Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 23
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
J Med Internet Res ; 23(9): e27098, 2021 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-34491204

RESUMO

BACKGROUND: Hemodialysis (HD) therapy is an indispensable tool used in critical care management. Patients undergoing HD are at risk for intradialytic adverse events, ranging from muscle cramps to cardiac arrest. So far, there is no effective HD device-integrated algorithm to assist medical staff in response to these adverse events a step earlier during HD. OBJECTIVE: We aimed to develop machine learning algorithms to predict intradialytic adverse events in an unbiased manner. METHODS: Three-month dialysis and physiological time-series data were collected from all patients who underwent maintenance HD therapy at a tertiary care referral center. Dialysis data were collected automatically by HD devices, and physiological data were recorded by medical staff. Intradialytic adverse events were documented by medical staff according to patient complaints. Features extracted from the time series data sets by linear and differential analyses were used for machine learning to predict adverse events during HD. RESULTS: Time series dialysis data were collected during the 4-hour HD session in 108 patients who underwent maintenance HD therapy. There were a total of 4221 HD sessions, 406 of which involved at least one intradialytic adverse event. Models were built by classification algorithms and evaluated by four-fold cross-validation. The developed algorithm predicted overall intradialytic adverse events, with an area under the curve (AUC) of 0.83, sensitivity of 0.53, and specificity of 0.96. The algorithm also predicted muscle cramps, with an AUC of 0.85, and blood pressure elevation, with an AUC of 0.93. In addition, the model built based on ultrafiltration-unrelated features predicted all types of adverse events, with an AUC of 0.81, indicating that ultrafiltration-unrelated factors also contribute to the onset of adverse events. CONCLUSIONS: Our results demonstrated that algorithms combining linear and differential analyses with two-class classification machine learning can predict intradialytic adverse events in quasi-real time with high AUCs. Such a methodology implemented with local cloud computation and real-time optimization by personalized HD data could warn clinicians to take timely actions in advance.

2.
Int J Mol Sci ; 22(5)2021 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-33670996

RESUMO

Human mesenchymal stem cells (hMSCs) possess potential of bone formation and were proposed as ideal material against osteoporosis. Although interrogation of directing effect on lineage specification by physical cues has been proposed, how mechanical stimulation impacts intracellular viscoelasticity during osteogenesis remained enigmatic. Cyto-friendly 3D matrix was prepared with polyacrylamide and conjugated fibronectin. The hMSCs were injected with fluorescent beads and chemically-induced toward osteogenesis. The mechanical properties were assessed using video particle tracking microrheology. Inverted epifluorescence microscope was exploited to capture the Brownian trajectory of hMSCs. Mean square displacement was calculated and transformed into intracellular viscoelasticity. Two different stiffness of microspheres (12 kPa, 1 kPa) were established. A total of 45 cells were assessed. hMSCs possessed equivalent mechanical traits initially in the first week, while cells cultured in rigid matrix displayed significant elevation over elastic (G') and viscous moduli (G") on day 7 (p < 0.01) and 14 (p < 0.01). However, after two weeks, soft niches no longer stiffened hMSCs, whereas the effect by rigid substrates was consistently during the entire differentiation course. Stiffness of matrix impacted the viscoelasticity of hMSCs. Detailed recognition of how microenvironment impacts mechanical properties and differentiation of hMSCs will facilitate the advancement of tissue engineering and regenerative medicine.


Assuntos
Diferenciação Celular , Proliferação de Células , Módulo de Elasticidade/fisiologia , Matriz Extracelular/fisiologia , Células-Tronco Mesenquimais/fisiologia , Osteogênese , Engenharia Tecidual , Células Cultivadas , Humanos , Células-Tronco Mesenquimais/citologia , Viscosidade
3.
Biochem Biophys Res Commun ; 526(3): 827-832, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32273088

RESUMO

Mechanical properties of biological tissues are increasingly recognized as an important parameter for the indication of disease states as well as tissue homeostasis and regeneration. Multipotent mesenchymal stromal/stem cells (MSCs), which play important roles in bone formation and remodeling, are potential cell sources for regenerative medicine. However, the cellular mechanical properties of differentiating MSCs corresponding to the substrate stiffness has not been sufficiently studied. In this study, we used Atomic Force Microscopy (AFM) to measure changes of stiffness of human MSCs cultured in rigid Petri dish and on polyacrylamide (PA) substrates during osteogenic differentiation. The results showed that the Young's modulus of MSC cytoplasmic outer region increased over time during osteogenesis. There is a strong linear correlation between the osteogenic induction time and the Young's modulus of the cells cultured in rigid Petri dishes in the first 15 days after the induction; the Young's modulus approaches to a plateau after day 15. On the other hand, the Young's moduli of MSCs cultured on PA gels with stiffness of 7 kPa and 42 kPa also increase over time during osteogenic differentiation, but the inclination of such increase is much smaller than that of MSCs differentiating in rigid dishes. Herein, we established a protocol of AFM measurement to evaluate the maturation of stem cell osteogenic differentiation at the single cell level and could encourage further AFM applications in tissue engineering related to mechanobiology.


