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1.
PLoS One ; 18(12): e0295684, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-38150443

RESUMO

Alveolar barrier dysfunction is one of the major pathophysiological changes in acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). In ALI/ARDS, tumor necrosis factor-alpha (TNFα) disrupts the barriers of alveolar epithelium and endothelium. Glucocorticoids (GCs) exert anti-inflammatory effects and ameliorate pulmonary edema in ALI/ARDS. However, the involvement of GCs in the restoration of alveolar epithelial barrier dysfunction has not been extensively studied. Here, we elucidated that dexamethasone (Dex) restored TNFα-induced alveolar epithelial barrier dysfunction in vitro using primary rat alveolar epithelial cells isolated from Sprague-Dawley rats. Moreover, Dex promoted the alveolar epithelial cell barrier integrity by initiating GC receptor-mediated signaling via the downregulation of myosin light chain kinase (MLCK) expression and the dephosphorylation of myosin light chain (MLC) 2. Further investigation revealed that Dex enhanced the expression of zonula occludens-1 (ZO-1), a tight junction-related protein, at intercellular junction sites. These findings suggest that GCs strengthen the integrity of the alveolar epithelial barrier in ALI/ARDS via the GR-MLCK-pMLC2 axis.


Assuntos
Células Epiteliais Alveolares , Síndrome do Desconforto Respiratório , Ratos , Animais , Células Epiteliais Alveolares/metabolismo , Fator de Necrose Tumoral alfa , Ratos Sprague-Dawley , Proteínas de Junções Íntimas/metabolismo , Dexametasona/farmacologia , Células Epiteliais/metabolismo
2.
PLoS One ; 15(11): e0242789, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33237957

RESUMO

There has been an increase in the usage of heat-not-burn (HNB) cigarette products. However, their effects on alveolar epithelial cells (AECs) remain unknown. AECs are the target cells of conventional cigarette smoking-related respiratory diseases such as chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis and lung cancer whose pathogenesis involves oxidative stress. In this study, primary rat AECs were isolated, cultured and stimulated by HNB cigarette smoke extract (CSE). Our data indicate that rat AECs exposed to HNB CSE induced oxidative stress response genes (e.g. Hmox-1, Gsta1, Gsta3 and Nqo1). We also compared the oxidative stress response between two different types of AECs, alveolar type I-like (ATI-like) cells and type II (ATII) cells, and between two different types of cigarette, HNB cigarettes and conventional cigarettes. The expressions of Gsta1, Gsta3 and Nqo1 were higher in ATII cells than ATI-like cells in response to HNB and conventional cigarettes, but there was no significant difference in their expression levels between HNB cigarette and conventional cigarette. Taken together, our results suggest that HNB cigarettes have the similar potential as conventional cigarette products to induce oxidative stress response in AECs.


Assuntos
Células Epiteliais Alveolares/efeitos dos fármacos , Fumar Cigarros/efeitos adversos , Estresse Oxidativo/efeitos dos fármacos , Alvéolos Pulmonares/efeitos dos fármacos , Células Epiteliais Alveolares/patologia , Animais , Modelos Animais de Doenças , Sistemas Eletrônicos de Liberação de Nicotina , Temperatura Alta/efeitos adversos , Humanos , Oxirredução/efeitos dos fármacos , Cultura Primária de Células , Alvéolos Pulmonares/patologia , Doença Pulmonar Obstrutiva Crônica/induzido quimicamente , Ratos , Fumaça/efeitos adversos , Nicotiana/efeitos adversos
3.
Biomaterials ; 28(29): 4294-302, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17602737

RESUMO

Current methods including the use of various biological and synthetic sealants are ineffective in the closure of intraoperative air leaks that often occur during cardiothoracic surgeries, resulting in a decreased quality of life for patients. We present the development of a novel lung air leak sealant using tissue engineered cell sheets. In contrast to previous materials such as fibrin glue, these bioengineered cell sheets immediately and permanently seal air leaks in a dynamic fashion that allows for the extensive tissue contraction and expansion involved in respiration, without any postoperative recurrences. Additionally, we demonstrate that mesothelial cells migrate to cover the transplanted cells sheets, thereby confirming excellent biocompatibility and integration with the host tissues. Finally, we present the use of skin fibroblasts as an effective and readily available autologous cell source that can be easily applied. This study shows for the first time, the development of an immediate and permanent lung air leak sealant, suitable for future clinical applications.


