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Id: biblio-1284288
Autor: Marzbany, Marzieh; Rasekhian, Mahsa.
Título: Effect of encephalomyocarditis virus 3'Untranslated region on GFP transient expression: implications in recombinant protein production / Efecto de la Región 3'-no traducida del virus de la encefalomiocarditis sobre la expresión transitoria de GFP: implicaciones en la producción de proteínas recombinantes
Fonte: Bol. latinoam. Caribe plantas med. aromát;19(6):542-554, 2020. ilus, tab.
Idioma: en.
Resumo: The enrichment of therapeutic protein production yield in mammalian cell cultures by modulating mRNA stability is a fairly new strategy in biotechnological applications. Here, we describe the application of 3'-untranslated region (3'UTR) from RNA viral genome to modulate mRNA stability.The data obtained showed that the use of the 3 'UTR sequence of the encephalomyocarditis virus (EMCV 3'UTR) downstream of the target gene was not able to significantly modulate the free energy density indicators of the RNA. However, the sequence influenced the stability of the mRNA (and, therefore, the amount of protein production) in a cell type and time-dependent manner, indicating a central role of mRNA-stabilizing binding sites/cellular factors in this process. Our data might be of interest for the biotechnology community to improve recombinant protein production in mammalian cell cultures and RNA-based therapy/vaccination approaches.

El enriquecimiento de la producción terapéutica de proteínas en cultivos de células de mamíferos mediante la modulación de la estabilidad del ARNm es una estrategia nueva en aplicaciones biotecnológicas. Se describe la aplicación de la región 3'-no traducida (3'UTR) del genoma viral ARN para modular la estabilidad del ARNm. Los datos obtenidos mostraron que el uso de la secuencia 3'UTR del virus de la encefalomiocarditis (EMCV 3'UTR) aguas abajo del gen objetivo no pudo modular significativamente los indicadores de densidad de energía libre del ARN. Sin embargo, la secuencia influyó en la estabilidad del ARNm (y, por lo tanto, en la cantidad de producción de proteínas) dependiente de la célula y del tiempo, lo que indica un papel central de los sitios de unión estabilizadores de ARNm/factores celulares en este proceso. Nuestros datos podrían ser de interés para la comunidad biotecnológica para mejorar la producción de proteínas recombinantes en cultivos de células de mamíferos y en enfoques de terapia/vacunación basados en ARN.
Descritores: Produtos Biológicos
Proteínas Recombinantes/biossíntese
Regiões não Traduzidas
Proteínas de Fluorescência Verde/metabolismo
Vírus da Encefalomiocardite/metabolismo
-Biotecnologia
Genoma Viral
Técnicas de Cultura de Células
Estabilidade de RNA
Vírus da Encefalomiocardite/genética
Responsável: CL1.1 - Biblioteca Central


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Id: biblio-953182
Autor: Rodrigues, Isabella Caroline Pereira; Kaasi, Andreas; Maciel Filho, Rubens; Jardini, André Luiz; Gabriel, Laís Pellizzer.
Título: Cardiac tissue engineering: current state-of-the-art materials, cells and tissue formation / Engenharia de tecidos cardíacos: atual estado da arte a respeito de materiais, células e formação tecidual
Fonte: Einstein (Säo Paulo);16(3):eRB4538, 2018. tab, graf.
Idioma: en.
Projeto: Instituto Nacional de Ciência e Tecnologia em Biofabricação (INCT-BIOFABRIS), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq); . Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP).
Resumo: ABSTRACT Cardiovascular diseases are the major cause of death worldwide. The heart has limited capacity of regeneration, therefore, transplantation is the only solution in some cases despite presenting many disadvantages. Tissue engineering has been considered the ideal strategy for regenerative medicine in cardiology. It is an interdisciplinary field combining many techniques that aim to maintain, regenerate or replace a tissue or organ. The main approach of cardiac tissue engineering is to create cardiac grafts, either whole heart substitutes or tissues that can be efficiently implanted in the organism, regenerating the tissue and giving rise to a fully functional heart, without causing side effects, such as immunogenicity. In this review, we systematically present and compare the techniques that have drawn the most attention in this field and that generally have focused on four important issues: the scaffold material selection, the scaffold material production, cellular selection and in vitro cell culture. Many studies used several techniques that are herein presented, including biopolymers, decellularization and bioreactors, and made significant advances, either seeking a graft or an entire bioartificial heart. However, much work remains to better understand and improve existing techniques, to develop robust, efficient and efficacious methods.