Assuntos
Módulo de Elasticidade/fisiologia , Células-Tronco Mesenquimais/metabolismo , Microscopia de Força Atômica/métodos , Osteogênese/fisiologia , Resinas Acrílicas/química , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Humanos , Propriedades de Superfície , Engenharia Tecidual
4.
Int J Mol Sci ; 18(10)2017 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-29064396

RESUMO

Human mesenchymal stem cells (hMSCs) can differentiate into osteoblasts and are regulated by chemical cues. The recombinant N-terminal (1-34 amino acids) fragment of the parathyroid hormone (PTH (1-34)) is identified to promote osteogenesis. The osteoanabolic effects of intermittent PTH (1-34) treatment are linked to a complex consisting of signaling pathways; additionally, protein kinase C (PKC) act as mediators of multifunctional signaling transduction pathways, but the role of PKC δ (PKCδ), a downstream target in regulating osteoblast differentiation during intermittent administration of PTH (1-34) is less studied and still remains elusive. The purpose of this study is to examine the role of PKCδ during intermittent and continuous PTH (1-34) administration using osteoblast-lineage-committed hMSCs. Relative gene expression of osteoblast-specific genes demonstrated significant upregulation of RUNX2, type I Collagen, ALP, and Osterix and increased alkaline phosphatase activity in the presence of PTH (1-34). Intermittent PTH (1-34) administration increased PKC activity at day 7 of osteogenic differentiation, whereas inhibition of PKC activity attenuated these effects. In addition, the specific isoform PKCδ was activated upon treatment. These findings demonstrate that intermittent PTH (1-34) treatment enhances the osteogenesis of hMSCs by upregulating osteoblast-specific genes via PKCδ activation.


Assuntos
Osteogênese/efeitos dos fármacos , Hormônio Paratireóideo/farmacologia , Proteína Quinase C-delta/metabolismo , Acetofenonas/farmacologia , Benzopiranos/farmacologia , Diferenciação Celular , Células Cultivadas , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , Proteína Quinase C-delta/antagonistas & inibidores , Transdução de Sinais , Fator de Transcrição Sp7/genética , Fator de Transcrição Sp7/metabolismo , Regulação para Cima/efeitos dos fármacos
5.
Stem Cell Res Ther ; 8(1): 39, 2017 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-28222767

RESUMO

BACKGROUND: Magnesium is essential for numerous physiological functions. Magnesium exists mostly in bone and the amount is dynamically regulated by skeletal remodeling. Accelerating bone mass loss occurs when magnesium intake is insufficient; whereas high magnesium could lead to mineralization defects. However, the underlying magnesium regulatory mechanisms remain elusive. In the present study, we investigated the effects of high extracellular magnesium concentration on osteogenic differentiation of mesenchymal stromal/stem cells (MSCs) and the role of magnesium transporter SLC41A1 in the mineralization process. METHODS: Murine MSCs derived from the bone marrow of BALB/c mouse or commercially purchased human MSCs were treated with osteogenic induction medium containing 5.8 mM magnesium chloride and the osteogenic differentiation efficiency was compared with that of MSCs in normal differentiation medium containing 0.8 mM magnesium chloride by cell morphology, gene expression profile of osteogenic markers, and Alizarin Red staining. Slc41a1 gene knockdown in MSCs was performed by siRNA transfection using Lipofectamine RNAiMAX, and the differentiation efficiency of siRNA-treated MSCs was also assessed. RESULTS: High concentration of extracellular magnesium ion inhibited mineralization during osteogenic differentiation of MSCs. Early osteogenic marker genes including osterix, alkaline phosphatase, and type I collagen were significantly downregulated in MSCs under high concentration of magnesium, whereas late marker genes such as osteopontin, osteocalcin, and bone morphogenetic protein 2 were upregulated with statistical significance compared with those in normal differentiation medium containing 0.8 mM magnesium. siRNA treatment targeting SLC41A1 magnesium transporter, a member of the solute carrier family with a predominant Mg2+ efflux system, accelerated the mineralization process and ameliorated the inhibition of mineralization caused by high concentration of magnesium. High concentration of magnesium significantly upregulated Dkk1 gene expression and the upregulation was attenuated after the Slc41a1 gene was knocked down. Immunofluorescent staining showed that Slc41a1 gene knockdown promoted the translocation of phosphorylated ß-catenin into nuclei. In addition, secreted MGP protein was elevated after Slc41a1 was knocked down. CONCLUSIONS: High concentration of extracellular magnesium modulates gene expression of MSCs during osteogenic differentiation and inhibits the mineralization process. Additionally, we identified magnesium transporter SLC41A1 that regulates the interaction of magnesium and MSCs during osteogenic differentiation. Wnt signaling is suggested to be involved in SLC41A1-mediated regulation. Tissue-specific SLC41A1 could be a potential treatment for bone mass loss; in addition, caution should be taken regarding the role of magnesium in osteoporosis and the design of magnesium alloys for implantation.


