Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 1.837
Filtrar
1.
Int J Mol Sci ; 22(4)2021 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-33670029

RESUMO

Hair follicle morphogenesis is heavily dependent on reciprocal, sequential, and epithelial-mesenchymal interaction (EMI) between epidermal stem cells and the specialized cells of the underlying mesenchyme, which aggregate to form the dermal condensate (DC) and will later become the dermal papilla (DP). Similar models were developed with a co-culture of keratinocytes and DP cells. Previous studies have demonstrated that co-culture with keratinocytes maintains the in vivo characteristics of the DP. However, it is often challenging to develop three-dimensional (3D) DP and keratinocyte co-culture models for long term in vitro studies, due to the poor intercellular adherence between keratinocytes. Keratinocytes exhibit exfoliative behavior, and the integrity of the DP and keratinocyte co-cultured spheroids cannot be maintained over prolonged culture. Short durations of culture are unable to sufficiently allow the differentiation and re-programming of the keratinocytes into hair follicular fate by the DP. In this study, we explored a microgel array approach fabricated with two different hydrogel systems. Using poly (ethylene glycol) diacrylate (PEGDA) and gelatin methacrylate (GelMA), we compare their effects on maintaining the integrity of the cultures and their expression of important genes responsible for hair follicle morphogenesis, namely Wnt10A, Wnt10B, and Shh, over prolonged duration. We discovered that low attachment surfaces such as PEGDA result in the exfoliation of keratinocytes and were not suitable for long-term culture. GelMA, on the hand, was able to sustain the integrity of co-cultures and showed higher expression of the morphogens overtime.


Assuntos
Derme/citologia , Queratinócitos/citologia , Microgéis/química , Polietilenoglicóis/farmacologia , Adesão Celular/efeitos dos fármacos , Agregação Celular/efeitos dos fármacos , Linhagem Celular , Técnicas de Cocultura , Proteínas de Fluorescência Verde/metabolismo , /efeitos dos fármacos , Humanos , Hidrogéis/farmacologia , Proteínas Luminescentes/metabolismo , Esferoides Celulares/citologia , Esferoides Celulares/efeitos dos fármacos , Proteínas Wnt/metabolismo
2.
Methods Mol Biol ; 2269: 175-201, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33687680

RESUMO

Bench-to-bedside axis of therapeutic product development is currently being oriented towards minimum invasiveness on both ends-not only clinical application but harvesting of the starting biological material as well. This is particularly relevant for Advanced Therapy Medicinal Products and their specific legislative requirements, even more so in skin regeneration. It is precisely the skin equivalents and grafts that benefit from the minimum-to-noninvasive approach to a noteworthy extent, taking in account the sensitive nature of both skin harvesting and grafting.This chapter includes protocols for two separate steps of generating skin equivalent from the cells cultured from hair follicle outer root sheath. The first step is a non-pigmented epidermal equivalent generated from human keratinocytes from the outer root sheath named non-pigmented epidermal graft. The second step consists of co-cultivating human keratinocytes and human melanocytes from the outer root sheath, hereby producing a pigmented epidermal graft.


Assuntos
Derme/metabolismo , Fibroblastos/metabolismo , Folículo Piloso/metabolismo , Queratinócitos/metabolismo , Melanócitos/metabolismo , Engenharia Tecidual , Técnicas de Cocultura , Derme/citologia , Fibroblastos/citologia , Folículo Piloso/citologia , Humanos , Queratinócitos/citologia , Melanócitos/citologia
3.
Int J Mol Sci ; 22(4)2021 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-33669634

RESUMO

Little is known about the effects on hyaluronan (HA) metabolism of UVA radiation. This study demonstrates that the secretion of HA by human dermal fibroblasts (HDFs) is downregulated by UVA, accompanied by the down- and upregulation of mRNA and protein levels of the HA-synthesizing enzyme (HAS2) and the HA-degrading protein, HYaluronan Binding protein Involved in HA Depolymerization(HYBID), respectively. Signaling analysis revealed that the exposure distinctly elicits activation of the p38/MSK1/CREB/c-Fos/AP-1 axis, the JNK/c-Jun axis, and the p38/ATF-2 axis, but downregulates the phosphorylation of NF-kB and JAK/STAT3. A signal inhibition study demonstrated that the inhibition of p38 significantly abrogates the UVA-accentuated mRNA level of HYBID. Furthermore, the inhibition of STAT3 significantly downregulates the level of HAS2 mRNA in non-UVA exposed HDFs. Analysis using siRNAs demonstrated that transfection of ATF-2 siRNA but not c-Fos siRNA abrogates the increased protein level of HYBID in UVA-exposed HDFs. An inhibitor of protein tyrosine phosphatase but not of protein serine/threonine phosphatase restored the diminished phosphorylation level of STAT3 at Tyr 705, accompanied by a significant abolishing effect on the decreased mRNA expression level of HAS2. Silencing with a protein tyrosine phosphatase PTP-Meg2 siRNA revealed that it abrogates the decreased phosphorylation of STAT3 at Tyr 705 in UVA-exposed HDFs. These findings suggest that the UVA-induced decrease in HA secretion by HDFs is attributable to the down- and upregulation of HAS2 and HYBID expression, respectively, changes that are mainly ascribed to the inactivated signaling of the STAT3 axis due to the activated tyrosine protein phosphatase PTP-Meg2 and the activated signaling of the p38/ATF2 axis, respectively.


