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Rev. bras. med. esporte ; 27(spe2): 73-78, Apr.-June 2021. graf
Article in English | LILACS | ID: biblio-1280080


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.

Animals , Cattle , Cell Differentiation/physiology , Adipocytes/metabolism , Myoblasts, Skeletal/metabolism , Cell Proliferation/physiology , Energy Metabolism , Myostatin/metabolism , Oligonucleotide Array Sequence Analysis
Rev. bras. oftalmol ; 80(1): 8-11, jan.-fev. 2021. graf
Article in Portuguese | LILACS | ID: biblio-1251324


RESUMO Objetivo: Avaliar a inibição da proliferação de fibroblastos in vitro das conjuntivas obtidas através de exérese de pterígios de pacientes utilizando mitomicina C (MMC) e ciclofosfamida (CF). Métodos: Os pterígios foram retirados de 7 pacientes e submetidos a cultivo celular. Após o cultivo, 3 fragmentos de dimensões iguais deste material foram colhidos de áreas adjacentes do pterígio removido de cada paciente. Eles foram randomicamente selecionados de tal forma que: um fragmento de cada paciente foi exposto: ao meio de cultura (grupo controle), a MMC e a CF por igual período de tempo nas concentrações de 0,4 mg/ml e 10 mg/ml respectivamente. Após este período realizou-se a contagem celular de fibroblastos destes 3 grupos. Cada grupo continha 7 fragmentos. Resultados: Com a utilização da MMC tivemos uma taxa de 95% da inibição da proliferação dos fibroblastos, enquanto com a CF 100%. Conclusões: Ambas as drogas apresentaram elevada taxa da inibição da proliferação de fibroblastos, porém a CF apresentou inibição maior que a MMC.

Abstract Objective: To evaluate the inhibition of fibroblast proliferation in vitro of conjunctiva obtained by excision of pterygium from patients using mitomycin (MMC) and cyclophosphamide (CF). Methods: Pterygiums were removed from 7 patients and subjected to cell culture. After cell cultivation, 3 fragments of equal dimensions of these tissues were collected from adjacent areas of each patient removed pterygium. They were randomly selected in such a way that one fragment of each patient was exposed to: the culture medium (group control), to MMC and to CF for an equal period of time at concentrations of 0,4 mg/dl and 10 mg/dl respectively. After this period, the fibroblast cell count of these groups were performed. Each group had seven fragments. Results: With the use of MMC we had a 95% rate of inhibition of fibroblast proliferation, while with CF 100%. Conclusion: Both drugs showed a high rate of inhibition of fibroblast proliferation, but CF showed greater inhibition than MMC.

Humans , Male , Female , Adult , Middle Aged , Wound Healing , Pterygium/surgery , Mitomycin/adverse effects , Cyclophosphamide/adverse effects , Cell Proliferation/physiology , Antimitotic Agents/adverse effects , Fibroblasts/physiology , In Vitro Techniques
Braz. j. med. biol. res ; 53(1): e8883, Jan. 2020. tab, graf
Article in English | LILACS | ID: biblio-1055486


Opa-interacting protein 5 antisense transcript 1 (OIP5-AS1) is one kind of cytoplasmic long non-coding RNA (lncRNA), which has been demonstrated to play a critical function in multiple cancers. However, the detailed mechanism of OIP5-AS1 in the regulation of cervical cancer progression is still obscure. Here, we demonstrated that lncRNA OIP5-AS1 was upregulated in cervical cancer and was correlated with poor prognosis by bioinformatics studies. OIP5-AS1 depletion inhibited cell proliferation and promoted cell apoptosis in cervical cancer cells. Furthermore, we clarified that ROCK1 was the downstream effector of OIP5-AS1 and OIP5-AS1 acted as a molecular sponge of miR-143-3p. Finally, we verified that OIP5-AS1 exerted its function in the regulation of cervical cancer progression via interacting with miR-143-3p to regulate ROCK1 expression. Our study revealed novel mechanisms about how lncRNA OIP5-AS1 executed its function in cervical cancer and thus provided potential therapeutic targets for the disease.