Assuntos
Calcificação Fisiológica/efeitos dos fármacos , Proteínas de Transporte de Cátions/genética , Magnésio/farmacologia , Células-Tronco Mesenquimais/metabolismo , Osteogênese/efeitos dos fármacos , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 2/metabolismo , Calcificação Fisiológica/genética , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Transporte de Cátions/antagonistas & inibidores , Proteínas de Transporte de Cátions/metabolismo , Diferenciação Celular , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Regulação da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Transporte de Íons , Magnésio/metabolismo , Masculino , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Osteocalcina/genética , Osteocalcina/metabolismo , Osteogênese/genética , Osteopontina/genética , Osteopontina/metabolismo , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Fator de Transcrição Sp7 , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Via de Sinalização Wnt
6.
Int J Mol Sci ; 18(1)2017 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-28106724

RESUMO

There is a growing interest in cell therapies using mesenchymal stromal cells (MSCs) for repairing bone defects. MSCs have the ability to differentiate into osteoprogenitors and osteoblasts as well as to form calcified bone matrix. However, the molecular mechanisms governing mineralization during osteogenic differentiation remain unclear. Non-collagenous proteins in the extracellular matrix are believed to control different aspects of the mineralization. Since osteocalcin is the most abundant non-collagenous bone matrix protein, the purpose of this study is to investigate the roles of osteocalcin in mineral species production during osteogenesis of MSCs. Using Raman spectroscopy, we found that the maturation of mineral species was affected by osteocalcin expression level. After osteocalcin was knocked down, the mineral species maturation was delayed and total hydroxyapatite was lower than the control group. In addition, the expression of osteogenic marker genes, including RUNX2, alkaline phosphatase, type I collagen, and osteonectin, was downregulated during osteogenic differentiation compared to the control group; whereas gene expression of osterix was upregulated after the knockdown. Together, osteocalcin plays an essential role for the maturation of mineral species and modulates osteogenic differentiation of MSCs. The results offer new insights into the enhancement of new bone formation, such as for the treatments of osteoporosis and fracture healing.


Assuntos
Calcificação Fisiológica/genética , Células-Tronco Mesenquimais/metabolismo , Osteocalcina/genética , Osteogênese/genética , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Antraquinonas , Diferenciação Celular/genética , Células Cultivadas , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Durapatita/metabolismo , Expressão Gênica , Células-Tronco Mesenquimais/citologia , Osteocalcina/metabolismo , Osteonectina/genética , Osteonectina/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise Espectral Raman , Coloração e Rotulagem/métodos
7.
Sci Rep ; 6: 37371, 2016 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-27901049

RESUMO

Lineage commitment and differentiation of mesenchymal stromal cells (MSCs) into osteoblasts in vitro is enhanced by a potent synthetic form of glucocorticoid (GC), dexamethasone (Dex). Paradoxically, when used chronically in patients, GCs exert negative effects on bone, a phenomenon known as glucocorticoid-induced osteoporosis in clinical practice. The mechanism on how GC differentially affects bone precursor cells to become mature osteoblasts during osteogenesis remains elusive. In this study, the dose and temporal regulation of Dex on MSC differentiation into osteoblasts were investigated. We found that continuous Dex treatment led to a net reduction of the maturation potential of differentiating osteoblasts. This phenomenon correlated with a decrease in glucocorticoid receptor (GR) expression, hastened degradation, and impaired sub cellular localization. Similarly, Histone Deacetylase 6 (HDAC6) expression was found to be regulated by Dex, co-localized with GR and this GR-HDAC6 complex occupied the promoter region of the osteoblast late marker osteocalcin (OCN). Combinatorial inhibition of HDAC6 and GR enhanced OCN expression. Together, the cross-talk between the Dex effector molecule GR and the inhibitory molecule HDAC6 provided mechanistic explanation of the bimodal effect of Dex during osteogenic differentiation of MSCs. These findings may provide new directions of research to combat glucocorticoid-induced osteoporosis.


Assuntos
Dexametasona/farmacologia , Desacetilase 6 de Histona/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Receptores de Glucocorticoides/metabolismo , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular , Linhagem Celular Tumoral , Células Cultivadas , Expressão Gênica/efeitos dos fármacos , Glucocorticoides/farmacologia , Desacetilase 6 de Histona/genética , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos Endogâmicos BALB C , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteogênese/genética , Receptores de Glucocorticoides/genética
8.
Stem Cell Res Ther ; 7(1): 120, 2016 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-27542358