Assuntos
Regulação para Baixo/efeitos da radiação , Fibroblastos/efeitos da radiação , Hialuronan Sintases/metabolismo , Ácido Hialurônico/metabolismo , Hialuronoglucosaminidase/metabolismo , Transdução de Sinais/efeitos da radiação , Raios Ultravioleta , Regulação para Cima/efeitos da radiação , Fator 2 Ativador da Transcrição/metabolismo , Morte Celular/efeitos dos fármacos , Morte Celular/efeitos da radiação , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Derme/citologia , Regulação para Baixo/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Janus Quinase 2/metabolismo , Masculino , Modelos Biológicos , Peso Molecular , Fosforilação/efeitos dos fármacos , Fosforilação/efeitos da radiação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Transcrição STAT3/metabolismo , Regulação para Cima/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
4.
J Vis Exp ; (169)2021 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-33749670

RESUMO

The genome is associated with several structures inside cell nuclei, in order to regulate its activity and anchor it in specific locations. These structures are collectively known as the nucleoskeleton and include the nuclear lamina, the nucleoli, and nuclear bodies. Although many variants of fluorescence in situ hybridization (FISH) exist to study the genome and its organization, these are often limited by resolution and provide insufficient information on the genome's association with nuclear structures. The DNA halo method uses high salt concentrations and nonionic detergents to generate DNA loops that remain anchored to structures within nuclei through attachment regions within the genome. Here, soluble nuclear proteins, such as histones, lipids, and DNA not tightly bound to the nuclear matrix, are extracted. This leads to the formation of a halo of unattached DNA surrounding a residual nucleus which itself contains DNA closely associated with internal nuclear structures and extraction-resistant proteins. These extended DNA strands enable increased resolution and can facilitate physical mapping. In combination with FISH, this method has the added advantage of studying genomic interactions with all the structures that the genome is anchored by. This technique, termed HALO-FISH, is highly versatile whereby DNA halos can be coupled with nucleic acid probes to reveal gene loci, whole chromosomes, alpha satellite, telomeres and even RNA. This technique provides an insight into nuclear organization and function in normal cells and in disease progression such as with cancer.


Assuntos
Cromossomos/metabolismo , DNA/metabolismo , Loci Gênicos , Hibridização in Situ Fluorescente , Telômero/metabolismo , Núcleo Celular/metabolismo , Células Cultivadas , Cromossomos Artificiais Bacterianos/metabolismo , Derme/citologia , Fibroblastos/metabolismo , Humanos , Processamento de Imagem Assistida por Computador
5.
Int J Mol Sci ; 22(3)2021 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-33498156

RESUMO

Excessive cross-linking is a major factor in the resistance to the remodelling of the extracellular matrix (ECM) during fibrotic progression. The role of TGFß signalling in impairing ECM remodelling has been demonstrated in various fibrotic models. We hypothesised that increased ECM cross-linking by TGFß contributes to skin fibrosis in Systemic Sclerosis (SSc). Proteomics was used to identify cross-linking enzymes in the ECM of primary human dermal fibroblasts, and to compare their levels following treatment with TGFß-1. A significant upregulation and enrichment of lysyl-oxidase-like 1, 2 and 4 and transglutaminase 2 were found. Western blotting confirmed the upregulation of lysyl hydroxylase 2 in the ECM. Increased transglutaminase activity in TGFß-1 treated ECM was revealed from a cell-based assay. We employed a mass spectrometry-based method to identify alterations in the ECM cross-linking pattern caused by TGFß-1. Cross-linking sites were identified in collagens I and V, fibrinogen and fibronectin. One cross-linking site in fibrinogen alpha was found only in TGFß-treated samples. In conclusion, we have mapped novel cross-links between ECM proteins and demonstrated that activation of TGFß signalling in cultured dermal fibroblasts upregulates multiple cross-linking enzymes in the ECM.


