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1.
Chinese Journal of Burns ; (6): 354-362, 2022.
Article in Chinese | WPRIM | ID: wpr-936018

ABSTRACT

Objective: To investigate the regulatory effects of bio-intensity electric field on the transformation of human skin fibroblasts (HSFs). Methods: The experimental research methods were used. HSFs were collected and divided into 200 mV/mm electric field group treated with 200 mV/mm electric field for 6 h and simulated electric field group placed in the electric field device without electricity for 6 h. Changes in morphology and arrangement of cells were observed in the living cell workstation; the number of cells at 0 and 6 h of treatment was recorded, and the rate of change in cell number was calculated; the direction of cell movement, movement velocity, and trajectory velocity within 3 h were observed and calculated (the number of samples was 34 in the simulated electric field group and 30 in 200 mV/mm electric field group in the aforementioned experiments); the protein expression of α-smooth muscle actin (α-SMA) in cells after 3 h of treatment was detected by immunofluorescence method (the number of sample was 3). HSFs were collected and divided into simulated electric field group placed in the electric field device without electricity for 3 h, and 100 mV/mm electric field group, 200 mV/mm electric field group, and 400 mV/mm electric field group which were treated with electric fields of corresponding intensities for 3 h. Besides, HSFs were divided into simulated electric field group placed in the electric field device without electricity for 6 h, and electric field treatment 1 h group, electric field treatment 3 h group, and electric field treatment 6 h group treated with 200 mV/mm electric field for corresponding time. The protein expressions of α-SMA and proliferating cell nuclear antigen (PCNA) were detected by Western blotting (the number of sample was 3). Data were statistically analyzed with Mann-Whitney U test, one-way analysis of variance, independent sample t test, and least significant difference test. Results: After 6 h of treatment, compared with that in simulated electric field group, the cells in 200 mV/mm electric field group were elongated in shape and locally adhered; the cells in simulated electric field group were randomly arranged, while the cells in 200 mV/mm electric field group were arranged in a regular longitudinal direction; the change rates in the number of cells in the two groups were similar (P>0.05). Within 3 h of treatment, the cells in 200 mV/mm electric field group had an obvious tendency to move toward the positive electrode, and the cells in simulated electric field group moved around the origin; compared with those in simulated electric field group, the movement velocity and trajectory velocity of the cells in 200 mV/mm electric field group were increased significantly (with Z values of -5.33 and -5.41, respectively, P<0.01), and the directionality was significantly enhanced (Z=-4.39, P<0.01). After 3 h of treatment, the protein expression of α-SMA of cells in 200 mV/mm electric field group was significantly higher than that in simulated electric field group (t=-9.81, P<0.01). After 3 h of treatment, the protein expressions of α-SMA of cells in 100 mV/mm electric field group, 200 mV/mm electric field group, and 400 mV/mm electric field group were 1.195±0.057, 1.606±0.041, and 1.616±0.039, respectively, which were significantly more than 0.649±0.028 in simulated electric field group (P<0.01). Compared with that in 100 mV/mm electric field group, the protein expressions of α-SMA of cells in 200 mV/mm electric field group and 400 mV/mm electric field group were significantly increased (P<0.01). The protein expressions of α-SMA of cells in electric field treatment 1 h group, electric field treatment 3 h group, and electric field treatment 6 h group were 0.730±0.032, 1.561±0.031, and 1.553±0.045, respectively, significantly more than 0.464±0.020 in simulated electric field group (P<0.01). Compared with that in electric field treatment 1 h group, the protein expressions of α-SMA in electric field treatment 3 h group and electric field treatment 6 h group were significantly increased (P<0.01). After 3 h of treatment, compared with that in simulated electric field group, the protein expressions of PCNA of cells in 100 mV/mm electric field group, 200 mV/mm electric field group, and 400 mV/mm electric field group were significantly decreased (P<0.05 or P<0.01); compared with that in 100 mV/mm electric field group, the protein expressions of PCNA of cells in 200 mV/mm electric field group and 400 mV/mm electric field group were significantly decreased (P<0.05 or P<0.01); compared with that in 200 mV/mm electric field group, the protein expression of PCNA of cells in 400 mV/mm electric field group was significantly decreased (P<0.01). Compared with that in simulated electric field group, the protein expressions of PCNA of cells in electric field treatment 1 h group, electric field treatment 3 h group, and electric field treatment 6 h group were significantly decreased (P<0.01); compared with that in electric field treatment 1 h group, the protein expressions of PCNA of cells in electric field treatment 3 h group and electric field treatment 6 h group were significantly decreased (P<0.05 or P<0.01); compared with that in electric field treatment 3 h group, the protein expression of PCNA of cells in electric field treatment 6 h group was significantly decreased (P<0.01). Conclusions: The bio-intensity electric field can induce the migration of HSFs and promote the transformation of fibroblasts to myofibroblasts, and the transformation displays certain dependence on the time and intensity of electric field.


Subject(s)
Humans , Actins/biosynthesis , Cell Differentiation/physiology , Cell Movement/physiology , Electric Stimulation Therapy , Electricity , Fibroblasts/physiology , Myofibroblasts/physiology , Proliferating Cell Nuclear Antigen/biosynthesis , Skin/cytology
2.
Chinese Journal of Burns ; (6): 296-300, 2022.
Article in Chinese | WPRIM | ID: wpr-936009

ABSTRACT

Sweat gland is one of the important appendage organs of the skin, which plays an important role in thermoregulation and homeostasis maintenance. Sweat glands are damaged and unable to self-repair after burns, resulting in perspiration disorders eventually. However, current clinical strategies cannot restore the function of the damaged sweat glands effectively. Therefore, it is urgent to seek treatments that can promote the regeneration of sweat glands and restore their normal functions. Stem cells have extensive sources, low immunogenicity, high proliferation capacity, and multi-directional differentiation potential, which have become a focus in the field of regenerative medicine. In recent years, a variety of stem cells have been induced to differentiate into sweat gland-like tissue with certain secretory function, which provides treatment direction for sweat gland regeneration after burns in clinic. This article reviews the recent research advances on the application of stem cells in sweat gland regeneration from the perspectives of the manner by which stem cells transform into sweat gland cells in different environments and their influencing factors.