RESUMO

BACKGROUND: Mesenchymal stromal cells (MSCs) are multipotent and have great potential in cell therapy. Previously we reported the differentiation potential of human MSCs into hepatocytes in vitro and that these cells can rescue fulminant hepatic failure. However, the conventional static culture method neither maintains growth factors at an optimal level constantly nor removes cellular waste efficiently. In addition, not only is the duration of differentiating hepatocyte lineage cells from MSCs required to improve, but also the need for a large number of hepatocytes for cell therapy has not to date been addressed fully. The purpose of this study is to design and develop an innovative microfluidic device to overcome these shortcomings. METHODS: We designed and fabricated a microfluidic device and a culture system for hepatic differentiation of MSCs using our protocol reported previously. The microfluidic device contains a large culture chamber with a stable uniform flow to allow homogeneous distribution and expansion as well as efficient induction of hepatic differentiation for MSCs. RESULTS: The device enables real-time observation under light microscopy and exhibits a better differentiation efficiency for MSCs compared with conventional static culture. MSCs grown in the microfluidic device showed a higher level of hepatocyte marker gene expression under hepatic induction. Functional analysis of hepatic differentiation demonstrated significantly higher urea production in the microfluidic device after 21 days of hepatic differentiation. CONCLUSIONS: The microfluidic device allows the generation of a large number of MSCs and induces hepatic differentiation of MSCs efficiently. The device can be adapted for scale-up production of hepatic cells from MSCs for cellular therapy.


Assuntos
Técnicas de Cocultura/métodos , Hepatócitos/citologia , Células-Tronco Mesenquimais/citologia , Microfluídica/métodos , Animais , Biomarcadores/metabolismo , Técnicas de Cultura de Células/métodos , Diferenciação Celular/fisiologia , Linhagem da Célula/fisiologia , Células Cultivadas , Expressão Gênica/fisiologia , Hepatócitos/metabolismo , Fígado/citologia , Fígado/metabolismo , Masculino , Células-Tronco Mesenquimais/metabolismo , Camundongos Endogâmicos C57BL
9.
Sci Rep ; 6: 31547, 2016 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-27526936

RESUMO

Cellular biophysical properties are novel biomarkers of cell phenotypes which may reflect the status of differentiating stem cells. Accurate characterizations of cellular biophysical properties, in conjunction with the corresponding biochemical properties could help to distinguish stem cells from primary cells, cancer cells, and differentiated cells. However, the correlated evolution of these properties in the course of directed stem cells differentiation has not been well characterized. In this study, we applied video particle tracking microrheology (VPTM) to measure intracellular viscoelasticity of differentiating human mesenchymal stromal/stem cells (hMSCs). Our results showed that osteogenesis not only increased both elastic and viscous moduli, but also converted the intracellular viscoelasticity of differentiating hMSCs from viscous-like to elastic-like. In contrast, adipogenesis decreased both elastic and viscous moduli while hMSCs remained viscous-like during the differentiation. In conjunction with bio- chemical and physical parameters, such as gene expression profiles, cell morphology, and cytoskeleton arrangement, we demonstrated that VPTM is a unique approach to quantify, with high data throughput, the maturation level of differentiating hMSCs and to anticipate their fate decisions. This approach is well suited for time-lapsed study of the mechanobiology of differentiating stem cells especially in three dimensional physico-chemical biomimetic environments including porous scaffolds.


Assuntos
Diferenciação Celular , Células-Tronco Mesenquimais/citologia , Actinas/metabolismo , Adipogenia , Adesões Focais , Perfilação da Expressão Gênica , Humanos , Células-Tronco Mesenquimais/metabolismo , Microscopia de Fluorescência , Osteogênese , Reologia , Viscosidade
10.
Sci Rep ; 6: 21253, 2016 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-26884253

RESUMO

The spatial boundary condition (SBC) arising from the surrounding microenvironment imposes specific geometry and spatial constraints that affect organogenesis and tissue homeostasis. Mesenchymal stromal cells (MSCs) sensitively respond to alterations of mechanical cues generated from the SBC. However, mechanical cues provided by a three-dimensional (3D) environment are deprived in a reductionist 2D culture system. This study investigates how SBC affects osteogenic differentiation of MSCs using 3D scaffolds with monodispersed pores and homogenous spherical geometries. MSCs cultured under SBCs with diameters of 100 and 150 µm possessed the greatest capability of osteogenic differentiation. This phenomenon was strongly correlated with MSC morphology, organization of actin cytoskeleton, and distribution of focal adhesion involving α2 and α5 integrins. Further silencing either α2 or α5 integrin significantly reduced the above mentioned mechanosensitivity, indicating that the α2 and α5 integrins as mechano-sensitive molecules mediate MSCs' ability to provide enhanced osteogenic differentiation in response to different spherical SBCs. Taken together, the findings provide new insights regarding how MSCs respond to mechanical cues from the surrounding microenvironment in a spherical SBC, and such biophysical stimuli should be taken into consideration in tissue engineering and regenerative medicine in conjunction with biochemical cues.