Assuntos
Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Aminoácido Oxirredutases/metabolismo , Células Cultivadas , Colágeno/química , Colágeno/metabolismo , Reagentes para Ligações Cruzadas/química , Derme/citologia , Matriz Extracelular/química , Matriz Extracelular/efeitos dos fármacos , Feminino , Fibrinogênio/química , Fibrinogênio/metabolismo , Fibronectinas/química , Fibronectinas/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Humanos , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Transglutaminases/metabolismo
6.
J Surg Res ; 257: 306-316, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32890866

RESUMO

BACKGROUND: A keloid is a type of pathological scar often caused by abnormal tissue repair after a skin injury and is more common in genetically susceptible individuals. cAMP is a universal second messenger and regulates critical physiological processes, including calcium homeostasis, secretion, cell fate, and gene transcription, by affecting the expression of the exchange protein directly activated by cAMP (Epac). Epac has two isoforms, Epac1 (cAMP-GEF-1) and Epac2 (cAMP-GEF-II), which show varying expression levels depending on the tissue and cell type. The expression of Epac1 in keloids has not yet been investigated. MATERIALS AND METHODS: Keloid tissue and normal dermal skin tissue were analyzed by hematoxylin and eosin staining and immunofluorescence. Primary human keloid fibroblasts (HKFs) and human normal dermal fibroblasts were studied using immunofluorescence, wound healing tests, reverse transcription polymerase chain reaction, and western blot analysis with different concentrations of the Epac1 inhibitor ESI-09. RESULTS: Downregulation of Epac was performed using ESI-09, a specific Epac inhibitor. The proliferation and migration capacities of HKFs and human normal dermal fibroblasts showed an ESI-09 concentration-dependent decrease. Furthermore, the apoptosis rates were significantly different between fibroblasts treated with ESI-09 and control fibroblasts. In addition, the phosphorylation level of Akt was significantly decreased, indicating that ESI-09 reduces fibrosis and induces apoptosis through Akt signaling in HKFs. CONCLUSIONS: Our results illustrate the role of Epac1 in regulating fibroblast function during keloid pathogenesis and indicate that Epac1 may be a potential therapeutic target in keloid treatment.


Assuntos
Fibroblastos/patologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Queloide/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Apoptose/efeitos dos fármacos , Células Cultivadas , Derme/citologia , Derme/patologia , Regulação para Baixo , Fibrose , Fatores de Troca do Nucleotídeo Guanina/antagonistas & inibidores , Humanos , Hidrazonas/farmacologia , Isoxazóis/farmacologia , Cultura Primária de Células , Isoformas de Proteínas/metabolismo , Transdução de Sinais/efeitos dos fármacos
7.
Methods Mol Biol ; 2147: 111-124, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32840814

RESUMO

Melt electrospinning writing (MEW) is a solvent-free fabrication method for making polymer fiber scaffolds with features which include large surface area, high porosity, and controlled deposition of the fibers. These scaffolds are ideal for tissue engineering applications. Here we describe how to produce scaffolds made from poly(ε-caprolactone) using MEW and the seeding of primary human-derived dermal fibroblasts to create cell-scaffold constructs. The same methodology could be used with any number of cell types and MEW scaffold designs.


Assuntos
Materiais Biocompatíveis/síntese química , Fibroblastos/citologia , Poliésteres/química , Impressão Tridimensional , Engenharia Tecidual/instrumentação , Tecidos Suporte/química , Células 3T3 , Animais , Materiais Biocompatíveis/química , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Células Cultivadas , Derme/citologia , Técnicas Eletroquímicas , Regeneração Tecidual Guiada/instrumentação , Regeneração Tecidual Guiada/métodos , Humanos , Camundongos , Engenharia Tecidual/métodos
8.
Methods Mol Biol ; 2158: 125-139, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32857370

RESUMO

Failure to regenerate myocardium after injury is a major cause of mortality and morbidity in humans. Direct differentiation of human induced pluripotent stem cells (iPSCs) into cardiomyocytes provides an invaluable resource to pursue cardiac regeneration based on cellular transplantation. Beyond the potential for clinical therapies, iPSC technology also enables the generation of cardiomyocytes to recapitulate patient-specific phenotypes, thus presenting a powerful in vitro cell-based model to understand disease pathology and guide precision medicine. Here, we describe protocols for reprogramming of human dermal fibroblasts and blood cells into iPSCs using the non-integrative Sendai virus system and for the monolayer differentiation of iPSCs to cardiomyocytes using chemically defined media.