Humans , Female , Uterine Cervical Neoplasms/pathology , Apoptosis/physiology , MicroRNAs/metabolism , Cell Proliferation/physiology , rho-Associated Kinases/metabolism , RNA, Long Noncoding/metabolism , Gene Expression Regulation, Neoplastic , Up-Regulation , Uterine Cervical Neoplasms/metabolism , Blotting, Western , Apoptosis/genetics , Reverse Transcriptase Polymerase Chain Reaction , MicroRNAs/genetics , Cell Line, Tumor , Cell Proliferation/genetics , rho-Associated Kinases/genetics , RNA, Long Noncoding/genetics
Braz. oral res. (Online) ; 34: e030, 2020. tab, graf
Article in English | LILACS | ID: biblio-1089389


Abstract: The abnormal increase in proliferation rate of human periodontal ligament stem cells (PDLSCs) is considered to be involved in the pathogenesis of periodontitis, a disease in which the IL-10-mediated anti-inflammatory pathway plays a critical role. This study aimed to investigate the involvement of microRNA-466l in periodontitis and to explore the possible interaction between IL-10 and microRNA-466l. PDLSCs were obtained from periodontitis-affected teeth and healthy control teeth. The expression of microRNA-466l and IL-10 mRNA was measured in PDLSCs using RT-qPCR. The proliferation ability of PDLSCs was analyzed using CCK-8 assays. Overexpression of microRNA-466l in a PDLSC cell line was established using two different types of PDLSCs, and the effect of microRNA-466l overexpression on IL-10 expression and cell proliferation were detected by western blot and CCK-8 assays, respectively. We found that expression levels of microRNA-466l and IL-10 mRNA were significantly lower (P < 0.05) in PDLSCs derived from periodontitis-affected teeth compared to those derived from healthy teeth. However, the cell proliferation ability was significantly higher in the PDLSCs derived from periodontitis-affected teeth. Meanwhile microRNA-466l overexpression decreased cell proliferation rates of both types of PDLSCs and upregulated IL-10 expression. Together, these data suggest that microRNA-466l can upregulate IL-10 and reduce the proliferation rate of PDLSCs.

Humans , Adult , Periodontitis/genetics , Stem Cells/metabolism , Up-Regulation , Interleukin-10/therapeutic use , MicroRNAs/metabolism , Cell Proliferation/physiology , Periodontitis/metabolism , Periodontitis/therapy , Cell Differentiation , Blotting, Western , Interleukin-10/metabolism
Int. j. morphol ; 37(3): 1132-1141, Sept. 2019. tab, graf
Article in English | LILACS | ID: biblio-1012409


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.

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
Int. j. odontostomatol. (Print) ; 13(2): 142-149, jun. 2019. tab, graf
Article in English | LILACS | ID: biblio-1002297


ABSTRACT: The present study aimed to investigate the occurrence of mandibular canal alterations in regions with dental inflammation by means of cone beam computed tomography (CBCT). A database of 2,484 CBCTs was reviewed for identifying dental inflammation in mandibular alveolar ridges. The final sample consisted of 150 CBCTs, including 91 females and 59 males, with ages ranging from 13 to 89 years (mean age of 47.06; ± SD=18.722). The presence and location of dental inflammation, gender, age, as well as presence and location of mandibular canal branching (MCB) were evaluated. The Kolmogorov-Smirnov, Chi-square, and T-test were applied to verify the statistical relationship of the data. There were 178 images of dental inflammation on 150 CBCTs, mainly located at molars' region (75 %). Apical lesions were the most common type of dental inflammation found (79 or 44.4 % of the sample), followed by pericoronitis (32; 18.0 %). This study identified 135 mandibular canal branches in the exams that presented dental inflammation. The MCB were also most commonly located at molars' region (74.07 %). No statistical difference was identified regarding the distribution of mandibular canal branching in relation to the sites with dental inflammation (p=0.370).The MCB found were mostly single (86 or 63.7 % of the total). Sex had no influence on mandibular canal branching occurrence (p=0.308), not did age (p=0.728). A high prevalence of mandibular canal branching was observed in the regions where dental inflammation were identified, most commonly found in the molar region.