Assuntos
Diferenciação Celular , Células-Tronco Mesenquimais/citologia , Osteogênese , Nicho de Células-Tronco , Actinas/metabolismo , Adesão Celular , Técnicas de Cultura de Células , Sobrevivência Celular , Adesões Focais , Humanos , Integrina alfa2/metabolismo , Integrina alfa5/metabolismo , Esferoides Celulares , Tecidos Suporte
11.
Acta Biomater ; 32: 210-222, 2016 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-26790775

RESUMO

UNLABELLED: Osteogenic potential of mesenchymal stromal cells (MSCs) is mechanosensitive. It's affected by the mechanical properties of the cellular microenvironment, particularly its mechanical modulus. To explore the effect of mechanical modulus on osteogenesis in the third dimension (3D), this study used a novel polyacrylamide (PA) scaffold whose pores are monodisperse and spherical, the mechanical moduli of which can be tuned across a wide range. It was found that MSCs have similar proliferation rates in PA scaffolds independent of the matrix stiffness. The contractile force exerted by MSCs inside PA scaffolds was strong enough to deform the pores of scaffolds made of more compliant PAs (whose shear modulus, G'scaffold<4 kPa). Only scaffolds of the highest stiffness (G'scaffold=12 kPa) can withhold the contraction from MSCs. After osteogenic induction for 21 days, the expression profiles of marker genes showed that PA scaffolds of G'scaffold=12 kPa promoted osteogenesis of MSCs. Confocal image analysis demonstrated that there are more F-actin cytoskeletons and bundled stress fibers at higher matrix moduli in 2D and 3D. Moreover, the 3D porous structure promotes osteogenesis of MSCs more than 2D flat substrates. Together, the differences of cellular behaviors when cultured in 2D and 3D systems are evident. The PA scaffolds developed in the present study can be used for further investigation into the mechanism of MSC mechanosensing in the 3D context. STATEMENT OF SIGNIFICANCE: Mechanical properties of the microenvironment affect cellular behaviors, such as matrix stiffness. Traditionally, cell biological investigations have mostly employed cells growing on 2D substrates. The 3D porous PA scaffolds with the same topological conformation and pore sizes but different stiffness generated in this study showed that the differences of cellular behaviors in 2D and 3D systems are evident. Our 3D scaffolds provide insights into tissue engineering when stem cells incorporated with 3D scaffolds and support the future studies of cellular mechanobiology as well as the elucidation the role mechanical factor plays on the physiology and fate determination of MSCs in the 3D context.


Assuntos
Matriz Extracelular/metabolismo , Células-Tronco Mesenquimais/citologia , Osteogênese , Resinas Acrílicas/farmacologia , Actinas/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Hidrogéis/química , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Microfluídica , Osteogênese/efeitos dos fármacos , Osteogênese/genética , Porosidade , Tecidos Suporte/química
12.
Sci Rep ; 5: 16522, 2015 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-26558702

RESUMO

Microenvironments that modulate fate commitments of mesenchymal stromal cells (MSCs) are composed of chemical and physical cues, but the latter ones are much less investigated. Here we demonstrate that intermittent fluid shear stress (IFSS), a potent and physiologically relevant mechanical stimulus, regulates osteogenic differentiation of MSCs through Transient receptor potential melastatin 7 (TRPM7)-Osterix axis. Immunostaining showed the localization of TRPM7 near or at cell membrane upon IFSS, and calcium imaging analysis demonstrated the transient increase of cytosolic free calcium. Expressions of osteogenic marker genes including Osterix, but not Runx2, were upregulated after three-hour IFSS. Phosphorylation of p38 and Smad1/5 was promoted by IFSS as well. TRPM7 gene knockdown abolished the promotion of bone-related gene expressions and phosphorylation. We illustrate that TRPM7 is mechanosensitive to shear force of 1.2 Pa, which is much lower than 98 Pa pressure loading reported recently, and mediates distinct mechanotransduction pathways. Additionally, our results suggest the differential roles of TRPM7 in endochondral and intramembranous ossification. Together, this study elucidates the mechanotransduction in MSCs fate commitments and displays an efficient mechano-modulation for MSCs osteogenic differentiation. Such findings should be taken into consideration when designing relevant scaffolds and microfluidic devices for osteogenic induction in the future.


Assuntos
Mecanotransdução Celular , Células-Tronco Mesenquimais/metabolismo , Osteogênese , Estresse Mecânico , Canais de Cátion TRPM/genética , Animais , Biomarcadores , Cálcio/metabolismo , Diferenciação Celular , Citoesqueleto/metabolismo , Expressão Gênica , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Mecanotransdução Celular/genética , Camundongos , Osteogênese/genética , Fosforilação , Transporte Proteico , Proteína Smad1/metabolismo , Proteína Smad5/metabolismo , Canais de Cátion TRPM/antagonistas & inibidores , Canais de Cátion TRPM/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
13.
PLoS One ; 10(2): e0117128, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25679525