Assuntos
Diferenciação Celular , Reprogramação Celular , Derme/citologia , Fibroblastos/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologia , Regeneração , Humanos
9.
Int J Mol Sci ; 21(24)2020 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-33352936

RESUMO

Pseudoxanthoma elasticum (PXE) is a rare autosomal-recessive disorder that is mainly caused by mutations in the ATP-binding cassette sub-family C member 6 (ABCC6) gene. Clinically PXE is characterized by a loss of skin elasticity, arteriosclerosis or visual impairments. It also shares some molecular characteristics with known premature aging syndromes like the Hutchinson-Gilford progeria syndrome (HGPS). However, little is known about accelerated aging processes, especially on a cellular level for PXE now. Therefore, this study was performed to reveal a potential connection between premature cellular aging and PXE pathogenesis by analyzing cellular senescence, a corresponding secretory phenotype and relevant factors of the cell cycle control in primary human dermal fibroblasts of PXE patients. Here, we could show an increased senescence-associated ß-galactosidase (SA-ß-Gal) activity as well as an increased expression of proinflammatory factors of a senescence-associated secretory phenotype (SASP) like interleukin 6 (IL6) and monocyte chemoattractant protein-1 (MCP1). We further observed an increased gene expression of the cyclin-dependent kinase inhibitor (CDKI) p21, but no simultaneous induction of p53 gene expression. These data indicate that PXE is associated with premature cellular senescence, which is possibly triggered by a p53-independent p21-mediated mechanism leading to a proinflammatory secretory phenotype.


Assuntos
Senescência Celular/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Derme/citologia , Fibroblastos/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/deficiência , Pseudoxantoma Elástico/etiologia , Pseudoxantoma Elástico/metabolismo , Biomarcadores , Inibidor de Quinase Dependente de Ciclina p27/genética , Expressão Gênica , Humanos , Lamina Tipo B/genética , Mutação , Fenótipo , Pseudoxantoma Elástico/patologia , RNA Mensageiro
10.
J Oleo Sci ; 69(11): 1487-1495, 2020 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-33055443

RESUMO

Photoaged skin is characterized by the appearance of pigmented spots such as solar lentigos, deep wrinkles and sags, and progresses due to chronic sun exposure. Among the wavelengths of sunlight, UVA is responsible for the appearance of wrinkles and sags that originate from structural alterations in the dermis of photoaged skin such as the depletion of collagen fibers. Thus, improving and restoring collagen fibers is an effective approach to reduce skin photoaging and maintain a youthful appearance. This study was conducted to evaluate the potential of an extract of Ocimum basilicum (OC), which contains rosmarinic acid (RA), as an anti-photoaging material focusing on the capacity to restore collagen fibers that are disrupted due to intracellular oxidative stress. In spite of their relatively low capacities for chemical scavenging of reactive oxygen species (ROS), both OC and RA showed efficient removal of biological oxidative stress by reducing levels of intracellular ROS and carbonylated proteins (CPs) in fibroblasts following exposure to single or repetitive UVA irradiations. Fibroblasts irradiated with repetitive UVA as a model for chronic sun-exposed cells showed significant increases in matrix metalloproteinase-1 and decreases in type I collagen synthesis and formed reduced numbers of collagen fibers. Since both OC and RA restored the adverse phenomena caused by repetitive UVA irradiation, we conclude that OC containing RA is an effective anti-photoaging material.


Assuntos
Cinamatos/farmacologia , Colágeno/metabolismo , Colágeno/efeitos da radiação , Depsídeos/farmacologia , Derme/citologia , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Ocimum basilicum/química , Extratos Vegetais/farmacologia , Envelhecimento da Pele/efeitos dos fármacos , Raios Ultravioleta/efeitos adversos , Células Cultivadas , Cinamatos/isolamento & purificação , Depsídeos/isolamento & purificação , Fibroblastos/patologia , Humanos , Estresse Oxidativo/efeitos dos fármacos , Extratos Vegetais/isolamento & purificação , Espécies Reativas de Oxigênio/metabolismo , Envelhecimento da Pele/patologia
11.
Adv Gerontol ; 33(2): 313-318, 2020.
Artigo em Russo | MEDLINE | ID: mdl-32593246