RESUMEN: El presente estudio tuvo como objetivo investigar la ocurrencia de ramificación del canal mandibular (RCM) en regiones con inflamación dental mediante tomografía computarizada de haz cónico (TCHC). Se revisó una base de datos de 2.484 TCHC para identificar la inflamación dental en las crestas alveolares mandibulares. La muestra final consistió en 150 TCHC, incluidas 91 mujeres y 59 hombres, con edades comprendidas entre 13 y 89 años (edad media de 47,06 ± DE = 18,722). Se evaluaron el sexo, la edad, la presencia y la ubicación de la inflamación dental, así como la presencia y ubicación de RCM. KolmogorovSmirnov, Chi-cuadrado y prueba-T se aplicaron para verificar la relación estadística de los datos. Hubo 178 imágenes de inflamación dental en 150 TCHC, ubicados principalmente en la región de los molares (75 %). Las lesiones apicales fueron el tipo más común de inflamación dental encontrada (79 o 44,4 % de la muestra), seguidas por pericoronitis (32; 18,0 %). Este estudio identificó 135 ramas del canal mandibular en las regiones que presentaron inflamación dental. El RCM también se localizó con mayor frecuencia en la región de los molares (74,07 %). No se identificaron diferencias estadísticas con respecto a la distribución de la ramificación del canal mandibular en relación con los sitios con inflamación dental (p = 0,370). Las RCM encontrados fueron en su mayoría solteros (86 o 63,7 % del total). El sexo no tuvo influencia en la ocurrencia de la ramificación del canal mandibular (p = 0,308), no la edad (p = 0,728). Se observó una alta prevalencia de ramificación del canal mandibular en las regiones donde se identificó la inflamación dental, que se encuentra con mayor frecuencia en la región molar.

Humans , Male , Female , Young Adult , Granuloma, Giant Cell/surgery , Ki-67 Antigen/metabolism , Immunohistochemistry , Granuloma, Giant Cell/diagnosis , Statistical Analysis , Analysis of Variance , Cell Proliferation/physiology , Guatemala , Mexico
Arq. bras. oftalmol ; 82(1): 78-84, Jan.-Feb. 2019. tab
Article in English | LILACS | ID: biblio-973874


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.

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
Biol. Res ; 52: 60, 2019. graf
Article in English | LILACS | ID: biblio-1100912


BACKGROUND: Recent studies have confirmed that RASAL1 has an antitumor effect in many cancers, but its functional role and the molecular mechanism underlying in colon cancer has not been investigated. RESULTS: We collected human colon cancer tissues and adjacent non-tumor tissues, human colon cancer cell lines LoVo, CaCo2, SW1116, SW480 and HCT-116, and normal colonic mucosa cell line NCM460. RT-qPCR was used to detect the RASAL1 level in the clinical tissues and cell lines. In LoVo and HCT-116, RASAL1 was artificially overexpressed. Cell viability and proliferation were measured using CCK-8 assays, and cell cycle was detected via PI staining and flow cytometry analysis. RASAL1 significantly inhibited the cell proliferation via inducing cell cycle arrest, suppressed cell cycle associated protein expression, and decreased the lipid content and inhibited the SCD1 expression. Moreover, SCD1 overexpression induced and downregulation repressed cell proliferation by causing cell cycle arrest. Additionally, luciferase reporter assays were performed to confirm the direct binding between SREBP1c, LXRα; and SCD1 promoter, we also demonstrated that RASAL1 inhibit SCD1 3'-UTR activity. RASAL1 inhibited tumor growth in xenograft nude mice models and shows inhibitory effect of SCD1 expression in vivo. CONCLUSION: Taken together, we concluded that RASAL1 inhibited colon cancer cell proliferation via modulating SCD1 activity through LXRα/SREBP1c pathway.

Humans , Animals , Mice , Stearoyl-CoA Desaturase/metabolism , Colonic Neoplasms/pathology , GTPase-Activating Proteins/metabolism , Cell Proliferation/physiology , Sterol Regulatory Element Binding Protein 1/metabolism , Liver X Receptors/metabolism , Stearoyl-CoA Desaturase/genetics , Down-Regulation , GTPase-Activating Proteins/genetics , Cell Line, Tumor , Sterol Regulatory Element Binding Protein 1/genetics , Liver X Receptors/genetics
Biol. Res ; 52: 59, 2019. graf
Article in English | LILACS | ID: biblio-1100911