RESUMO

Addition of menthol to cigarettes may be associated with increased initiation of smoking. The potential mechanisms underlying this association are not known. Menthol, likely due to its effects on cold-sensing peripheral sensory neurons, is known to inhibit the sensation of irritation elicited by respiratory irritants. However, it remains unclear whether menthol modulates cigarette smoke irritancy and nicotine absorption during initial exposures to cigarettes, thereby facilitating smoking initiation. Using plethysmography in a C57Bl/6J mouse model, we examined the effects of L-menthol, the menthol isomer added to cigarettes, on the respiratory sensory irritation response to primary smoke irritants (acrolein and cyclohexanone) and smoke of Kentucky reference 2R4 cigarettes. We also studied L-menthol's effect on blood levels of the nicotine metabolite, cotinine, immediately after exposure to cigarette smoke. L-menthol suppressed the irritation response to acrolein with an apparent IC50 of 4 ppm. Suppression was observed even at acrolein levels well above those necessary to produce a maximal response. Cigarette smoke, at exposure levels of 10 mg/m³ or higher, caused an immediate and marked sensory irritation response in mice. This response was significantly suppressed by L-menthol even at smoke concentrations as high as 300 mg/m³. Counterirritation by L-menthol was abolished by treatment with a selective inhibitor of Transient Receptor Potential Melastatin 8 (TRPM8), the neuronal cold/menthol receptor. Inclusion of menthol in the cigarette smoke resulted in roughly a 1.5-fold increase in plasma cotinine levels over those observed in mice exposed to smoke without added menthol. These findings document that, L-menthol, through TRPM8, is a strong suppressor of respiratory irritation responses, even during highly noxious exposures to cigarette smoke or smoke irritants, and increases blood cotinine. Therefore, L-menthol, as a cigarette additive, may promote smoking initiation and nicotine addiction.


Assuntos
Cotinina/sangue , Mentol/farmacologia , Fumar/efeitos adversos , Acroleína/administração & dosagem , Animais , Feminino , Irritantes/administração & dosagem , Mentol/administração & dosagem , Camundongos , Respiração/efeitos dos fármacos , Canais de Cátion TRPM/agonistas , Canais de Cátion TRPM/antagonistas & inibidores
14.
Tissue Eng Part A ; 21(7-8): 1444-54, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25625545

RESUMO

In vitro differentiation of stem cells into specific cell lineages provides a stable cell supply for cell therapy and tissue engineering. Therefore, understanding the mechanisms underlying such differentiation processes is critical for generating committed lineage-specific cell progenies effectively. We previously developed a two-step protocol to differentiate mesenchymal stromal cells (MSCs) into hepatocyte-like cells. Since hepatic differentiation involves mesenchymal-epithelial transition (MET), we hypothesize that promoting MET could further accelerate the differentiation process. Ras-related C3 botulinum toxin substrate 1 (Rac1) is involved in actin polymerization and its role in MET was investigated in the study. Our results showed that inhibition of Rac1 activation by Rac1-specific inhibitor, NSC23766, led to cells favoring epithelial morphology and being more packed during hepatic differentiation. In addition, Rac1 inhibition accelerated the upregulation of hepatic marker genes accompanied by more mature hepatic functions. Taken together, promotion of MET by inhibiting Rac1 accelerates the hepatic differentiation of MSCs. Our findings open a new prospect of directing the commitment of MSCs by manipulating cell morphology and cytoskeleton arrangement through small molecules. The results provide further insight into scaffold design for rapid production of MSC-differentiated hepatocytes.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Transição Epitelial-Mesenquimal , Fígado/citologia , Células-Tronco Mesenquimais/citologia , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas rac1 de Ligação ao GTP/antagonistas & inibidores , Actinas/metabolismo , Animais , Biomarcadores/metabolismo , Caderinas/metabolismo , Movimento Celular/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Fenótipo , Regulação para Cima/efeitos dos fármacos , Proteínas rac1 de Ligação ao GTP/metabolismo
15.
J Cancer Res Clin Oncol ; 141(1): 11-21, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25060070

RESUMO

PURPOSE: Lysine-specific demethylase 1 (LSD1) was highly expressed in several malignancies and had been implicated in pathological processes of cancer cells. However, the role of LSD1 in colorectal cancer (CRC) carcinogenesis, prognosis and treatment remains uncharacterized. METHODS: In this study, we examined LSD1 expression in paraffin-embedded CRC specimens from 295 patients, including 65 patients with paired samples of colorectal carcinoma, adjacent adenoma and normal colorectal tissues. Using an LSD1 inhibitor, CBB1003, as a probe, we studied the association between LSD1 and leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5), a CRC stem cell marker involved in carcinogenesis. The anti-tumor effects of CBB1003 on CRC cells were also examined. RESULTS: LSD1 expression was significantly elevated in colorectal tumor tissues compared with adjacent adenoma and normal colorectal tissues (P < 0.001), and LSD1 levels were significantly correlated with an advanced AJCC T stage (P = 0.012) and distant metastasis (P = 0.004). CBB1003 inhibited CRC cell proliferation and colony formation. In cultured CRC cells, inhibiting LSD1 activity by CBB1003 caused a decrease in LGR5 levels while overexpression of LGR5 reduced CBB1003-induced cell death. We also observed the inactivation of ß-catenin/TCF signaling after CBB1003 treatment, consistent with the positive correlations among LSD1, LGR5, ß-catenin and c-Myc expression in human colon tumor and adenoma tissues. CONCLUSION: Our result suggested that LSD1 overexpression promotes CRC development and that the LSD1 inhibitor inhibits CRC cell growth by down-regulating LGR5 levels and inactivates the Wnt/ß-catenin pathway. Thus, LSD1 and its inhibitor might provide a new target or a useful strategy for therapy of CRC.