RESUMO Doenças cardiovasculares são responsáveis pelo maior número de mortes no mundo. O coração possui capacidade de regeneração limitada, e o transplante, por consequência, representa a única solução em alguns casos, apresentando várias desvantagens. A engenharia de tecidos tem sido considerada a estratégia ideal para a medicina cardíaca regenerativa. Trata-se de uma área interdisciplinar, que combina muitas técnicas as quais buscam manter, regenerar ou substituir um tecido ou órgão. A abordagem principal da engenharia de tecidos cardíacos é criar enxertos cardíacos, sejam substitutos do coração inteiro ou de tecidos que podem ser implantados de forma eficiente no organismo, regenerando o tecido e dando origem a um coração completamente funcional, sem desencadear efeitos colaterais, como imunogenicidade. Nesta revisão, apresentase e compara-se sistematicamente as técnicas que ganharam mais atenção nesta área e que geralmente focam em quatro assuntos importantes: seleção do material a ser utilizado como enxerto, produção do material, seleção das células e cultura de células in vitro. Muitos estudos, fazendo uso de várias das técnicas aqui apresentadas, incluindo biopolímeros, descelularização e biorreatores, têm apresentado avanços significativos, seja para obter um enxerto ou um coração bioartifical inteiro. No entanto, ainda resta um grande esforço para entender e melhorar as técnicas existentes, para desenvolver métodos robustos, eficientes e eficazes.
Descritores: Transplante de Coração/métodos
Engenharia Tecidual/métodos
Miocárdio/citologia
-Biopolímeros
Transplante de Coração/tendências
Técnicas de Cultura de Células/métodos
Reatores Biológicos
Engenharia Tecidual/tendências
Tecidos Suporte
Limites: Humanos
Responsável: BR1.1 - BIREME


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Id: biblio-1056032
Autor: Mamani, Javier Bustamante; Marinho, Bruna Souto; Rego, Gabriel Nery de Albuquerque; Hospital das ClínicasNucci, Mariana Penteado; Alvieri, Fernando; Santos, Ricardo Silva dos; Ferreira, João Victor Matias; Oliveira, Fernando Anselmo de; Gamarra, Lionel Fernel.
Título: Magnetic hyperthermia therapy in glioblastoma tumor on-a-Chip model / Terapia de magneto-hipertermia no modelo de tumor de glioblastoma on-a-Chip
Fonte: Einstein (Säo Paulo);18:eAO4954, 2020. graf.
Idioma: en.
Projeto: CNPq; . FAPESP.
Resumo: ABSTRACT Objective: To evaluate the magnetic hyperthermia therapy in glioblastoma tumor-on-a-Chip model using a microfluidics device. Methods: The magnetic nanoparticles coated with aminosilane were used for the therapy of magnetic hyperthermia, being evaluated the specific absorption rate of the magnetic nanoparticles at 300 Gauss and 305kHz. A preculture of C6 cells was performed before the 3D cells culture on the chip. The process of magnetic hyperthermia on the Chip was performed after administration of 20μL of magnetic nanoparticles (10mgFe/mL) using the parameters that generated the specific absorption rate value. The efficacy of magnetic hyperthermia therapy was evaluated by using the cell viability test through the following fluorescence staining: calcein acetoxymethyl ester (492/513nm), for live cells, and ethidium homodimer-1 (526/619nm) for dead cells dyes. Results: Magnetic nanoparticles when submitted to the alternating magnetic field (300 Gauss and 305kHz) produced a mean value of the specific absorption rate of 115.4±6.0W/g. The 3D culture of C6 cells evaluated by light field microscopy imaging showed the proliferation and morphology of the cells prior to the application of magnetic hyperthermia therapy. Fluorescence images showed decreased viability of cultured cells in organ-on-a-Chip by 20% and 100% after 10 and 30 minutes of the magnetic hyperthermia therapy application respectively. Conclusion: The study showed that the therapeutic process of magnetic hyperthermia in the glioblastoma on-a-chip model was effective to produce the total cell lise after 30 minutes of therapy.