OBJECTIVES: In varicose veins, vascular smooth muscle cells (VSMCs) often shows phenotypic transition and abnormal proliferation and migration. Evidence suggests the FOXC2-Notch pathway may be involved in the pathogenesis of varicose veins. Here, this study aimed to explore the role of long non-coding RNA FOXC2-AS1 (FOXC2 antisense RNA 1) in phenotypic transition, proliferation, and migration of varicose vein-derived VSMCs and to explore whether the FOXC2-Notch pathway was involved in this process. METHODS: The effect of FOXC2-AS1 on the proliferation and migration of human great saphenous vein smooth muscle cells (SV-SMCs) was analyzed using MTT assay and Transwell migration assay, respectively. The levels of contractile marker SM22α and synthetic marker osteopontin were measured by immunohistochemistry and Western blot to assess the phenotypic transition. RESULTS: The human varicose veins showed thickened intima, media and adventitia layers, increased synthetic VSMCs, as well as upregulated FOXC2-AS1 and FOXC2 expression. In vitro assays showed that FOXC2-AS1 overexpression promoted phenotypic transition, proliferation, and migration of SV-SMCs. However, the effect of FOXC2-AS1 overexpression could be abrogated by both FOXC2 silencing and the Notch signaling inhibitor FLI-06. Furthermore, FOXC2-AS1 overexpression activated the Notch pathway by upregulating FOXC2. CONCLUSION: FOXC2-AS1 overexpression promotes phenotypic transition, proliferation, and migration of SV-SMCs, at least partially, by activating the FOXC2-Notch pathway.

Humans , Saphenous Vein/metabolism , Cell Movement/physiology , Myocytes, Smooth Muscle/metabolism , Cell Proliferation/physiology , Forkhead Transcription Factors/metabolism , Phenotype , Saphenous Vein/pathology , Signal Transduction , Up-Regulation , Cells, Cultured , Myocytes, Smooth Muscle/pathology
Einstein (Säo Paulo) ; 17(2): eRB4733, 2019. graf
Article in English | LILACS | ID: biblio-1001908


ABSTRACT Healthy aging is partly related to appropriate function of the immune system. As already reported, some changes in this system are observed, including reduced number and repertoire of T cells due to thymic involution, accumulation of memory T cells by chronic infections, homeostatic proliferation compensating for the number of naïve T cells, decreased proliferation of T cells against a stimulus, telomere shortening, replicative senescence of the T cells, and inflammaging, besides the accumulation of myeloid-derived suppressor cells. The purpose of this article is to clarify each of these changes, aiming to minimize limitations of immunosenescence. If such associations can be established, these cells may be used as early and less invasive markers of aging-related diseases, as well as to indicate interventions, evaluate the efficacy of interventions and be a tool to achieve longevity with quality of life.

RESUMO O envelhecimento saudável está relacionado, pelo menos em parte, com a função adequada do sistema imunológico. Isso porque já foi relatado que, com o envelhecimento, algumas mudanças desse sistema são observadas, como a diminuição da percentagem e do repertório de células T pela involução tímica, acúmulo de células T de memória por infecções crônicas, compensação do número de células T naïve por proliferação homeostática, diminuição da capacidade de proliferação das células T frente a um estímulo, encurtamento dos telômeros, senescência replicativa das células T, e inflammaging, além do acúmulo de células mieloides supressoras. Este artigo visa esclarecer cada uma das mudanças, mencionadas, com o intuito de buscar meios de minimizar as limitações da imunosenescência. Caso seja possível estabelecer tais relações, essas células podem ser utilizadas como marcadores precoces e pouco invasivos de doenças relacionadas ao envelhecimento, além da possibilidade de serem utilizadas para indicar intervenções, avaliar a eficácia das intervenções e como ferramenta para alcance da longevidade com qualidade de vida.

Humans , Aging/immunology , T-Lymphocytes/physiology , Immunosenescence/immunology , Myeloid-Derived Suppressor Cells/physiology , Adaptation, Physiological/immunology , Cell Proliferation/physiology
Braz. j. med. biol. res ; 52(5): e8412, 2019. graf
Article in English | LILACS | ID: biblio-1001528


Multiple myeloma (MM) is a malignant neoplasm of plasma, and exhibits several harmful effects including osteolytic injuries, hypercalcemia, and immune dysfunction. Many patients with MM succumb to the underlying malignancy. An S-phase kinase-related protein 2 (Skp2) inhibitor, designated SKPin C1, has been developed and confirmed to have an inhibitory effect on metastatic melanoma cells. This study aimed to determine the effect of SKPin C1 on MM. Normal B lymphocytes, THP-1 cells, and MM U266 and RPMI 8226 cells were exposed to various dosages of SKPin C1 for 48 h. Cell proliferation was determined by MTT, EdU staining, and cell cycle assays. Western blot assays were performed to assess intracellular protein levels of Skp2, p27, and cleaved caspase-3. The amount of ubiquitin attached to p27 was determined using an immunoprecipitation assay. The viability of U266 and RPMI 8226 cells was significantly inhibited by 10 μM SKPin C1 and the inhibitory effect was enhanced with increasing doses of SKPin C1. In contrast, 50 μM SKPin C1 only marginally decreased viability of normal B lymphocytes in 12 h. Skp2 and p27 expression in U266 and RPMI 8226 cells was higher and lower, respectively, than that in the normal B lymphocytes. Treatment with SKPin C1 or Skp2 knockdown increased p27 protein levels in U266 and RPMI 8226 cells by preventing p27 from being ubiquitinated, which slowed the cell cycle, inhibited cell proliferation, and triggered apoptosis. Therefore, this study suggested SKPin C1 as a potent inhibitor against aberrant proliferation and immortalization of MM.