Assuntos
Adenoma/tratamento farmacológico , Amidinas/farmacologia , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Histona Desmetilases/antagonistas & inibidores , Piperazinas/farmacologia , Receptores Acoplados a Proteínas G/metabolismo , Adenoma/metabolismo , Adenoma/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose/efeitos dos fármacos , Western Blotting , Ciclo Celular/efeitos dos fármacos , Colo/efeitos dos fármacos , Colo/metabolismo , Colo/patologia , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Feminino , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Humanos , Técnicas Imunoenzimáticas , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores Acoplados a Proteínas G/genética , Reto/efeitos dos fármacos , Reto/metabolismo , Reto/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas
16.
Cell Signal ; 26(11): 2333-42, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25025569

RESUMO

Colorectal cancer (CRC) is one of the most common causes of cancer-related death worldwide. The leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) is a newly identified surface marker of colorectal cancer stem cells (CSCs). Expression level of LGR5 is commonly elevated in human CRCs. Our previous study demonstrated that the elevated expression of LGR5 is associated with CRC initiation and progression. However, the role of LGR5 in CRC pathogenesis has not been sufficiently established. In this study, we aimed to characterize the role of LGR5 in CRC pathogenesis using the loss-of-function approach. Depletion of LGR5 suppressed the growth of several cultured CRC cells and caused an increase in the fraction of apoptotic cells, which were analyzed using Annexin V/PI staining and DNA fragmentation assay. Furthermore, depleting LGR5 induced apoptosis through the loss of mitochondrial membrane potential. Additionally, depletion of LGR5 suppressed ß-catenin nuclear translocation and blocked the activity of Wnt/ß-catenin signaling as manifested in the reduced expression of c-myc and cyclin D, two Wnt/ß-catenin targets in CRC cells. Treatment with Wnt3a considerably alleviated the growth inhibition and apoptotic cell death induced by LGR5 depletion in CRC cells. These data suggested that LGR5 regulates cell proliferation and survival by targeting the Wnt/ß-catenin signaling pathway. Thus, the findings of this study suggest that LGR5 plays a vital role in CRC pathogenesis and has the potential to serve as a diagnostic marker and a therapeutic target for CRC patients.


Assuntos
Apoptose , Biomarcadores Tumorais/metabolismo , Neoplasias Colorretais/metabolismo , Proteínas de Neoplasias/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Via de Sinalização Wnt , Anexina A5/genética , Anexina A5/metabolismo , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Fragmentação do DNA , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Humanos , Proteínas de Neoplasias/genética , Receptores Acoplados a Proteínas G/genética , beta Catenina/genética , beta Catenina/metabolismo
17.
Cell Transplant ; 23(1): 1-11, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24439034

RESUMO

Stem cells are undifferentiated cells with the ability to self-renew and to differentiate into diverse specialized cell types; hence, they have great potential in tissue engineering and cell therapies. In addition to biochemical regulation, the physical properties of the microenvironments, such as scaffold topography, substrate stiffness, and mechanical forces, including fluid shear stress, compression, and tensile strain, can also regulate the proliferation and differentiation of stem cells. Upon physical stimuli, cytoskeleton rearrangements are expected to counterbalance the extracellular mechanical forces, trigger signaling cascades, and eventually cause epigenetic modifications. This article mainly focuses on the mechanosensing, which is the upstream event of stem cell mechanotransduction and the downstream one of physical stimuli. Putative mechanosensors such as ion channels, integrins, and cell membrane as well as primary cilia are discussed. Because mechanical environment is an important stem cell niche, identification of mechanosensors not only can elucidate the mechanisms of mechanotransduction and fate commitments but also bring new prospects of the mechanical control as well as drug development for clinical application.


Assuntos
Mecanotransdução Celular/fisiologia , Células-Tronco Mesenquimais/fisiologia , Animais , Cálcio/fisiologia , Diferenciação Celular/fisiologia , Humanos , Estresse Mecânico , Relação Estrutura-Atividade , Engenharia Tecidual
18.
Biomed Pharmacother ; 67(8): 791-9, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24138824