Subject(s)
Cell Differentiation/physiology , Regeneration/physiology , Skin , Stem Cells , Sweat Glands/physiology
3.
Article in English | WPRIM | ID: wpr-929148

ABSTRACT

Multiple signaling pathways are involved in the regulation of cell proliferation and differentiation in odontogenesis and dental tissue renewal, but the details of these mechanisms remain unknown. Here, we investigated the expression patterns of a transcription factor, Krüppel-like factor 6 (KLF6), during the development of murine tooth germ and its function in odontoblastic differentiation. KLF6 was almost ubiquitously expressed in odontoblasts at various stages, and it was co-expressed with P21 (to varying degrees) in mouse dental germ. To determine the function of Klf6, overexpression and knockdown experiments were performed in a mouse dental papilla cell line (iMDP-3). Klf6 functioned as a promoter of odontoblastic differentiation and inhibited the proliferation and cell cycle progression of iMDP-3 through p21 upregulation. Dual-luciferase reporter assay and chromatin immunoprecipitation showed that Klf6 directly activates p21 transcription. Additionally, the in vivo study showed that KLF6 and P21 were also co-expressed in odontoblasts around the reparative dentin. In conclusion, Klf6 regulates the transcriptional activity of p21, thus promoting the cell proliferation to odontoblastic differentiation transition in vitro. This study provides a theoretical basis for odontoblast differentiation and the formation of reparative dentine regeneration.


Subject(s)
Animals , Mice , Cell Differentiation/physiology , Cell Proliferation , Odontoblasts/metabolism , Odontogenesis , Tooth Germ
4.
Article in English | WPRIM | ID: wpr-929135

ABSTRACT

Therapeutic dentin regeneration remains difficult to achieve, and a majority of the attention has been given to anabolic strategies to promote dentinogenesis directly, whereas, the available literature is insufficient to understand the role of inflammation and inflammatory complement system on dentinogenesis. The aim of this study is to determine the role of complement C5a receptor (C5aR) in regulating dental pulp stem cells (DPSCs) differentiation and in vivo dentin regeneration. Human DPSCs were subjected to odontogenic differentiation in osteogenic media treated with the C5aR agonist and C5aR antagonist. In vivo dentin formation was evaluated using the dentin injury/pulp-capping model of the C5a-deficient and wild-type mice. In vitro results demonstrate that C5aR inhibition caused a substantial reduction in odontogenic DPSCs differentiation markers such as DMP-1 and DSPP, while the C5aR activation increased these key odontogenic genes compared to control. A reparative dentin formation using the C5a-deficient mice shows that dentin regeneration is significantly reduced in the C5a-deficient mice. These data suggest a positive role of C5aR in the odontogenic DPSCs differentiation and tertiary/reparative dentin formation. This study addresses a novel regulatory pathway and a therapeutic approach for improving the efficiency of dentin regeneration in affected teeth.


Subject(s)
Animals , Mice , Cell Differentiation/physiology , Cells, Cultured , Complement C5a/metabolism , Dental Pulp/physiology , Dentin , Receptor, Anaphylatoxin C5a , Stem Cells
5.
Rev. méd. Maule ; 36(2): 8-19, dic. 2021. graf
Article in Spanish | LILACS | ID: biblio-1377868

ABSTRACT

Skeletal muscle appears to play a central role in the development of insulin resistance (IR) and consequently the metabolic syndrome due to high-fat diets, obesity, and aging. Recent evidence suggests that some bioactive compounds present in natural products can affect blood glucose, possibly due to interactions between the compounds and glucose transporters. As an objective, to evaluate the effect of the extract of the green bean (PV, Phaseolus vulgaris) and apple of small fruit of thinning (Malus domestica, MAF and MIT extracts) on the incorporation of glucose in C2C12 muscle cells. For this, the cytotoxic effect of the extracts on the cells was determined by detecting formazan. Subsequently, glucose incorporation was determined using a fluorescent glucose analog in cells treated with the extracts. Finally, the effect of the extracts on IL-6 and TNFα production was evaluated by ELISA. Results: PV and MAF decreased 50% of viability at 1000µg / mL while MIT only decreased 10% at that concentration. PV had no significant effect on glucose incorporation and the MAF and MIT extract extracts significantly increased glucose incorporation at 100 µg / mL (13500 and 18000 URF respectively). PV increases the secretion of IL-6 and TNF-α, MAF and MIT only increase the expression of IL-6. Conclusion: These results make it possible to establish natural extracts derived from thinning small fruit apple can be used as a possible treatment for pathologies with high blood glucose levels.


Subject(s)
Humans , Cell Differentiation/physiology , Obesity/epidemiology , Insulin Resistance , Interleukin-6 , Tumor Necrosis Factor-alpha , Phaseolus , Malus , Glucose
6.
Rev. bras. med. esporte ; 27(spe2): 73-78, Apr.-June 2021. graf
Article in English | LILACS | ID: biblio-1280080

ABSTRACT

ABSTRACT Myoblasts fuse into multinucleated muscle fibers to form and promote the growth of skeletal muscle. In order to analyze the role of myostatin (MSTN) in body fat, skeletal muscle cell proliferation and differentiation and energy metabolism, this study will use the antisense RNA technology of gene chip technology to study it. The results showed that the MSTN gene regulated the growth and proliferation of myoblasts and affected the development of skeletal muscle by affecting the expression of Cdc42, bnip2, p38 and other genes; knockout or overexpression of the MSTN gene would lead to a trend of fat-related genes from fat synthesis to fat decomposition; after the MSTN gene was knocked down, the expression levels of cpti-b, PPARG and other genes in the cells were corresponding after MSTN overexpression, the relative expression of the PPARG gene decreased. It is suggested that the knockout or overexpression of MSTN may affect lipid accumulation, and cpti-b and PPARG may directly regulate lipid level. It is hoped that this experiment can provide a reference for the study of MSTN effect on fat deposition.


