selective activity against brain and bone cancers. Results: Nanoparticle uptake and intracellular localization were prerequisites for reduction of cancer viability in alternate magnetic fields of extremely low power. Sheer adsorption onto the outer membrane was not sufficient to produce this effect, which was extremely significant for Mag@CaP and iron-doped hydroxyapatite, but negligible for Mag, demonstrating benefits of combining magnetic iron with calcium phosphates. Conclusion: Such selective effects are important in the global effort to rejuvenate clinical prospects of magnetic hyperthermia.


Assuntos
Neoplasias Ósseas/terapia , Neoplasias Encefálicas/terapia , Fosfatos de Cálcio/química , Nanocompostos/química , Animais , Antibacterianos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular , Durapatita/química , Fibroblastos/citologia , Humanos , Hipertermia Induzida , Ferro/química , Campos Magnéticos , Nanopartículas de Magnetita/química
15.
Mol Med Rep ; 19(6): 5203-5210, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31059039

RESUMO

The aim of the current study was to investigate the expression and role of microRNA­486­5p (miR­486­5p) in hypertrophic scar (HS) formation, and to examine the associated mechanisms. First, miR­486­5p expression was detected in HS tissues and human hypertrophic scar fibroblasts (hHSFs) by reverse transcription­quantitative polymerase chain reaction. Target genes of miR­486­5p were predicted using TargetScan and verified by dual­luciferase reporter assays. To investigate the role of miR­486­5p in HS formation, miR­486­5p was overexpressed in hHSFs through transfection with miR­486­5p mimics. MTT, cell apoptosis and cell cycle assays were preformed to investigate the proliferation, cell apoptosis and cell cycle distribution of hHSFs, respectively. Additionally, protein expression was measured by western blot analysis. The results demonstrated that miR­486­5p expression was significantly decreased in HS tissues and cells. Mothers against decapentaplegic homolog (Smad)2 was a target gene of miR­486­5p, and it was negatively regulated by miR­486­5p. It was also found that Smad2 expression was significantly increased in HS tissues and cells. Further analysis indicated that miR­486­5p mimic transfection inhibited the proliferation, induced cell apoptosis and increased G1/S phase arrest in hHSFs. Furthermore, the expression of cyclin­dependent kinase (CDK)2, CDK4 and apoptosis regulator Bcl­2 was repressed, while apoptosis regulator BAX expression was enhanced by miR­486­5p mimic transfection. Notably, the effects of miR­486­5p mimic on hHSFs were significantly eliminated by Smad2 plasmid transfection. Taken together, these results demonstrated that miR­486­5p inhibited the proliferation, induced apoptosis and increased G1/S phase arrest of hHSFs by targeting Smad2. miR­486­5p may be a promising therapeutic target for HS management.


Assuntos
Cicatriz Hipertrófica/patologia , MicroRNAs/metabolismo , Proteína Smad2/metabolismo , Regiões 3' não Traduzidas , Adulto , Antagomirs/metabolismo , Apoptose , Proliferação de Células , Cicatriz Hipertrófica/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Quinase 4 Dependente de Ciclina/metabolismo , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular , Humanos , Masculino , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína Smad2/química , Proteína Smad2/genética , Proteína X Associada a bcl-2/metabolismo
16.
Mol Med Rep ; 19(6): 5251-5262, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31059100

RESUMO

Keloids are benign fibrous overgrowths that occur as a result of abnormal wound healing following cutaneous injury. MicroRNAs (miRNAs/miRs) are short non­coding RNAs that serve critical roles in numerous important biological processes, such as cell proliferation, differentiation and apoptosis. However, their role in keloid development remains largely unknown. In the present study, the role of miR­30a­5p, a miRNA regulated by Trichostatin A (TSA), in apoptosis within cultured keloid fibroblasts was investigated. An MTT assay was used to detect the proliferation of cultured keloid fibroblasts treated with TSA. Cell apoptosis and cell cycle phases were analyzed using flow cytometry. In addition, an miRNA microarray was performed to compare expression profiles between cultured keloid fibroblasts treated with or without 1,000 nM TSA. Reverse transcription­quantitative polymerase chain reaction analysis was conducted to estimate miRNA expression levels. The direct target of miR­30a­5p was identified using a dual­luciferase reporter assay. Western blotting was employed to assess protein expression levels in keloid fibroblasts. The results demonstrated that TSA inhibited the proliferation of keloid fibroblasts in a time­ and dose­dependent manner. The miRNA microarray revealed alterations in the expression of numerous miRNA sequences in response to TSA when compared with controls. Notably, the expression of miR­30a­5p was downregulated in keloid tissues. In addition, overexpression of miR­30a­5p induced apoptosis by targeting B­cell lymphoma 2, which was similar to that observed in response to TSA. These results provide important information regarding a novel miR­30a­5p­mediated signaling pathway induced by TSA treatment, and suggest a potential use for TSA and miR­30a­5p as effective therapeutic strategies for keloids.