Humans , Apoptosis , S-Phase Kinase-Associated Proteins/metabolism , Cell Proliferation/physiology , Cyclin-Dependent Kinase Inhibitor p27/metabolism , Multiple Myeloma/metabolism , Cell Cycle , S-Phase Kinase-Associated Proteins/antagonists & inhibitors , Cell Proliferation/drug effects , Cyclin-Dependent Kinase Inhibitor p27/pharmacology , Ubiquitination/physiology , Ubiquitinated Proteins/metabolism , Multiple Myeloma/physiopathology
Braz. j. med. biol. res ; 52(5): e8026, 2019. tab, graf
Article in English | LILACS | ID: biblio-1001526


Carassius auratus is a teleost fish that has been largely used in behavioral studies. However, little is known about potential environmental influences on its performance of learning and memory tasks. Here, we investigated this question in C. auratus, and searched for potential correlation between exercise and visuospatial enrichment with the total number of telencephalic glia and neurons. To that end, males and females were housed for 183 days in either an enriched (EE) or impoverished environment (IE) aquarium. EE contained toys, natural plants, and a 12-hour/day water stream for voluntary exercise, whereas the IE had none of the above. A third plus-maze aquarium was used for spatial and object recognition tests. Different visual clues in 2 of its 4 arms were used to guide fish to reach the criteria to complete the task. The test consisted of 30 sessions and was concluded when each animal performed three consecutive correct choices or seven alternated, each ten trials. Learning rates revealed significant differences between EE and IE fish. The optical fractionator was used to estimate the total number of telencephalic cells that were stained with cresyl violet. On average, the total number of cells in the subjects from EE was higher than those from subjects maintained in IE (P=0.0202). We suggest that environmental enrichment significantly influenced goldfish spatial learning and memory abilities, and this may be associated with an increase in the total number of telencephalic cells.

Animals , Male , Female , Telencephalon/metabolism , Cell Proliferation/physiology , Fishes/physiology , Spatial Learning/physiology , Spatial Memory/physiology , Physical Conditioning, Animal , Behavior, Animal/physiology , Cell Count
Braz. oral res. (Online) ; 33: e059, 2019. graf
Article in English | LILACS | ID: biblio-1039303


Abstract We recently demonstrated that a co-culture system of human umbilical vein endothelial cells (HUVECs) and human dental pulp stem cells (hDPSCs) could enhance angiogenesis ability in vitro. However, whether tumor necrosis factor α (TNF-α) could promote blood vessel formation during pulp regeneration remained unknown. The aim of this study was to investigate the effects of TNF-α on the formation of endothelial tubules and vascular networks in a co-culture system of hDPSCs and HUVECs. hDPSCs were co-cultured with HUVECs at a ratio of 1:5. The Matrigel assay was performed to detect the total tubule branching lengths and numbers of branches, and the Cell-Counting Kit 8 assay was performed to examine the effect of TNF-α on cell proliferation. Real-time polymerase chain reactions and western blot were used to detect vascular endothelial growth factor (VEGF) mRNA and protein expression. The Matrigel assay showed significantly greater total branching lengths and numbers of branches formed in the experimental groups treated with different concentrations of TNF-α compared with the control group. The decomposition times of the tubule structures were also significantly prolonged (P < 0.05). Treatment with 50 ng/ml TNF-α did not significantly change the proliferation of co-cultured cells, but it significantly increased the VEGF mRNA and protein expression levels (p < 0.05). In addition, the migration abilities of HUVECs and hDPSCs increased after co-culture with TNF-α (p < 0.05). TNF-α enhanced angiogenic ability in vitro in the co-culture system of hDPSCs and HUVECs.