RESUMO Os mioblastos se fundem eM fibras musculares multinucleadas para formar e promover o crescimento do músculo esquelético. A fim de analisar o papel da miostatina (MSTN) na gordura corporal, proliferação de células musculares esqueléticas e diferenciação e metabolismo energético, este estudo utilizará a tecnologia anti-RNA de chips genéticos para estudá-la. Os resultados mostraram que o gene MSTN regulava o crescimento e a proliferação de mioblastos e afetava o desenvolvimento do músculo esquelético, afetando a expressão de Cdc42, bnip2, p38 e outros genes; a eliminação ou sobrexpressão do gene MSTN conduziria a uma tendência de os genes adiposos sintetizarem a gordura até sua decomposição; após a eliminação do gene MSTN, os níveis de expressão de cpti-b, PPARG e outros genes nas células mostraram-se correspondentes após a sobrexpressão do gene MSTN, e a expressão relativa do gene PPARG diminuiu. Sugere-se que a eliminação ou sobrexpressão da MSTN possa afetar a acumulação de lipídeos, e o cpti-b e o PPARG podem regular diretamente o nível lipídico. Espera-se que esta experiência possa fornecer uma referência para o estudo do efeito da MSTN sobre a deposição de gordura.


RESUMEN Los mioblastos se funden en fibras musculares multinucleadas para formar y promover el crecimiento del músculo esquelético. A fin de analizar el papel de la miostatina (MSTN) en la grasa corporal, proliferación de células musculares esqueléticas y diferenciación y metabolismo energético, este estudio utilizará la tecnología anti-RNA de chips genéticos para estudiarla. Los resultados mostraron que el gen MSTN regulaba el crecimiento y la proliferación de mioblastos y afectaba el desarrollo del músculo esquelético, afectando la expresión de Cdc42, bnip2, p38 y otros genes; la eliminación o sobreexpresión del gen MSTN conduciría a una tendencia de que los genes adiposos sinteticen la grasa hasta su descomposición; después de la eliminación del gen MSTN, los niveles de expresión de cpti-b, PPARG y otros genes en las células se mostraron correspondientes después de la sobreexpresión del gen MSTN, y la expresión relativa del gen PPARG disminuyó. Se sugiere que la eliminación o sobreexpresión de la MSTN pueda afectar la acumulación de lipídos, y el cpti-b y el PPARG pueden regular directamente el nivel lipídico. Se espera que esta experiencia pueda proveer una referencia para el estudio del efecto de la MSTN sobre el depósito de grasa.


Subject(s)
Animals , Cattle , Cell Differentiation/physiology , Adipocytes/metabolism , Myoblasts, Skeletal/metabolism , Cell Proliferation/physiology , Energy Metabolism , Myostatin/metabolism , Oligonucleotide Array Sequence Analysis
7.
Braz. oral res. (Online) ; 34: e006, 2020. tab, graf
Article in English | LILACS | ID: biblio-1055522

ABSTRACT

Abstract Induced pluripotent stem (iPS) cells could be induced into ameloblast-like cells by ameloblasts serum-free conditioned medium (ASF-CM), and bone morphogenetic proteins (BMPs) might be essential during the regulation of this process. The present study investigates the signal transduction that regulates the ameloblastic differentiation of iPS cells induced by ASF-CM. Mouse iPS cells were characterized and then cultured for 14 days in epithelial cell medium (control) or ASF-CM. Bone morphogenetic protein receptor II (BMPR-II) siRNA, inhibitor of Smad1/5 phosphorylation activated by activin receptor-like kinase (ALK) receptors, and inhibitors of mitogen-activated protein kinases (MAPKs) phosphorylation were used to treat the iPS cells in combination with ASF-CM. Real-time PCR, western blotting, and immunofluorescent staining were used to evaluate the expressions of ameloblast markers ameloblastin, enamelin, and cytokeratin-14. BMPR-II gene and protein levels increased markedly in ASF-CM-treated iPS cells compared with the controls, while the mRNA levels of Bmpr-Ia and Bmpr-Ib were similar between the ASF-CM and control groups. ASF-CM stimulation significantly increased the gene and protein expression of ameloblastin, enamelin and cytokeratin-14, and phosphorylated SMAD1/5, p38 MAPK, and ERK1/2 MAPK compared with the controls. Knockdown of BMPR-II and inhibition of Smad1/5 phosphorylation both could significantly reverse the increased expression of ameloblastin, enamelin, and cytokeratin-14 induced by ASF-CM, while neither inhibition of p38 nor ERK1/2 phosphorylation had significant reversing effects. We conclude that smad1/5 signaling transduction, activated by ALK receptors, regulates the ameloblastic differentiation of iPS cells induced by ameloblast-conditioned medium.


Subject(s)
Signal Transduction/physiology , Smad1 Protein/physiology , Induced Pluripotent Stem Cells/cytology , Ameloblasts/cytology , Phosphorylation , Time Factors , Gene Expression , Cell Differentiation/physiology , Cell Differentiation/genetics , Cells, Cultured , Blotting, Western , Fluorescent Antibody Technique , Culture Media, Serum-Free , Reverse Transcriptase Polymerase Chain Reaction , MAP Kinase Signaling System/physiology , Activin Receptors/analysis , Activin Receptors/physiology , RNA Interference , p38 Mitogen-Activated Protein Kinases/analysis , p38 Mitogen-Activated Protein Kinases/physiology , Bone Morphogenetic Protein Receptors, Type II/analysis , Bone Morphogenetic Protein Receptors, Type II/physiology , Smad1 Protein/analysis
8.
Braz. oral res. (Online) ; 34: e006, 2020. tab, graf
Article in English | LILACS | ID: biblio-1089380