Assuntos
Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , MicroRNAs/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Regiões 3' não Traduzidas , Antagomirs/metabolismo , Análise por Conglomerados , Colágeno/metabolismo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Humanos , Queloide/metabolismo , Queloide/patologia , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/genética , Pele/metabolismo , Pele/patologia
17.
Int J Mol Sci ; 20(9)2019 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-31067675

RESUMO

BACKGROUND: We have characterized a new reconstructed full-thickness skin model, T-Skin™, compared to normal human skin (NHS) and evaluated its use in testing anti-aging compounds. METHODS: The structure and layer-specific markers were compared with NHS using histological and immunohistological staining. In anti-aging experiments, T-SkinTM was exposed to retinol (10 µM) or vitamin C (200 µM) for 5 days, followed by immunohistological staining evaluation. RESULTS: T-Skin™ exhibits a well stratified, differentiated and self-renewing epidermis with a dermal compartment of functional fibroblasts. Epidermal (cytokeratin 10, transglutaminase 1), dermo-epidermal junction (DEJ) (laminin 5, collagen-IV, collagen VII) and dermally-located (fibrillin 1, procollagen I) biomarkers were similar to those in NHS. Treatment of T-Skin™ with retinol decreased the expression of differentiation markers, cytokeratin 10 and transglutaminase 1 and increased the proliferation marker, Ki67, in epidermis basal-layer cells. Vitamin C increased the expression of DEJ components, collagen IV and VII and dermal procollagen 1. CONCLUSIONS: T-Skin™ exhibits structural and biomarker location characteristics similar to NHS. Responses of T-Skin™ to retinol and vitamin C treatment were consistent with those of their known anti-aging effects. T-Skin™ is a promising model to investigate responses of epidermal, DEJ and dermal regions to new skin anti-ageing compounds.


Assuntos
Ácido Ascórbico/farmacologia , Envelhecimento da Pele , Pele/efeitos dos fármacos , Vitamina A/farmacologia , Vitaminas/farmacologia , Moléculas de Adesão Celular/metabolismo , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Colágeno/metabolismo , Fibrilina-1/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Humanos , Queratina-10/metabolismo , Queratinócitos/efeitos dos fármacos , Pele/citologia
18.
BMC Cancer ; 19(1): 402, 2019 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-31035967

RESUMO

BACKGROUND: Different 3D-cell culture approaches with varying degrees of complexity have been developed to serve as melanoma models for drug testing or mechanistic studies. While these 3D-culture initiatives are already often superior to classical 2D approaches, they are either composed of only melanoma cells or they are so complex that the behavior of individual cell types is hard to understand, and often they are difficult to establish and expensive. METHODS: This study used low-attachment based generation of spheroids composed of up to three cell types. Characterization of cells and spheroids involved cryosectioning, immunofluorescence, FACS, and quantitative analyses. Statistical evaluation used one-way ANOVA with post-hoc Tukey test or Student's t-test. RESULTS: The tri-culture model allowed to track cellular behavior in a cell-type specific manner and recapitulated different characteristics of early melanoma stages. Cells arranged into a collagen-IV rich fibroblast core, a ring of keratinocytes, and groups of highly proliferating melanoma cells on the outside. Regularly, some melanoma cells were also found to invade the fibroblast core. In the absence of melanoma cells, the keratinocyte ring stratified into central basal-like and peripheral, more differentiated cells. Conversely, keratinocyte differentiation was clearly reduced upon addition of melanoma cells. Treatment with the cytostatic drug, docetaxel, restored keratinocyte differentiation and induced apoptosis of external melanoma cells. Remaining intact external melanoma cells showed a significantly increased amount of ABCB5-immunoreactivity. CONCLUSIONS: In the present work, a novel, simple spheroid-based melanoma tri-culture model composed of fibroblasts, keratinocytes, and melanoma cells was described. This model mimicked features observed in early melanoma stages, including loss of keratinocyte differentiation, melanoma cell invasion, and drug-induced increase of ABCB5 expression in external melanoma cells.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Técnicas de Cultura de Células/métodos , Técnicas de Cocultura/métodos , Esferoides Celulares/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Docetaxel/farmacologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Queratinócitos/citologia , Queratinócitos/metabolismo , Melanoma/metabolismo , Melanoma/patologia , Esferoides Celulares/citologia
19.
Artif Cells Nanomed Biotechnol ; 47(1): 1693-1701, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31062610