Humans , Adolescent , Adult , Young Adult , Tumor Necrosis Factor-alpha/pharmacology , Neovascularization, Physiologic/drug effects , Dental Pulp/cytology , Dental Pulp/drug effects , Angiogenesis Inducing Agents/pharmacology , Human Umbilical Vein Endothelial Cells/drug effects , Proteoglycans , Reference Values , Time Factors , Cell Count , Cells, Cultured , Blotting, Western , Reproducibility of Results , Collagen , Laminin , Neovascularization, Physiologic/physiology , Dental Pulp/physiology , Vascular Endothelial Growth Factor A/analysis , Vascular Endothelial Growth Factor A/drug effects , Cell Proliferation/drug effects , Cell Proliferation/physiology , Drug Combinations , Cell Migration Assays , Human Umbilical Vein Endothelial Cells/physiology , Real-Time Polymerase Chain Reaction
Braz. j. med. biol. res ; 52(9): e8551, 2019. graf
Article in English | LILACS | ID: biblio-1019565


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.

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
Rev. méd. Chile ; 146(2): 150-159, feb. 2018. tab, graf
Article in English | LILACS | ID: biblio-961372


ABSTRACT Background: The dual potential to promote tolerance or inflammation when facing self-antigens makes dendritic cells (DCs) fundamental players in autoimmunity. There is an association between smoking and DCs maturation in patients with rheumatoid arthritis (RA). However, ethnicity is a key factor in autoimmune disorders. Aim: To evaluate phenotypic and functional alterations of DCs obtained from Chilean patients with RA as compared to healthy controls (HC). In second term, to compare the inflammatory behaviour of DCs between smoker and non-smoker patients. Material and Methods: Monocyte-derived DCs and T-cells were obtained from blood samples isolated from 30 HC and 32 RA-patients, 14 of which were currently smokers and 18 non-smokers. Several maturation surface markers were evaluated in DCs, including HLA-DR, CD40, CD80, CD83 and CD86. Furthermore, autologous co-cultures of DCs and T-cells were carried out and then T-cell proliferation, and expansion of Th1, Th17 and Tregs were analysed. Results: Compared with HC, RA-patients displayed increased HLA-DR expression in DCs, which was manifested mainly in patients with moderate-to- high disease activity scores (DAS28). Furthermore, RA-patients presented a stronger Th17-expansion and a correlation between DAS28 and Th1-expansion. Both effects were mainly observed in patients in remission or with a low DAS28. Moreover, smoker RA-patients displayed enhanced HLA-DR and CD83 expression in DCs as well as an exacerbated Th17-expansion and a correlation between DAS28 and Th1-expansion. Conclusions: These findings suggest that smoking enhances the inflammatory behaviour of DCs and the consequent Th1 and Th17-mediated response in patients with RA

Introducción: El potencial dual que poseen para promover tolerancia o inflamación ante antígenos propios, hace de las células dendríticas (CDs) actores fundamentales en el desarrollo de autoinmunidad. Existe una asociación entre fumar y la maduración de las CDs en pacientes con artritis reumatoide (AR). No obstante, la etnicidad es un factor clave a considerar en desórdenes autoinmunes. Objetivos: Comparar las alteraciones fenotípicas y funcionales de las CDs obtenidas desde pacientes Chilenos con AR y controles sanos (CS). Además, analizamos las diferencias en el comportamiento inflamatorio que existe entre las CDs obtenidas de pacientes fumadores y CS. Materiales y Métodos: Se obtuvieron CDs derivadas de monocitos y células T desde muestras de sangre aisladas de 30 CS y 32 pacientes con AR, 14 de los cuales eran fumadores y 18 no fumadores. Se evaluaron marcadores de maduración en la superficie de las CDs: HLA-DR, CD40, CD80, CD83 y CD86. Además, se realizaron co-cultivos autólogos de células T y CDs, analizando la proliferación de células T, y la expansión de células Th1, Th17 y Tregs. Resultados: En comparación con los CS, los pacientes AR mostraron un aumento de la expresión de HLA-DR en las CDs, principalmente en los individuos con DAS28 moderado-alto. Los pacientes con AR presentaron una mayor expansión de células Th17 y una correlación entre el DAS28 y la expansión de células Th1, ambos efectos manifestados principalmente en los individuos con un DAS28 bajo o en remisión. Además, los pacientes con AR fumadores mostraron un aumento en la expresión de HLA-DR y CD83 en las CDs y una expansión de células Th17 exacerbada así como una correlación entre el DAS28 y la expansión de células Th1. Conclusiones: Nuestros resultados sugieren que fumar favorece el comportamiento inflamatorio de las CDs y en consecuencia la inducción de respuestas mediadas por células Th1 y Th17 en los pacientes Chilenos con AR.