ABSTRACT

Abstract Induced pluripotent stem (iPS) cells could be induced into ameloblast-like cells by ameloblasts serum-free conditioned medium (ASF-CM), and bone morphogenetic proteins (BMPs) might be essential during the regulation of this process. The present study investigates the signal transduction that regulates the ameloblastic differentiation of iPS cells induced by ASF-CM. Mouse iPS cells were characterized and then cultured for 14 days in epithelial cell medium (control) or ASF-CM. Bone morphogenetic protein receptor II (BMPR-II) siRNA, inhibitor of Smad1/5 phosphorylation activated by activin receptor-like kinase (ALK) receptors, and inhibitors of mitogen-activated protein kinases (MAPKs) phosphorylation were used to treat the iPS cells in combination with ASF-CM. Real-time PCR, western blotting, and immunofluorescent staining were used to evaluate the expressions of ameloblast markers ameloblastin, enamelin, and cytokeratin-14. BMPR-II gene and protein levels increased markedly in ASF-CM-treated iPS cells compared with the controls, while the mRNA levels of Bmpr-Ia and Bmpr-Ib were similar between the ASF-CM and control groups. ASF-CM stimulation significantly increased the gene and protein expression of ameloblastin, enamelin and cytokeratin-14, and phosphorylated SMAD1/5, p38 MAPK, and ERK1/2 MAPK compared with the controls. Knockdown of BMPR-II and inhibition of Smad1/5 phosphorylation both could significantly reverse the increased expression of ameloblastin, enamelin, and cytokeratin-14 induced by ASF-CM, while neither inhibition of p38 nor ERK1/2 phosphorylation had significant reversing effects. We conclude that smad1/5 signaling transduction, activated by ALK receptors, regulates the ameloblastic differentiation of iPS cells induced by ameloblast-conditioned medium.


Subject(s)
Signal Transduction/physiology , Smad1 Protein/physiology , Induced Pluripotent Stem Cells/cytology , Ameloblasts/cytology , Phosphorylation , Time Factors , Gene Expression , Cell Differentiation/physiology , Cell Differentiation/genetics , Cells, Cultured , Blotting, Western , Fluorescent Antibody Technique , Culture Media, Serum-Free , Reverse Transcriptase Polymerase Chain Reaction , MAP Kinase Signaling System/physiology , Activin Receptors/analysis , Activin Receptors/physiology , RNA Interference , p38 Mitogen-Activated Protein Kinases/analysis , p38 Mitogen-Activated Protein Kinases/physiology , Bone Morphogenetic Protein Receptors, Type II/analysis , Bone Morphogenetic Protein Receptors, Type II/physiology , Smad1 Protein/analysis
9.
Braz. j. med. biol. res ; 53(4): e9282, 2020. graf
Article in English | LILACS | ID: biblio-1089351

ABSTRACT

Vitiligo is an acquired pigmentary disorder resulting from selective destruction of melanocytes. Emerging studies have suggested that T helper cell 17 (Th17) is potentially implicated in vitiligo development and progression. It was recently discovered that metabotropic glutamate receptor 4 (mGluR4) can modulate Th17-mediated adaptive immunity. However, the influence of mGluR4 on melanogenesis of melanocytes has yet to be elucidated. In the present study, we primarily cultured mouse bone marrow-derived dendritic cells (BMDC) and then knocked down and over-expressed mGluR4 using transfection. Transduced BMDC were co-cultured with CD4+ T cells and the expression of Th17-related cytokines were measured. The morphology and melanogenesis of B16 cells were observed after being treated with co-culture medium of CD4+ T cells and transduced BMDC. We found that mGluR4 knockdown did not affect the co-stimulatory CD80 and CD86 upregulation after lipopolysaccharide stimulation but did increase the expression of Th17-related cytokines, and further down-regulated the expression of microphthalmia-associated transcription factor (MITF) and the downstream genes, decreased melanin production, and destroyed the morphology of B16 cells. Conversely, over-expression of mGluR4 reduced the expression of CD80 and CD86, suppressed the production of Th17-related cytokines, increased the expression of MITF, and did not destroy the morphology of B16 cells. Our study confirmed that mGluR4 modulated the Th17 cell polarization and resulted in the alteration of melanogenesis and morphology of B16 cells. Collectively, these findings suggest mGluR4 might be a potent target involved in the immune pathogenesis of vitiligo.


Subject(s)
Animals , Male , Vitiligo/immunology , Dendritic Cells/cytology , Bone Marrow Cells/cytology , Cell Differentiation/physiology , Receptors, Metabotropic Glutamate/physiology , Th17 Cells/immunology , Vitiligo/genetics , RNA, Small Interfering/immunology , Th17 Cells/cytology , Flow Cytometry , Melanins/biosynthesis , Melanocytes/cytology , Mice, Inbred C57BL
10.
Int. j. morphol ; 37(3): 1132-1141, Sept. 2019. tab, graf
Article in English | LILACS | ID: biblio-1012409

ABSTRACT

Spermatogonial stem cells (SSCs) have self-renewal and differentiation capacity essential for sperm production throughout the male reproductive life. The electrospun polycaprolactone/gelatin (PCL/Gel) nanofibrous scaffold mimics important features of the extracellular matrix (ECM), which can provide a promising technique for the proliferation and differentiation of SSCs in vitro. The goal of the present study was to investigate the effects of PCL/Gel nanofibrous scaffold on the propagation and differentiation of neonate mouse SSCs (mSSCs). mSSCs were enzymatically isolated, and the cells were purified by differential plating method and seeded on scaffold. After 2 weeks, viability, colony number and diameter, and expression of specific spermatogonial cell genes were investigated. After mSSCs propagation, the cells were cultivated in a differentiation medium on the scaffold for another 2 weeks, and differentiating cells were analyzed by real-time PCR. The number of mSSC colony (P<0.01) and expression levels of specific spermatogonial genes Plzf and Inga6 (P<0.01) and also differentiation genes c-Kit, Tp1 and Ptm1 (P<0.05) were higher in scaffold group compared with control during the culture period. We conclude that mSSCs can be expanded and can differentiate toward spermatid cells on PCL/Gel nanofibrous scaffold with improved developmental parameters.


Las células madre espermatogónicas (CME) tienen capacidad de auto renovación y diferenciación esenciales para la producción de esperma a lo largo de la vida reproductiva masculina. El «scaffold¼ nanofibroso de policaprolactona / gelatina (PCL / Gel) electrohilado imita características importantes de la matriz extracelular (MEC), que puede proporcionar una técnica prometedora para la proliferación y diferenciación de CME in vitro. El objetivo del presente estudio fue investigar los efectos del «scaffold¼ nanofibroso PCL / Gel en la propagación y diferenciación de CME de ratones neonatos (mSSC). Los mSSC se aislaron enzimáticamente y las células se purificaron mediante un método de siembra diferencial y se sembraron en un «scaffold¼. Después de 2 semanas, se investigaron la viabilidad, el número y el diámetro de las colonias y la expresión de genes específicos de células espermatogónicas. Después de la propagación de mSSC, las células se cultivaron en un medio de diferenciación en el «scaffold¼ durante otras 2 semanas, y las células se analizaron mediante PCR en tiempo real. El número de colonias mSSC (P <0,01) y los niveles de expresión de los genes espermatogónicos específicos Plzf e Inga6 (P <0,01) y también los genes de diferenciación c-Kit, Tp1 y Ptm1 (P <0,05) fueron mayores en el grupo de «scaffold¼ en comparación con el control durante el período de cultivo. Concluimos que los mSSC pueden expandirse y diferenciarse en células espermátidas en un «scaffold¼ de nanofibras PCL / Gel con parámetros de desarrollo mejorados.