RESUMO

The aim of this work was to examine the content of arylhydrocarbon receptor nuclear translocator (ARNT) in fibroblasts of human dermis from 20 weeks of pregnancy until 85 years old, and defining of a role of ARNT in age-dependent changes in the number of fibroblasts in the dermis. ARNT, proliferating cells nuclear antigen (PCNA) were detected with indirect immunohistochemical technique. Results showed that a portion of fibroblasts with positive staining for ARNT in the dermis is decreased from 20 weeks of pregnancy to 40 years old. Percent of ARNT positive fibroblasts in dermis is increased sufficiently since 41 year old until 60-85 years old group. A total number and percent of PCNA positive fibroblasts in dermis decreased with progression of age. Most sufficient age-dependent reduction in a total and PCNA positive number of dermal fibroblast was observed from antenatal until 40 years of life. Age-related changes in the content of ARNT in fibroblasts is not associated with an age-related decrease in total number and percent of PCNA positive fibroblasts the dermis.


Assuntos
Envelhecimento/metabolismo , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Envelhecimento da Pele , Pele/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Derme/citologia , Derme/metabolismo , Feto/metabolismo , Fibroblastos/metabolismo , Humanos , Lactente , Recém-Nascido , Pele/citologia , Adulto Jovem
12.
Arch Biochem Biophys ; 689: 108437, 2020 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-32526201

RESUMO

Skin aging is influenced by several genetic, physiological, and environmental factors. In particular, ultraviolet (UV) exposure is an important factor involved in inducing skin photoaging. Autophagy controlling homeostatic balance between the synthesis, degradation, and recycling of cellular organelles and proteins plays important regulatory roles in several biological processes, including aging. The opioid neuropeptide α-neoendorphin (named NEP) is an endogenous decapeptide (N-YGGFLRKYPK-C) that activates the kappa opioid receptor and exhibits certain anti-aging and anti-wrinkling effects on skin cells; however, its action mechanism has not yet been elucidated. Therefore, the aim of this study was to determine the effects of NEP on anti-skin aging and autophagy activation in human dermal fibroblast cells. Western blot results showed that NEP down-regulates the production of phospho-mammalian target of rapamycin (p-mTOR), whereas increases the expression of key autophagy-related molecules such as Beclin-1, Atg5-Atg12, and LC3-II. The immunocytochemical analysis performed with anti-LC3-II antibody also showed that the autophagic indicators, autophagosomes are formed by NEP. These results suggest that NEP can activate cellular autophagy through mTOR-Beclin-1-mediated signaling pathway. It was also revealed by CM-H2DCF-DA assay and Western blottings that NEP can reduce the production of ultraviolet B (UVB)-induced reactive oxygen species (ROS) like with N-acetylcysteine (NAC), resulting in decreasing the expression levels of skin aging-related proteins, such as phospho-ERK (p-ERK), phospho-p38 (p-p38), and phospho-JNK (p-JNK). Furthermore, NEP could increase the type I procollagen production, while decreasing MMP-1, MMP-2, and MMP-9 activities. Taken together, the results demonstrate that NEP can reduce UVB-induced photoaging by activating autophagy.


Assuntos
Autofagia , Endorfinas/metabolismo , Precursores de Proteínas/metabolismo , Envelhecimento da Pele/efeitos da radiação , Raios Ultravioleta/efeitos adversos , Linhagem Celular , Derme/citologia , Derme/metabolismo , Derme/efeitos da radiação , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Humanos , Pró-Colágeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo
13.
Exp Mol Pathol ; 115: 104468, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32445750

RESUMO

OBJECTIVE: Exosomes originated from mesenchymal stem cells (MSCs) benefit wound healing. This study investigated effects of exosomes originated from human umbilical cord MSCs (hUC-MSCs) on dermal fibroblasts-myofibroblasts transition via the TGF-ß1/Smad2/3 signaling pathway. METHODS: Firstly, hUC-MSCs were collected and identified. Alizarin red, oil red O staining and toluidine blue staining were used to determine the osteogenic, adipogenic and chondrogenic differentiation abilities of hUC-MSCs. Then exosomes from hUC-MSCs were extracted and identified. To figure out the roles of exosomes and TGF-ß1 in dermal fibroblasts-myofibroblasts transition, dermal fibroblasts were treated with TGF-ß1 or/and exosomes at different concentrations. RT-qPCR, Western blot analyses were employed to examine levels of Collagen I, Collagen III, α-smooth muscle actin (α-SMA), and Smad2/3 phosphorylation, and immunofluorescence was employed to test α-SMA content and the localization and nucleation of Smad2/3 protein in cells. RESULTS: hUC-MSCs and exosomes were successfully cultured and extracted. Levels of Collagen I, Collagen III, α-SMA, and Smad2/3, and Smad2/3 phosphorylation in fibroblasts treated with exosomes decreased markedly. After treatment with exosomes and TGF-ß1 together, levels of Collagen I, Collagen III, α-SMA, and Smad2/3, and Smad2/3 phosphorylation in fibroblasts decreased significantly as compared to TGF-ß1-treated fibroblasts. Exosome treatment reduced the entry of Smad2/3 into fibroblasts. CONCLUSION: Our data suggested that hUC-MSCs-derived exosomes could inhibit dermal fibroblasts-myofibroblasts transition by inhibiting the TGF-ß1/Smad2/3 signaling pathway.