Humans , Female , Middle Aged , Arthritis, Rheumatoid/metabolism , Dendritic Cells/immunology , Smoking/adverse effects , Cell Proliferation/physiology , Phenotype , Arthritis, Rheumatoid/physiopathology , Arthritis, Rheumatoid/immunology , Smoking/physiopathology , Antigens, Differentiation, B-Lymphocyte/immunology , HLA-DR Antigens/immunology , Case-Control Studies , Chile , T-Lymphocyte Subsets/immunology , Disease Progression , Flow Cytometry , Inflammation/physiopathology , Inflammation/drug therapy
Braz. j. med. biol. res ; 51(1): e6536, 2018. tab, graf
Article in English | LILACS | ID: biblio-889004


Kidney stone disease is a major cause of chronic renal insufficiency. The role of long non-coding RNAs (lncRNAs) in calcium oxalate-induced kidney damage is unclear. Therefore, we aimed to explore the roles of lncRNAs in glyoxylate-exposed and healthy mouse kidneys using microarray technology and bioinformatics analyses. A total 376 mouse lncRNAs were differentially expressed between the two groups. Using BLAST, 15 lncRNA homologs, including AU015836 and CHCHD4P4, were identified in mice and humans. The AU015836 expression in mice exposed to glyoxylate and the CHCHD4P4 expression in human proximal tubular epithelial (HK-2) cells exposed to calcium oxalate monohydrate were analyzed, and both lncRNAs were found to be upregulated in response to calcium oxalate. To further evaluate the effects of CHCHD4P4 on the cell behavior, we constructed stable CHCHD4P4-overexpressing and CHCHD4P4-knockdown HK-2 cells. The results showed that CHCHD4P4 inhibited cell proliferation and promoted the epithelial-mesenchymal transition in kidney damage and fibrosis caused by calcium oxalate crystallization and deposition. The silencing of CHCHD4P4 reduced the kidney damage and fibrosis and may thus be a potential molecular target for the treatment of kidney stones.

Humans , Animals , Rabbits , Kidney Calculi/genetics , Mitochondrial Membrane Transport Proteins/physiology , Cell Proliferation/genetics , Epithelial-Mesenchymal Transition/genetics , RNA, Long Noncoding/physiology , Fibrosis , Calcium Oxalate , Kidney Calculi/physiopathology , Up-Regulation , Cell Fractionation , Cell Line , Blotting, Western , Microarray Analysis , Cell Proliferation/physiology , Epithelial-Mesenchymal Transition/physiology , Real-Time Polymerase Chain Reaction
J. appl. oral sci ; 26: e20180077, 2018. graf
Article in English | LILACS, BBO | ID: biblio-954491


Abstract Objective This study evaluated the influence of platelet-rich plasma (PRP) on the behaviour of human gingival fibroblasts (hGFs), including fibroblast proliferation, migration and colony formation. Methods PRP was obtained from the human peripheral blood of a healthy volunteer and then was diluted into platelet concentrations of 1%, 2% and 5%. The proliferation of hGFs was determined by two methods: (1) Cell-number counting with a haemocytometer method at days 1, 3, 5 and 7; (2) Colony-forming unit-fibroblast (CFU-F) assay at 2 weeks. The migration of hGFs was evaluated with scratch assay, then recorded digital images were analysed by Image-Analysis J 1.51j8 software to compare the remaining artificial wound areas between PRP groups at 0, 24 and 48 hours. Results All hGFs that were cultivated in media with 1%, 2% and 5% PRP showed their ability to proliferate and migrate. Cell numbers incubated with 1% PRP increased significantly during the first three days and peaked at day 5, tending to be similar to their proliferation in complete medium. With concentrations of 2% and 5% PRP, hGFs outgrew and peaked at day 3, which was faster than with those in medium with 1% PRP. Especially, hGFs in the group 5% PRP proliferated with higher cell numbers than those in the other remaining groups at day 3. The hGF colony number that was formed in the group 5% PRP was significantly higher than those in the groups 1% and 2% PRP. Scratch assay showed hGFs in the groups 2% and 5% PRP almost filled the artificial wound and migrated more effectively than in the group 1% PRP at 24 hours, which was significant. Conclusion In this study, perhaps the medium with 5% PRP is the dominant option, promoting the abilities of hGFs to heal wounds, because of its fast and effective impact on cell proliferation, colony formation and migration.