Subject(s)
Animals , Male , Mice , Spermatogonia/cytology , Spermatogonia/metabolism , Cell Differentiation/physiology , Cell Proliferation/physiology , Polyesters/chemistry , Cell Differentiation/genetics , Cell Survival , Fluorescent Antibody Technique , Cell Proliferation/genetics , Tissue Scaffolds , Nanofibers/chemistry , Real-Time Polymerase Chain Reaction , Animals, Newborn
11.
Arq. bras. oftalmol ; 82(1): 78-84, Jan.-Feb. 2019. tab
Article in English | LILACS | ID: biblio-973874

ABSTRACT

ABSTRACT The transparency and maintenance of corneal epithelial integrity are essential for its optical properties and, to preserve these characteristics, the epithelium undergoes continuous renewal. This renewal depends on the control of cell proliferation and differentiation mediated by mitogenic factors responsible for increasing mitoses and stimulating cellular migration. Cell-cell communication plays a pivotal role in epithelial healing process, and several cytokines and growth factors are involved in this process. Understanding the cross-talk and paracrine effects of these cytokines and growth factors released can help in the search for new therapeutic strategies to treat ocular surface diseases.


RESUMO A transparência e a manutenção da integridade epitelial da córnea são essenciais para suas propriedades ópticas e, para preservar tais características, o epitélio sofre renovação contínua. Essa renovação depende do controle da proliferação e diferenciação celular mediadas por fatores mitogênicos responsáveis pelo aumento das mitoses e estímulo à migração celular. A comunicação célula-célula desempenha um papel fundamental no processo de cicatrização epitelial, e várias citocinas e fatores de crescimento estão envolvidos neste processo. Compreender os efeitos cruzados e paracrinos dessas citocinas e fatores de crescimento liberados pode ajudar na busca de novas estratégias terapêuticas para o tratamento de doenças da superfície ocular.


Subject(s)
Humans , Wound Healing/physiology , Epithelium, Corneal/physiology , Intercellular Signaling Peptides and Proteins/therapeutic use , Cell Differentiation/physiology , Epithelium, Corneal/cytology , Corneal Diseases/therapy , Intercellular Signaling Peptides and Proteins/physiology , Cell Proliferation/physiology , Epithelial Cells/physiology , Fibroblasts/physiology
12.
Braz. j. med. biol. res ; 52(9): e8551, 2019. graf
Article in English | LILACS | ID: biblio-1019565

ABSTRACT

Fibroblasts are a highly heterogeneous population of cells, being found in a large number of different tissues. These cells produce the extracellular matrix, which is essential to preserve structural integrity of connective tissues. Fibroblasts are frequently engaged in migration and remodeling, exerting traction forces in the extracellular matrix, which is crucial for matrix deposition and wound healing. In addition, previous studies performed on primary myoblasts suggest that the E3 ligase MuRF2 might function as a cytoskeleton adaptor. Here, we hypothesized that MuRF2 also plays a functional role in skeletal muscle fibroblasts. We found that skeletal muscle fibroblasts express MuRF2 and its siRNA knock-down promoted decreased fibroblast migration, cell border accumulation of polymerized actin, and down-regulation of the phospho-Akt expression. Our results indicated that MuRF2 was necessary to maintain the actin cytoskeleton functionality in skeletal muscle fibroblasts via Akt activity and exerted an important role in extracellular matrix remodeling in the skeletal muscle tissue.


Subject(s)
Animals , Rats , Cell Differentiation/physiology , Muscle, Skeletal/physiology , Ubiquitin-Protein Ligases/physiology , Cell Proliferation/physiology , Fibroblasts/physiology , Muscle Proteins/physiology , Blotting, Western , Fluorescent Antibody Technique , Muscle, Skeletal/metabolism , Ubiquitin-Protein Ligases/metabolism , Fibroblasts/metabolism , Muscle Proteins/metabolism
13.
J. appl. oral sci ; 27: e20180317, 2019. tab, graf
Article in English | LILACS, BBO | ID: biblio-984571

ABSTRACT

Abstract Bone morphogenetic protein type 2 (BMP-2) and retinoic acid (RA) are osteoinductive factors that stimulate endogenous mechanisms of bone repair which can be applied on management of osseous defects in oral and maxillofacial fields. Objective Considering the different results of RA on osteogenesis and its possible use to substitute/potency BMP-2 effects, this study evaluated the outcomes of BMP-2, RA, and BMP-2+RA treatments on in vitro osteogenic differentiation of human adipose-derived stem cells (ASCs) and the signaling pathway(s) involved. Material and Methods ASCs were treated every other day with basic osteogenic medium (OM) alone or supplemented with BMP-2, RA, or BMP-2+RA. Alkaline phosphatase (ALP) activity was determined using the r-nitrophenol method. Extracellular matrix mineralization was evaluated using von Kossa staining and calcium quantification. Expression of osteonectin and osteocalcin mRNA were determined using qPCR. Smad1, Smad4, phosphorylated Smad1/5/8, BMP-4, and BMP-7 proteins expressions were analyzed using western blotting. Signaling pathway was evaluated using the IPA® software. Results RA promoted the highest ALP activity at days 7, 14, 21, and 28, in comparison to BMP-2 and BMP-2+RA. BMP-2+RA best stimulated phosphorylated Smad1/5/8 protein expression at day 7 and Smad4 expression at days 7, 14, 21, and 28. Osteocalcin and osteonectin mRNA expressions were best stimulated by BMP-2+RA at day 7. Matrix mineralization was most improved by BMP-2+RA at days 12 and 32. Additionally, BMP-2+RA promoted the highest BMP signaling pathway activation at days 7 and 14, and demonstrated more activation of differentiation of bone-forming cells than OM alone. Conclusions In summary, RA increased the effect of BMP-2 on osteogenic differentiation of human ASCs.