RESUMO

INTRODUCTION: Chemotherapy failure is a major problem in patients with advanced colorectal carcinoma (CRC). Leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5) is a well-established target gene of the Wnt pathway and is a bona fide marker of CRC cancer stem cells (CSCs). Our previous study showed that CRC patients with higher Lgr5 level are associated with poor response to 5-fluoracil-based treatment. In this study, we investigated the mechanisms underlying Lgr5-associated chemoresistance in cancer stem cells derived from cultured CRC cells. MATERIALS AND METHODS: Cancer stem cells were isolated from CRC cell lines by spheroid culture. The effect of Lgr5 on CRC cancer stem cell was investigated using both gain- and loss-of-function approaches. Stemness property was evaluated using sphere formation assay, side population analysis, and stem cell marker expression. Lgr5 and ABCB1 expression in CRC tissues was determined using immunohistochemical staining. RESULTS: Forced expression of Lgr5 increased the CRC sphere-forming efficiency and spheroid size while depletion of Lgr5 reduced the stem cell property in cultured CRC cells. Over-expression of Lgr5 also reduced the sensitivity of cultured CRC cells, including adherent and spheroids, towards 5-fluoracil and oxalipatin. In addition, Lgr5 positively regulates the expression of ABCB1 in both adherent and spheroid CRC cells. Finally, in human CRC tissues, higher expression levels of Lgr5 were associated with higher ABCB1 expression. CONCLUSIONS: The present study demonstrated that Lgr5 plays an active role in promoting the cancer stem cell property and that Lgr5 confers chemoresistance to CRC cells via ABCB1 induction.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/biossíntese , Neoplasias Colorretais/patologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Células-Tronco Neoplásicas/efeitos dos fármacos , Receptores Acoplados a Proteínas G/biossíntese , Esferoides Celulares/efeitos dos fármacos , Subfamília B de Transportador de Cassetes de Ligação de ATP , Antineoplásicos/farmacologia , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/metabolismo , Citometria de Fluxo , Fluoruracila/farmacologia , Humanos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Compostos Organoplatínicos/farmacologia , Oxaliplatina , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia
19.
Int J Colorectal Dis ; 28(11): 1535-46, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23784051

RESUMO

BACKGROUND: The leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5) is an adult intestinal stem cell marker frequently detected in human colorectal cancers (CRCs). However, the value of Lgr5 level in CRC prognosis and treatment prediction has not been well characterized. METHODS: We examined Lgr5 expression in 384 formalin-fixed paraffin-embedded CRC specimens from 296 CRC patients, including 64 patients treated with 5-fluorouracil (5-FU)-based chemotherapy. The effects of Lgr5 on cell proliferation, survival, and drug resistance were examined in cultured CRC cells. RESULTS: Elevated expression of Lgr5 was observed in CRC tissues, and Lgr5 protein levels were significantly correlated with an advanced American Joint Committee on Cancer stage (P < 0.001), T stage (P < 0.001), N stage (P < 0.001), and distant metastasis (P < 0.001). High expression levels of Lgr5 were significantly associated with shorter disease-free survival (P < 0.001) and shorter cancer-specific survival (P = 0.007) in CRC patients. Among the chemotherapy-treated subgroups, patients with low Lgr5 level showed a better response rate (65 %) than patients with high Lgr5 level (37 %) towards 5-FU-based treatment (P = 0.025). In cultured CRC cell lines, knocking down Lgr5 suppressed cell proliferation and colony formation ability, while it enhanced apoptosis and rendered cells more sensitive to chemotherapeutic agents. In contrast, overexpression of Lgr5 increased cell proliferation and enhanced chemoresistance. CONCLUSION: These results suggest that elevated Lgr5 level is associated with CRC progression and treatment response and has the potential to serve as a therapeutic target in CRC patients.


Assuntos
Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Resistencia a Medicamentos Antineoplásicos , Fluoruracila/uso terapêutico , Receptores Acoplados a Proteínas G/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Neoplasias Colorretais/patologia , Intervalo Livre de Doença , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Fluoruracila/farmacologia , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Prognóstico , Análise de Regressão , Resultado do Tratamento
20.
Chang Gung Med J ; 32(3): 276-82, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19527606

RESUMO

BACKGROUND: Extracorporeal shock wave treatment (ESWT) has been proven effective in enhancing spinal fusion in a preliminary animal study. However, biomechanical tests were not performed. METHODS: All 12 rabbits in this study underwent decortication at the bilateral L5 and L6 transverse processes. Bone was chipped off and placed onto the intertransverse space. The rabbits were divided into two groups, a study group (n = 6) and a control group (n = 6). In the study group, the bilateral L5 and L6 transverse processes were treated with 1000 impulses of ESWT at 14 kilovolts (KV) (equivalent to 0.18 mJ/mm(2)) at 12 and 18 weeks after surgery. The control group rabbits did not undergo ESWT. A series of radiographic examinations on each rabbit were subsequently performed. All rabbits were killed at 21 weeks, and their spines were harvested for biomechanical tests. RESULTS: Radiographic examination showed 5 of the 6 rabbits in the study group had callus formation in the fusion masses. Biomechanical tests of the fusion segments showed that the mean flexion stiffness (with internal control) in the study group was 2.11 +/- 0.46, while that in the control group was 1.17 +/- 0.19. The mean extension stiffness (with internal control) in the study group was 1.70 +/- 0.39, while that in the control group was 1.23 +/- 0.29. Statistical analysis showed that the fusion segments in the study group had significantly better flexion and extension stiffness than those in the control group (p < 0.05). CONCLUSION: In this animal study, radiographic examinations showed that ESWT stimulated new bone growth. Biomechanical tests showed that ESWT significantly increased the flexion and extension stiffness of spinal fusion segments.


Assuntos
Ondas de Choque de Alta Energia , Fusão Vertebral , Animais , Fenômenos Biomecânicos , Vértebras Lombares/cirurgia , Masculino , Coelhos
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...