Assuntos
Fibroblastos/transplante , Regeneração Tecidual Guiada/métodos , Pneumonectomia/efeitos adversos , Pneumonectomia/métodos , Complicações Pós-Operatórias/etiologia , Complicações Pós-Operatórias/cirurgia , Engenharia Tecidual/métodos , Animais , Masculino , Coelhos , Ratos , Ratos Nus , Resultado do Tratamento
4.
FASEB J ; 20(2): 392-4, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16339916

RESUMO

This study reports a new method for fabricating bioengineered human corneal endothelial cell sheets suitable for ocular surgery and repair. We have initially cultured human corneal endothelial cells on type IV collagen-coated dishes and, after several passages, expanded cells were then seeded onto novel temperature-responsive culture dishes. Four weeks after reaching confluence, these cultured endothelial cells were harvested as intact monolayer cell sheets by simple temperature reduction without enzymatic treatment. Scanning electron microscopy indicated that these cells were primarily hexagonal with numerous microvilli and cilia, similar to the native corneal endothelium. The Na+, K+-ATPase pump sites were located at the cell borders as in vivo. Moreover, cell densities and numbers of pump sites were identical to those of in vivo human corneal endothelium under optimized conditions. A 3H-ouabain binding analysis demonstrated a linear proportionality for cell pump density between confluent cell densities of 575 cells/mm2 and 3070 cells/mm2. We also confirmed Na+, K+-ATPase activity in the sheets in vitro. Xenograft transplantation results showed that the fabricated sheets retain their function of maintaining proper stromal hydration in vivo. We have established a regimen to culture and proliferate human corneal endothelial cells and fabricate endothelial sheets ex vivo morphologically and functionally similar to the native corneal endothelium. Our results support the value of harvested cell sheets for clinical applications in ocular reconstructive surgery in patients with ocular endothelial decompensation.


Assuntos
Técnicas de Cultura de Células/métodos , Endotélio Corneano/citologia , Temperatura , Engenharia Tecidual/métodos , Células Cultivadas , Endotélio Corneano/enzimologia , Humanos , ATPase Trocadora de Sódio-Potássio/metabolismo
5.
Tissue Eng ; 12(5): 1275-83, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16771640

RESUMO

To successfully engineer a bioartificial tracheal replacement, it is believed that the regeneration of a functional epithelial lining is a key requirement. In the present study, rabbit tracheal epithelial cells were cultured on temperature-responsive culture dishes, under normal culture conditions at 37 degrees C. By simple temperature reduction to 20 degrees C, the cultured epithelial cells were noninvasively harvested as intact sheets, without the use of any proteolytic enzymes. Support Dacron grafts that had been subcutaneously implanted for 4 weeks to allow for host tissue and vessel infiltration were then opened, and the tracheal epithelial cell sheets were transplanted to the luminal surface without sutures. These fabricated constructs were then used as tracheal replacements, in a rabbit model. Four weeks after transplantation, results showed that the tracheal grafts were covered by a mature, pseudostratified columnar epithelium. In contrast, control constructs that did not receive cell sheet transplantation demonstrated only a thin, immature epithelium at the center of the replacement graft. These results therefore demonstrate that these tracheal epithelial cell sheets can create an epithelial lining on the luminal surface of a bioartificial trachea.


Assuntos
Órgãos Bioartificiais , Células Epiteliais , Próteses e Implantes , Engenharia Tecidual , Traqueia , Animais , Células Cultivadas , Células Epiteliais/citologia , Neovascularização Fisiológica , Coelhos , Propriedades de Superfície , Traqueia/citologia , Traqueia/cirurgia
6.
FEBS Lett ; 579(29): 6569-74, 2005 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-16297384

RESUMO

The side population (SP) phenotype is shared by stem cells in various tissues and species. Here we demonstrate SP cells with Hoechst dye efflux were surprisingly collected from the epithelia of both the rat limbus and central cornea, unlike in human and rabbit eyes. Our results show that rat limbal SP cells have a significantly higher expression of the stem cell markers ABCG2, nestin, and notch 1, compared to central corneal SP cells. Immunohistochemistry also revealed that ABCG2 and the epithelial stem/progenitor cell marker p63 were expressed only in basal limbal epithelial cells. These results demonstrate that ABCG2 expression is closely linked to the stem cell phenotype of SP cells.