Subject(s)
Humans , Osteogenesis/drug effects , Tretinoin/pharmacology , Cell Differentiation/drug effects , Bone Morphogenetic Protein 2/drug effects , Mesenchymal Stem Cells/drug effects , Osteoblasts/drug effects , Osteogenesis/physiology , Reference Values , Time Factors , Osteocalcin/analysis , Osteocalcin/drug effects , Osteonectin/analysis , Osteonectin/drug effects , Cell Differentiation/physiology , Cells, Cultured , Blotting, Western , Reproducibility of Results , Analysis of Variance , Alkaline Phosphatase/analysis , Alkaline Phosphatase/adverse effects , Bone Morphogenetic Protein 2/metabolism , Mesenchymal Stem Cells/metabolism
14.
Arq. bras. oftalmol ; 81(5): 376-383, Sept.-Oct. 2018. tab, graf
Article in English | LILACS | ID: biblio-950491

ABSTRACT

ABSTRACT Purposes: To develop an efficient and xeno-free standard eye-derived induced pluripotent stem cell reprogramming protocol for use during induced pluripotent stem cell-based cell therapies in treating retinal degenerative diseases and to compare the relative effectiveness of both animal- and non-animal-derived culture systems in the generation of induced pluripotent stem cells. Methods: Primary cultured human pterygium fibroblasts and human Tenon's capsule fibroblasts were induced to induced pluripotent stem cells using a non-in­tegrated virus under two xeno-free systems; as part of this study, a traditional non-xeno-free reprogramming system was also assessed. Induced pluripotent stem cell clones were selected and counted by live staining. Reprogramming efficiencies were evaluated between the fibroblasts and among different culture systems. In a series of experiments, such as PCR and immunofluorescence staining, the induced pluripotent stem cells were characterized. Results: Human pterygium fibroblast- and human Tenon's capsule fibroblast-derived induced pluripotent stem cells were successfully established using different reprogramming systems, under which they exhibited properties of induced pluripotent stem cells. Reprogramming efficiencies of induced pluripotent stem cells using the cell therapy system, the traditional system, and the E6/E8 system were 0.014%, 0.028%, and 0.001%, respectively, and those of human pterygium fibroblast- and human Tenon's capsule fibroblast-derived induced pluripotent stem cells-using the aforementioned systems-were 0.018% and 0.017%, respectively. Conclusions: Sendai virus facilitates induced pluripotent stem cell reprogramming of ocular fibroblasts-both human pterygium and human Tenon's capsule fibroblasts being safe and efficient for induced pluripotent stem cell reprogramming. Although the reprogramming efficiencies of ocular-derived induced pluripotent stem cells under xeno-free conditions were not superior to those observed using the traditional reprogramming system, the cell therapy system reprogramming system is a good option when induced pluripotent stem cells are to be induced under xeno-free conditions.


RESUMO Objetivos: Desenvolver um protocolo padrão, eficiente e xeno-livre, para a reprogramação de células-tronco pluripotentes induzidas, que possa ser usado durante as terapias de células-tronco pluripotentes induzidas para o tratamento de doenças degenerativas da retina, e comparar a eficácia relativa de sistemas de cultivo de origem animal e de origem não animal na geração de células-tronco pluripotentes induzidas. Métodos: Cultivos primários de fibroblastos de pterígio humano e de fibroblastos da cápsula de Tenon humanos foram induzidos a células-tronco pluripotentes induzidas usando um vírus não integrado sob dois sistemas xeno-livres; um sistema tradicional de reprogramação não xeno-livre também foi avaliado como parte deste estudo. Os clones de células-tronco pluripotentes induzidas foram selecionados e contados por coloração de células vivas. As eficiências de reprogramação foram avaliadas entre os diferentes fibroblastos e entre os diferentes sistemas de cultivo. Uma série de experimentos, como o PCR e a coloração por imunofluorescência, foram conduzidos para caracterizar as células-tronco pluripotentes induzidas. Resultados: Célu­las-tronco pluripotentes induzidas derivadas de fibroblastos de pterígio humano e fibroblastos da cápsula de Tenon humanos foram estabelecidas com sucesso sob diferentes sistemas de reprogramação e exibiram propriedades de células-tronco pluripotentes induzidas. As eficiências de reprogramação das células-tronco pluripotentes induzidas usando o sistema de terapia celular, o sistema tradicional e o sistema E6/E8 foram 0,014, 0,028% e 0,001%, respectivamente. Além disso, as efi­ciências de reprogramação de células-tronco pluripotentes induzidas derivadas de fibroblastos de pterígio humano e de fibroblastos da cápsula de Tenon humanos usando todos os sistemas acima foram de 0,018% e 0,017%, respectivamente. Conclusões: O vírus Sendai pode ser usado para facilitar a reprogramação de fibroblastos oculares pelas células-tronco pluripotentes induzidas. Tanto os fibroblastos de pterígio humano quanto os fibroblastos da cápsula de Tenon humanos são seguros e eficientes para a reprogramação de células-tronco pluripotentes induzidas. Embora as eficiências de reprogramação das células-tronco pluripotentes induzidas de origem ocular sob condições xeno-livres não tenham sido superiores às eficiências observadas para o sistema tradicional de reprogramação, o sistema de reprogramação sistema de terapia celular é uma boa opção para a indução de células-tronco pluripotentes induzidas sob condições xeno-livres.