Humans , Cell Movement/physiology , Reproducibility of Results , Cell Proliferation/physiology , Fibroblasts/drug effects , Fibroblasts/physiology , Time Factors , Cell Count , Cell Movement/drug effects , Cells, Cultured , Culture Media , Cell Proliferation/drug effects , Platelet-Rich Plasma , Gingiva/cytology
Braz. oral res. (Online) ; 32: e48, 2018. tab, graf
Article in English | LILACS | ID: biblio-952159


Abstract The aim was to investigate the angiogenic effects of concentrated growth factors on human dental pulp cells and human umbilical vein endothelial cells. Cells were treated with concentrated growth factor extracts. The CCK-8 assay and cell cycle assay were conducted to evaluate cell growth. Cell migration was evaluated by the Transwell migration assay. Angiogenesis-associated mRNA and protein expression levels were determined using quantitative real-time PCR and Western blotting, respectively. A tube formation assay was conducted to evaluate the angiogenic capacity in vitro. The data showed that compared with the control, concentrated growth factor extracts significantly promoted dental pulp cell proliferation and differentiation and endothelial cell proliferation and migration in a dose-dependent manner (p < 0.05). Concentrated growth factor extracts also promoted the tube-like structure formation of endothelial cells in vitro. The RT-PCR and Western blot results showed that concentrated growth factor extracts upregulated the expression of angiogenesis-related genes - chemokine receptor-4, platelet-derived growth factor, and vascular endothelial growth factor - in dental pulp cells. In conclusion, concentrated growth factors showed proangiogenic effects on dental pulp cells and endothelial cells and have good application potential for dental pulp revascularization.

Humans , Male , Adult , Neovascularization, Physiologic/physiology , Intercellular Signaling Peptides and Proteins/physiology , Dental Pulp/cytology , Human Umbilical Vein Endothelial Cells/physiology , Reference Values , Time Factors , Platelet-Derived Growth Factor/analysis , Platelet-Derived Growth Factor/physiology , Cell Cycle/physiology , Cells, Cultured , Blotting, Western , Reproducibility of Results , Analysis of Variance , Receptors, CXCR4/analysis , Receptors, CXCR4/physiology , Intercellular Signaling Peptides and Proteins/analysis , Vascular Endothelial Growth Factor A/analysis , Vascular Endothelial Growth Factor A/physiology , Cell Proliferation/physiology , Cell Migration Assays , Real-Time Polymerase Chain Reaction
Braz. j. med. biol. res ; 51(12): e7574, 2018. graf
Article in English | LILACS | ID: biblio-974257


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.

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
An. acad. bras. ciênc ; 89(3): 1719-1727, July-Sept. 2017. tab, graf
Article in English | LILACS | ID: biblio-886728


ABSTRACT This study aimed to determine the histological features of the endometrium of bitches, as well as the cell proliferation at specific moments of diestrus, 10, 20, 30, 40, 50 and 60 days post ovulation, correlating the endometrial thickness with the uterine cell proliferation and the metabolic state (weight, blood glucose and plasma cholesterol) of the animals. Therefore, the right and left uterine horns of 26 clinically healthy bitches submitted to ovariohysterectomy were histologically analyzed 10, 20, 30, 40, 50 and 60 days post ovulation. The hematoxylin-eosin and AgNOR staining techniques were performed. All parameters were evaluated by ANOVA and post-hoc Tukey test (p<0.05). The correlation between endometrial thickness and uterine cell proliferation, weight, blood glucose and plasma cholesterol of animals was observed using the Pearson method (p<0.05). In the present study, it is concluded that endometrial thickness does not differ at any of the moments analyzed in diestrus. The endometrial thickness is not influenced by hormones, weight, blood glucose or serum cholesterol of bitches in this phase of the estrous cycle. However, there is greater cell proliferation in the endometrium at day 40 compared to day 60 post ovulation under the influence of the endocrine profile.

Animals , Female , Dogs , Diestrus/physiology , Cholesterol/blood , Cell Proliferation/physiology , Endometrium/cytology , Glucose/analysis , Time Factors , Diestrus/metabolism , Endometrium/physiology