Assuntos
Exossomos/metabolismo , Células-Tronco Mesenquimais/metabolismo , Miofibroblastos/metabolismo , Transdução de Sinais , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Cordão Umbilical/citologia , Células Cultivadas , Derme/citologia , Exossomos/ultraestrutura , Humanos , Recém-Nascido
14.
Adv Gerontol ; 33(1): 40-45, 2020.
Artigo em Russo | MEDLINE | ID: mdl-32362082

RESUMO

The aim of this work was to examine the content of heat shock protein 90 (HSP90) in fibroblasts of human dermis from the development until deep aging (from 20 weeks of pregnancy until 85 years old), and defining of a role of HSP90 in age-dependent changes in the number of fibroblasts in the dermis. HSP90, proliferating cells nuclear antigen (PCNA) were detected with indirect immunohistochemical technique. Results showed that a portion of fibroblasts with positive staining for HSP90 in the dermis is not changed from 20 weeks of development to 20 years old. Percent of HSP90 positive fibroblasts in dermis is decreased from 21 to 60 years old. From 61 year, the number of HSP90 positive fibroblasts in dermis is increased. Age-related changes in the number of HSP90 positive fibroblasts is not statistically associated with an age-related decrease in a total number and percent of PCNA positive fibroblasts the dermis.


Assuntos
Envelhecimento , Derme/citologia , Fibroblastos/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Recém-Nascido , Pessoa de Meia-Idade , Gravidez , Adulto Jovem
15.
Life Sci ; 252: 117667, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32304761

RESUMO

AIMS: Pantothenic acid (PA) has been applied to treat alopecia, but the underlying mechanism is still unclear. Our study aims to explore the underlying mechanism of PA in regulating hair follicle (HF) growth. MAIN METHODS: Mink HFs and dermal papilla (DP) cells were isolated and cultured in vitro. HFs and DP cells were treated with 0, 10, 20, 40 µg/ml PA. The effect of PA on HF growth, DP cell proliferation, cell cycle distribution, cell migration, and insulin-like growth factor-1 (IGF-1) and vascular endothelial growth factor (VEGF) expressions in DP cells was measured. Moreover, the effect of PA on inhibitor of DNA binding 3 (ID3)/Notch signaling pathway was analyzed. Subsequently, ID3 was silenced to validate whether ID3/Notch signaling pathway was involved in regulating DP cell proliferation by PA. KEY FINDINGS: Both 20 µg/ml and 40 µg/ml PA promoted HF growth, G1/S transition of DP cells and IGF-1 and VEGF expressions in DP cells, while only 20 µg/ml PA promoted cell viability and the migration of DP cells. Thus 20 µg/ml PA was chosen for the following experiments. PA treatment was found to up-regulate ID3 expression but down-regulate Notch receptor 1 (Notch1) and Notch signaling targets expressions. Furthermore, ID3 knockdown reversed PA-induced cell proliferation and inhibition of Notch1 and Notch signaling targets expressions, indicating that PA-induced DP cell proliferation and inhibition of Notch signaling were mediated via up-regulation of ID3. SIGNIFICANCE: This study provides an underlying mechanism related to the effect of PA on stimulating DP cell proliferation.


Assuntos
Proliferação de Células/efeitos dos fármacos , Derme/efeitos dos fármacos , Folículo Piloso/efeitos dos fármacos , Ácido Pantotênico/farmacologia , Animais , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Derme/citologia , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Folículo Piloso/citologia , Proteínas Inibidoras de Diferenciação/metabolismo , Masculino , Vison , Ácido Pantotênico/administração & dosagem , Receptores Notch/metabolismo , Transdução de Sinais/efeitos dos fármacos
16.
Hum Cell ; 33(3): 479-489, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32277427