RESUMO

Adipose tissue has the therapeutic capacity in the form of a fat graft, for example, for treatment of irradiation-induced scars and difficult to heal dermal wounds. For large-scale clinical application, an off-the-shelf product is warranted. In recent years, ECM-derived hydrogels are postulated to harbour therapeutic capacity and might even replicate the beneficial effects of adipose tissue. In normal homeostasis, the natural ECM acts as a deposit of growth factors, that releases them over time. In the healing of lesions, this might promote cell accumulation and proliferation which in turn stimulates angiogenesis and repair. The decellularization of tissue and the generation of hydrogels may leave cytotoxic traces. Therefore, our research assessed the cytotoxic effect of human adipose tissue-derived ECM hydrogels on connective tissue cells i.e. fibroblasts. The results showed no cytotoxicity, meaning the hydrogels caused no cell death. Cell migration and survival were observed when cultured in ECM hydrogels and followed for 7 days. Cell survival in the hydrogel was confirmed with CFDA staining and also cells showed the ability to penetrate and migrate throughout the gel. We conclude that ECM hydrogels are promising to use as innovative therapy for wound healing.


Assuntos
Tecido Adiposo/citologia , Materiais Biocompatíveis/farmacologia , Matriz Extracelular/metabolismo , Hidrogéis/farmacologia , Tecidos Suporte/química , Materiais Biocompatíveis/metabolismo , Diferenciação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Fibroblastos/citologia , Humanos , Hidrogéis/metabolismo , Miócitos de Músculo Liso/citologia , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos
20.
Nat Commun ; 10(1): 2129, 2019 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-31086189

RESUMO

De novo heterozygous missense variants in the γ-tubulin gene TUBG1 have been linked to human malformations of cortical development associated with intellectual disability and epilepsy. Here, we investigated through in-utero electroporation and in-vivo studies, how four of these variants affect cortical development. We show that TUBG1 mutants affect neuronal positioning, disrupting the locomotion of new-born neurons but without affecting progenitors' proliferation. We further demonstrate that pathogenic TUBG1 variants are linked to reduced microtubule dynamics but without major structural nor functional centrosome defects in subject-derived fibroblasts. Additionally, we developed a knock-in Tubg1Y92C/+ mouse model and assessed consequences of the mutation. Although centrosomal positioning in bipolar neurons is correct, they fail to initiate locomotion. Furthermore, Tubg1Y92C/+ animals show neuroanatomical and behavioral defects and increased epileptic cortical activity. We show that Tubg1Y92C/+ mice partially mimic the human phenotype and therefore represent a relevant model for further investigations of the physiopathology of cortical malformations.


Assuntos
Malformações do Desenvolvimento Cortical/genética , Microtúbulos/metabolismo , Neurogênese/genética , Neurônios/fisiologia , Tubulina (Proteína)/genética , Animais , Comportamento Animal , Movimento Celular/genética , Centrossomo/metabolismo , Córtex Cerebral/anormalidades , Córtex Cerebral/citologia , Córtex Cerebral/diagnóstico por imagem , Modelos Animais de Doenças , Embrião de Mamíferos , Epilepsia/genética , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Técnicas de Introdução de Genes , Predisposição Genética para Doença , Células HeLa , Humanos , Microscopia Intravital , Masculino , Camundongos , Camundongos Transgênicos , Microscopia Confocal , Microscopia Eletrônica , Microtúbulos/genética , Mutação de Sentido Incorreto
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