Assuntos
Córnea/citologia , Células Epiteliais/citologia , Sistema Límbico/citologia , Células-Tronco/citologia , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/análise , Animais , Biomarcadores/análise , Imuno-Histoquímica , Imunofenotipagem , Proteínas de Filamentos Intermediários/análise , Masculino , Proteínas do Tecido Nervoso/análise , Nestina , Coelhos , Ratos , Ratos Wistar , Receptor Notch1/análise
7.
Biomaterials ; 26(33): 6415-22, 2005 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16011847

RESUMO

While tissue engineering has long been thought to possess enormous potential, conventional applications using biodegradable scaffolds have limited the field's progress, demonstrating a need for new methods. We have previously developed cell sheet engineering using temperature-responsive culture dishes in order to avoid traditional tissue engineering approaches, and their related shortcomings. Using temperature-responsive dishes, cultured cells can be harvested as intact sheets by simple temperature changes, thereby avoiding the use of proteolytic enzymes. Cell sheet engineering therefore allows for tissue regeneration by either direct transplantation of cell sheets to host tissues or the creation of three-dimensional structures via the layering of individual cell sheets. By avoiding the use of any additional materials such as carrier substrates or scaffolds, the complications associated with traditional tissue engineering approaches such as host inflammatory responses to implanted polymer materials, can be avoided. Cell sheet engineering thus presents several significant advantages and can overcome many of the problems that have previously restricted tissue engineering with biodegradable scaffolds.


Assuntos
Materiais Biocompatíveis/química , Técnicas de Cultura de Células/métodos , Engenharia Tecidual/métodos , Animais , Proliferação de Células , Córnea/patologia , Humanos , Inflamação , Miócitos de Músculo Liso/citologia , Polímeros/química , Temperatura , Transplante de Tecidos
8.
J Tissue Eng Regen Med ; 5(4): e63-73, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20740688

RESUMO

Keratinocyte culture medium (KCM) has been used for the in vitro culture of keratinocytes and other types of epithelial cells, and the medium includes various ingredients. In this study, two modified KCMs were prepared. In the first, insulin, hydrocortisone and antibiotics that are normally included in KCM were replaced with clinically approved pharmaceutical agents, except transferrin and selenium; in the second, cholera toxin (CT) was replaced by L-isoproterenol (ISO). The modified KCMs were then compared to conventional KCM containing laboratory-grade reagents. Induced cell colony formations of canine oral mucosal epithelial cells cultured in both modified KCMs were found to be nearly equivalent to that in the control KCM, and there was no significant difference between the effect of CT and ISO. Canine oral mucosal cells proliferated to confluence in all three KCM formulations, with or without the use of 3T3 feeder layers. Cultured epithelial cells were harvested from temperature-responsive culture surfaces as an intact cell sheet, and the immunohistochemical analysis of the sheets showed that p63 and cytokeratin were expressed in the epithelial cell sheets cultured in all KCMs. Eventually, in the modified KCM formula, fetal bovine serum was replaced by autologous human serum, and the formula was found to be able to fabricate human oral mucosal epithelial cell sheets. These results indicated that the modified KCM was equally efficient as conventional KCM in the fabrication of transplantable stratified epithelial cell sheets.


Assuntos
Meios de Cultura/farmacologia , Queratinócitos/citologia , Medicina Regenerativa/métodos , Animais , Bovinos , Proliferação de Células/efeitos dos fármacos , Ensaio de Unidades Formadoras de Colônias , Cães , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/transplante , Humanos , Imuno-Histoquímica , Queratinas/metabolismo , Camundongos , Microscopia de Contraste de Fase , Mucosa Bucal/citologia , Células NIH 3T3 , Propriedades de Superfície/efeitos dos fármacos , Temperatura , Proteínas Supressoras de Tumor/metabolismo , Vimentina/metabolismo
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