RESUMO

The paracrine secretion of angiogenic cytokines from adipose-derived stem cells (ADSCs) might promote endothelial cell angiogenesis, therefore promoting wound healing in injured tissues. Hypoxia is one of the common occurrence in injured tissues, during which angiogenesis is enhanced to improve the oxygen supply. In the present study, miR-590-3p, an anti-angiogenic miRNA, was predicted to target VEGFA, a key factor that can be transcriptionally upregulated by HIF1A during ADSC proliferation and tubule formation in response to hypoxic stimulation. Herein, we found that in response to hypoxic stimuli, HIF1A and VEGFA protein expressions were remarkably induced. In addition, ADSC viability was promoted. Incubation with conditioned medium from ADSCs stimulated by hypoxia significantly enhanced the angiogenic ability of human dermal microvascular endothelial cells (HDMECs), while the conditioned medium from VEGFA-silenced ADSCs significantly reversed the angiogenic ability of HDMECs. Regarding the molecular mechanism, it was verified that miR-590-3p binds to VEGFA; miR-590-3p inhibited VEGFA to affect the paracrine regulation by ADSCs, subsequently hindering the HDMEC angiogenesis. More importantly, the consequences of miR-590-3p-overexpressing conditioned medium on HDMEC angiogenesis were partially reversed by VEGFA-overexpressing conditioned medium. In conclusion, miR-590-3-5p/VEGFA axis modulates the paracrine secretion of VEGFA by ADSCs to affect the angiogenesis of HDMECs.


Assuntos
Derme/citologia , Células Endoteliais/fisiologia , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/fisiologia , Neovascularização Fisiológica/genética , Comunicação Parácrina/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/fisiologia , Células Cultivadas , Expressão Gênica/genética , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , MicroRNAs/genética , MicroRNAs/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Cicatrização
17.
PLoS One ; 15(4): e0230428, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32240195

RESUMO

Cryopreservation is a method used for preserving living cells by cooling them to very low temperatures. Although cryopreservation methods for oocytes and embryos have been developed for use in reproductive medicine, there are no established methods yet for preserving cell aggregates (spheroids) in regenerative medicine. We have developed a bio-three-dimensional (3D) printer that can fabricate scaffold-free 3D constructs by loading spheroids onto a needle array. We fabricated several constructs such as blood vessels, liver, diaphragm, and a conduit for nerves by using this method. These constructs have the potential to be applied in patients. However, the process of fabricating tissue constructs (harvesting cells, expanding cells, making spheroids using cultured cells, printing constructs, and maturing constructs) is time-consuming. Therefore, cryopreservation methods for spheroids or constructs should be developed to increase the efficiency of this method for clinical use. Here, we developed a method for cryopreserving spheroids, which were then used to fabricate constructs. Fibroblast cell-based spheroids were cryopreserved in phosphate-buffered saline or cryopreservation solution at -80°C for 1 week. After thawing, spheroids in cryopreservation solution began to fuse on day 1. Cryopreserved spheroids were printed onto a needle array to fabricate a scaffold-free tubular construct using a bio-3D printer. After 7 days, the printed spheroids fused and formed scaffold-free constructs. We confirmed the viability of cells in the cryopreserved spheroids and fabricated tubular constructs. Our results indicate that spheroids can be cryopreserved and used to prepare scaffold-free constructs for clinical use.


Assuntos
Proliferação de Células , Criopreservação/métodos , Derme/citologia , Fibroblastos/citologia , Esferoides Celulares/citologia , Engenharia Tecidual/métodos , Tecidos Suporte/química , Matriz Extracelular , Humanos
18.
Arch Biochem Biophys ; 683: 108322, 2020 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-32113875

RESUMO

Post-burn hypertrophic scar (HTS) is a form of excessive dermal fibrosis characterized by cutaneous scarring, which is common in patients following burn injury. Moreover, at least 50% of HTS are accompanied by inflammation. Cytoplasmic polyadenylation element binding (CPEB) proteins are key mRNA-binding proteins that control the translation of several mRNAs. However, their potential roles in treating dermal fibrosis and scarring remain unknown. Therefore, in this study, we aimed to investigate the effects of small interfering RNA (siRNA)-mediated knockdown of CPEB1 or CPEB4 in human THP-1 macrophages and dermal fibroblasts treated with LPS and TGF-ß1. We found significantly increased CPEB1 and CPEB4 mRNA and protein levels in LPS-treated THP-1 cells and TGF-ß1-treated fibroblasts. CPEB1 and CPEB4 knockdowns suppressed LPS-activated TAK1 signaling cascades by reducing the levels of TNF-α and phosphorylated TAK1, p38, ERK, JNK, and NF-κB-p65 in THP-1 cells. CPEB1 and CPEB4 knockdowns also attenuated TGF-ß1-activated Smad-dependent and -independent signaling cascades by reducing the levels of TAK1, p38, ERK, JNK, and phosphorylated Smad 2 and Smad 1/5/8 in fibroblasts. Furthermore, CPEB1 or CPEB4 knockdown markedly decreased the levels of fibrosis markers, including α-SMA, type I collagen, and fibronectin in fibroblasts. Our findings indicate that CPEB1 and CPEB4 are involved in the regulation of the TAK1 and Smad signalings in human macrophages and dermal fibroblasts. These activities may play a role in cutaneous scarring responses.