Subject(s)
Humans , Pterygium/pathology , Cell Culture Techniques/methods , Eye/cytology , Cellular Reprogramming/physiology , Induced Pluripotent Stem Cells/cytology , Fibroblasts/cytology , Cell Differentiation/physiology , Cell Transdifferentiation
15.
Braz. j. med. biol. res ; 51(12): e7574, 2018. graf
Article in English | LILACS | ID: biblio-974257

ABSTRACT

Bone fracture is a common medical condition, which may occur due to traumatic injury or disease-related conditions. Evidence suggests that microRNAs (miRNAs) can regulate osteoblast differentiation and function. In this study, we explored the effects and mechanism of miR-221 on the growth and migration of osteoblasts using MC3T3-E1 cells. The expression levels of miR-221 in the different groups were measured by qRT-PCR. Then, miR-221 mimic and inhibitor were transfected into MC3T3-E1 cells, and cell viability and migration were measured using the CCK-8 assay and the Transwell migration assay. Additionally, the expression levels of differentiation-related factors (Runx2 and Ocn) and ZFPM2 were measured by qRT-PCR. Western blot was used to measure the expression of cell cycle-related proteins, epithelial-mesenchymal transition (EMT)-related proteins, ZFPM2, and Wnt/Notch, and Smad signaling pathway proteins. miR-221 was significantly up-regulated in the patients with lumbar compression fracture (LCM) and trochanteric fracture (TF). miR-221 promoted ALP, Runx2, and OPN expressions in MC3T3-E1 cells. miR-221 overexpression significantly increased cell proliferation, migration, differentiation, and matrix mineralization, whereas suppression of miR-221 reversed these effects. Additionally, the results displayed that ZFPM2 was a direct target gene of miR-221, and overexpression of ZFPM2 reversed the promoting effects of miR-221 overexpression on osteoblasts. Mechanistic study revealed that overexpression of miR-221 inactivated the Wnt/Notch and Smad signaling pathways by regulating ZFPM2 expression. We drew the conclusions that miR-221 overexpression promoted osteoblast proliferation, migration, and differentiation by regulation of ZFPM2 expression and deactivating the Wnt/Notch and Smad signaling pathways.


Subject(s)
Humans , Animals , Rabbits , Cell Differentiation/physiology , Cell Movement/physiology , MicroRNAs/physiology , Cell Proliferation/physiology , DNA-Binding Proteins/physiology , Fractures, Bone/blood , Osteoblasts/physiology , Reference Values , Transcription Factors/blood , Cell Survival/physiology , Blotting, Western , Analysis of Variance , 3T3 Cells , MicroRNAs/blood , DNA-Binding Proteins/blood
16.
Arch. cardiol. Méx ; 88(5): 483-495, dic. 2018. graf
Article in Spanish | LILACS | ID: biblio-1142160

ABSTRACT

Resumen Actualmente las enfermedades cardiovasculares se han convertido en un serio problema para los sistemas de salud de todo el mundo, ya que son la principal causa de muerte y representan una enorme carga económica. Este problema ha sido abordado con diferentes estrategias, entre ellas con la ayuda de terapia celular, aunque sin resultados contundentes. Durante más de 20 años, se ha utilizado una gran variedad de células madre en diferentes modelos de infarto del miocardio. El uso de células madre cardiacas (CSC) parece ser la mejor opción, pero la inaccesibilidad y la escasez de estas células hacen que su uso sea muy limitado. Además, existe un riesgo elevado pues tienen que obtenerse directamente del corazón del paciente. A diferencia de las CSC, las células madre adultas derivadas de médula ósea o tejido adiposo, entre otras, representan una opción atractiva debido a su fácil accesibilidad y abundancia, pero sobre todo a la probable existencia de progenitores cardiacos entre sus diferentes subpoblaciones. En esta revisión hacemos un análisis de los marcadores de superficie presentes en CSC en comparación con otras células madre adultas, y sugerimos la preexistencia de células que comparten marcadores de superficie específicos con CSC, la presencia de un inmunofenotipo predecible, aunque en proporciones bajas, pero con un potencial de diferenciación cardiaca similar a las CSC, lo cual podría aumentar su valor terapéutico. Este estudio revela las nuevas perspectivas con respecto a la presencia de dichos marcadores, los cuales comprometerían algunas de estas subpoblaciones a diferenciarse a tejido cardiaco.


Abstract It is well-known that cardiovascular diseases are the leading cause of death world- wide, and represent an important economic burden to health systems. In an attempt to solve this problem, stem cell therapy has emerged as a therapeutic option. Within the last 20 years, a great variety of stem cells have been used in different myocardial infarction models. Up until now, the use of cardiac stem cells (CSCs) has seemed to be the best option, but the inaccessibility and scarcity of these cells make their use unreliable. Additionally, there is a high risk as they have to be obtained directly from the heart of the patient. Unlike CSCs, adult stem cells originating from bone marrow or adipose tissue, among others, appear to be an attractive option due to their easier accessibility and abundance, but particularly due to the probable existence of cardiac progenitors among their different sub-populations. In this review an analysis is made of the surface markers present in CSCs compared with other adult stem cells. This suggested the pre-existence of cells sharing specific surface markers with CSCs, a predictable immunophenotype present in some cells, although in low proportions, and with a potential of cardiac differentiation that could be similar to CSCs, thus increasing their therapeutic value. This study highlights new perspectives regarding MSCs that would enable some of these sub-populations to be differentiated at cardiac tissue level.


Subject(s)
Humans , Animals , Stem Cells/cytology , Cardiovascular Diseases/therapy , Stem Cell Transplantation/methods , Cardiovascular Diseases/physiopathology , Cell Differentiation/physiology , Immunophenotyping , Myocardial Infarction/physiopathology , Myocardial Infarction/therapy
17.
Acta cir. bras ; 32(11): 984-994, Nov. 2017. tab, graf
Article in English | LILACS | ID: biblio-886180

ABSTRACT

Abstract Purpose: To investigate the use Aldefluor® and N, N - Dimethylaminobenzaldehyde (DEAB) to design a protocol to sort keratinocyte stem cells from cultured keratinocytes from burned patients. Methods: Activated Aldefluor® aliquots were prepared and maintained at temperature between 2 to 8°C, or stored at -20°C. Next, the cells were collected following the standard protocol of sample preparation. Results: Best results were obtained with Aldefluor® 1.5µl and DEAB 15 µl for 1 x 106 cells, incubated at 37°C for 15 minutes. Flow cytometer range for keratinocyte stem cells separation was evaluated. There were 14.8% of stem cells separated in one sample of keratinocyte culture used to pattern the protocol. After being defined the ideal concentration, the same test pattern was performed in other keratinocyte samples. We observed a final mean of 10.8%. Conclusion: Aldefluor® has been shown as a favorable marking of epidermal keratinocyte stem cells for subsequent separation on a flow cytometer, with detection of 10.8% of epidermal keratinocyte stem cells, in this protocol.