Assuntos
MAP Quinase Quinase Quinases/metabolismo , Proteínas de Ligação a RNA/genética , Transdução de Sinais , Proteínas Smad/metabolismo , Fatores de Transcrição/genética , Fatores de Poliadenilação e Clivagem de mRNA/genética , Animais , Derme/citologia , Fibroblastos/citologia , Regulação da Expressão Gênica , Humanos , Inflamação , Macrófagos/citologia , Camundongos , Fosforilação , Células RAW 264.7 , RNA Interferente Pequeno/metabolismo , Células THP-1 , Fator de Crescimento Transformador beta1/farmacologia , Regulação para Cima
19.
Vet Res Commun ; 44(2): 41-49, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32130648

RESUMO

Mesenchymal stromal cells (MSCs) have attracted great attention for therapeutic applications. Since cells derived from different tissues have different properties, using the right tissue source may impact their efficiency in regenerative medicine. This study describes for the first time the isolation and characterization of MSCs derived from the equine coronary corium, which may be useful for treating diseases such as laminitis. Seven coronary corium samples were used for isolation of cells (ccMSCs). Adherent cells were characterized for morphology, immunophenotype, proliferation and differentiation potential, in vitro migration and colony-forming capacity. The cells displayed the characteristic fibroblastoid morphology, with population doubling time increasing until passage 7 and reaching a plateau in passage 10. Cells were negative for CD14 and CD45, and positive for CD73 and CD90. ccMSCs showed chondrogenic and osteogenic, but not adipogenic differentiation, and migrated with nearly total closing of the empty area in 48 h, in the scratch assay. The clonogenic potential was in average 18% to 23%. This study describes for the first time the establishment of mesenchymal stromal cell cultures from the equine coronary corium. The results are similar to MSCs isolated from many other equine tissues, except for restricted differentiation potential. As coronary corium stem cell regulation may contribute to the pathogenesis of equine chronic laminitis, the use of ccMSCs in cell therapy for this significantly debilitating disease should be further investigated.


Assuntos
Derme/citologia , Cavalos , Células-Tronco Mesenquimais/citologia , Animais , Células Cultivadas , Medicina Regenerativa , Dermatopatias/terapia
20.
Sci Rep ; 10(1): 3775, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-32111895

RESUMO

Brown adipocytes coordinate systemic energy metabolism associated with the pathogenesis of obesity and related metabolic diseases including type 2 diabetes. We have previously reported chemical compound-induced brown adipocytes (ciBAs) converted from human dermal fibroblasts without using transgenes. In this study, to reveal a precise molecular mechanism underlying the direct conversion and human adipocyte browning, we developed serum-free brown adipogenic medium (SFBAM) with an optimized chemical cocktail consisting of Rosiglitazone, Forskolin, and BMP7. During the direct conversion, treatment with BMP7 enhanced Ucp1 expression rather than the conversion efficiency in the absence of BMP signalling inhibitors. Moreover, treatment with a TGF-ß signalling pathway inhibitor was no longer required in the serum-free medium, likely because the TGF-ß pathway was already suppressed. SFBAM and the chemical cocktail efficiently converted human dermal fibroblasts into ciBAs within four weeks. The ciBAs exhibited increased mitochondrial levels, elevated oxygen consumption rate, and a response to ß-adrenergic receptor agonists. Thus the ciBAs converted by the serum-free medium and the chemical cocktail provide a novel model of human brown (beige) adipocytes applicable for basic research, drug screening, and clinical applications.


Assuntos
Adipócitos Marrons/metabolismo , Diferenciação Celular , Derme/metabolismo , Fibroblastos/metabolismo , Transdução de Sinais , Adipócitos Marrons/citologia , Proteína Morfogenética Óssea 7/química , Proteína Morfogenética Óssea 7/farmacologia , Colforsina/química , Colforsina/farmacologia , Meios de Cultura Livres de Soro/química , Meios de Cultura Livres de Soro/farmacologia , Derme/citologia , Fibroblastos/citologia , Humanos , Rosiglitazona/química , Rosiglitazona/farmacologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...