Subject(s)
Humans , Animals , Stem Cells/cytology , Keratinocytes/cytology , Cell Differentiation/physiology , Flow Cytometry/methods , Skin/cytology , Biomarkers/analysis , Cells, Cultured , Clinical Protocols , Cell Culture Techniques
18.
Arq. bras. oftalmol ; 80(4): 268-272, July-Aug. 2017. tab, graf
Article in English | LILACS | ID: biblio-888124

ABSTRACT

ABSTRACT Various approaches have been taken to improve our knowledge of the microenvironmental regulation of limbal epithelial stem cells. Researchers have extensively investigated the roles of growth factors, survival factors, cytokines, enzymes, and permeable molecules secreted by the limbal cells. However, recent evidence suggests that stem cell fate (i.e., self-renewal or differentiation) can also be influenced by biophysical and mechanical cues related to the supramolecular organization and the liquid crystalline (mesophase) nature of the stromal extracellular matrix. These cues can be sensed by stem cells and transduced into intracellular biochemical and functional responses, a process known as mechanotransduction. The objective of this review is to offer perspectives on the supramolecular microenvironmental regulation of limbal epithelial stem cells and the differentiation of their progeny.


RESUMO Muitas abordagens têm sido utilizadas para ampliar entendimentos sobre a regulação microambiental das células tronco epiteliais limbais. Neste contexto, pesquisadores têm exaustivamente investigado a participação de fatores de crescimento, fatores de sobrevida, citocinas, enzimas e moléculas permeáveis secretadas pelas células limbais. Entretanto, evidências recentes sugerem que o destino (ie. autorrenovação ou recrutamento para a via de diferenciação) das células tronco também sofre influência de estímulos biofísicos ou mecânicos relacionados à organização supramolecular e à natureza liquido-cristalina (mesofases) da matriz extracelular estromal. Esses estímulos podem ser percebidos e traduzidos pelas células tronco em sinais bioquímicos que geram respostas funcionais, através de um processo designado de mecanotransdução. Objetiva-se, com a presente revisão, oferecer ao leitor perspectivas supramoleculares sobre a regulação microambiental das células tronco epiteliais limbais e a diferenciação de sua progênie.


Subject(s)
Humans , Stem Cells/physiology , Cell Differentiation/physiology , Limbus Corneae/cytology , Epithelium, Corneal/cytology , Mechanotransduction, Cellular/physiology , Extracellular Matrix/physiology , Epithelium, Corneal/physiology , Stem Cell Niche/physiology
19.
Medicina (B.Aires) ; 77(4): 314-320, ago. 2017. ilus
Article in Spanish | LILACS | ID: biblio-894485

ABSTRACT

La autofagia es un proceso de reciclado de partes de la célula. Como se describe en esta revisión, ocurre naturalmente preservando a las células de la acumulación de toxinas, moléculas y organelas dañadas y además permite los procesos de desarrollo y diferenciación de los tejidos. En el transcurso de la autofagia, el procesamiento de los sustratos a reciclar genera ATP, lo que constituye una fuente alternativa de energía en situaciones de estrés. En este sentido, bajo condiciones hostiles como hipoxia o falta de nutrientes, el proceso puede dispararse de modo exacerbado llevando a la muerte celular. Algunas alteraciones en su funcionamiento pueden involucrar el desarrollo de diversas patologías, tales como el daño hepático, el cáncer y las enfermedades neurodegenerativas.


Autophagy is a process of recycling parts of the cell. As described in this review, it occurs naturally in order to preserve cells from the accumulation of toxins, damaged molecules and organelles, and to allow processes of tissue development and differentiation. In the course of autophagy, the processing of the substrates to be recycled generates ATP, thus providing an alternative source of energy in stress situations. In this sense, under hostile conditions such as hypoxia or lack of nutrients, the autophagy process can be exacerbated leading to cell death. Some alterations in its functioning may involve the development of various pathologies, including liver damage, cancer and neurodegenerative diseases.


Subject(s)
Humans , Autophagy/physiology , Cell Differentiation/physiology , Cell Survival/physiology , Neurodegenerative Diseases/pathology , Energy Metabolism/physiology , Neoplasms/pathology , Cell Hypoxia , Adenosine Triphosphate/metabolism , Neurodegenerative Diseases/physiopathology , Neoplasms/physiopathology
20.
Braz. oral res. (Online) ; 31: e112, 2017. graf
Article in English | LILACS | ID: biblio-952076

ABSTRACT

Abstract: Cellular retinoic acid-binding protein 2 (CRABP2) has been detected in several organs during embryonic development. Recent studies have demonstrated that CRABP2 plays important roles in the retinoic acid, β-catenin and Notch signaling pathways, as well as in the interaction between epithelial and mesenchymal cells, which are important for human dental pulp stem cells (hDPSCs) and tooth development. In the present study, the expression of CRABP2 during mouse molar development and the role of CRABP2 in hDPSC odontoblastic differentiation were evaluated. CRABP2 was gradually decreased during the development of the first maxillary molar, which exhibited the same trend as the expression of CRABP2 during the odontoblastic induction of hDPSCs. CRABP2 knockdown inhibited the proliferative ability of hDPSCs, while it enhanced odontoblastic differentiation via promoting mineralization nodule formation and upregulating the activity of alkaline phosphatase and the expression of mineralization-related genes. The present study uncovered a novel function of CRABP2 in hDPSCs. Our data suggest that CRABP2 may act as a regulator during the proliferation and differentiation of hDPSCs.


Subject(s)
Humans , Animals , Male , Female , Stem Cells/physiology , Cell Differentiation/physiology , Receptors, Retinoic Acid/physiology , Dental Pulp/cytology , Cell Proliferation/physiology , Odontoblasts/physiology , Reference Values , Time Factors , Immunohistochemistry , Down-Regulation/physiology , Cell Communication , Cells, Cultured , Blotting, Western , Analysis of Variance , Anthraquinones , Receptors, Retinoic Acid/analysis , Reverse Transcriptase Polymerase Chain Reaction , Coloring Agents , Alkaline Phosphatase , Mice, Inbred C57BL
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