RESUMO Objetivo: Avaliar a terapia de magneto-hipertermia em modelo de tumor de glioblastoma on-a-Chip. Métodos: As nanopartículas magnéticas recobertas com aminosilana foram utilizadas para a terapia da magneto-hipertermia, sendo avaliada a taxa de absorção específica das nanopartículas magnéticas em 300 Gauss e 305kHz. Uma pré-cultura de células C6 foi realizada e, seguidamente, foi feito o cultivo das células 3D no chip. O processo de magneto-hipertermia no chip foi realizado após administração de 20μL de nanopartículas magnéticas (10mgFe/mL), utilizando os parâmetros que geraram o valor da taxa de absorção específica. A eficácia da terapia de magneto-hipertermia foi avaliada pela viabilidade celular por meio dos corantes fluorescentes acetoximetiléster de calceína (492/513nm), para células vivas, e etídio homodímero-1 (526/619nm), para células mortas. Resultados: As nanopartículas magnéticas, quando submetidas ao campo magnético alternado (300 Gauss e 305kHz), produziram um valor médio da taxa de absorção específica de 115,4±6,0W/g. A cultura 3D das células C6 avaliada por imagem de microscopia de campo claro mostrou a proliferação e a morfologia das células antes da aplicação da terapia de magneto-hipertermia. As imagens de fluorescência mostraram diminuição da viabilidade das células cultivadas no organ-on-a-Chip em 20% e 100% após 10 e 30 minutos, respectivamente, da aplicação da terapia de magneto-hipertermia. Conclusão: O processo terapêutico da magneto-hipertermia no modelo de tumor glioblastoma on-a-chip foi eficaz para produzir lise total das células após 30 minutos de terapia.
Descritores: Glioblastoma/terapia
Técnicas de Cultura de Células/métodos
Dispositivos Lab-On-A-Chip
Nanopartículas de Magnetita/uso terapêutico
Hipertermia Induzida/métodos
-Temperatura
Fatores de Tempo
Sobrevivência Celular
Reprodutibilidade dos Testes
Resultado do Tratamento
Linhagem Celular Tumoral
Campos Magnéticos
Fluorescência
Limites: Animais
Ratos
Tipo de Publ: Estudo de Avaliação
Responsável: BR1.1 - BIREME


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Mantovani, Mário Sérgio
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Id: biblio-1283485
Autor: Biazi, Bruna Isabela; Zanetti, Thalita Alves; Marques, Lilian Areal; Baranoski, Adrivanio; Coatti, Giuliana Castello; Mantovani, Mário Sérgio.
Título: mRNAs biomarker related to the control of proliferation and cell death in HepG2/C3A spheroid and monolayer cultures treated with piperlongumine
Fonte: Appl. cancer res;40:1-13, Oct. 19, 2020. ilus.
Idioma: en.
Resumo: Background: Cell culture (spheroid and 2D monolayer cultures) is an essential tool in drug discovery. Piperlongumine (PLN), a naturally occurring alkaloid present in the long pepper (Piper longum), has been implicated in the regulation of GSTP1 activity. In vitro treatment of cancer cells with PLN increases ROS (reactive oxygen species) levels and induces cell death, but its molecular mode of action has not been entirely elucidated. Methods: In this study, we correlated the antiproliferative effects (2D and 3D cultures) of PLN (CAS 20069­09-4, Sigma-Aldrich) with morphological and molecular analyses in HepG2/C3A cell line. We performed assays for cytotoxicity (MTT), comet assays for genotoxicity, induction of apoptosis, analysis of the cell cycle phase, and analysis of the membrane integrity by flow cytometry. Relative expression of mRNA of genes related to proliferation, apoptosis, cell cycle control, metabolism of xenobiotics, and reticulum endoplasmic stress. Results: PLN reduced the cell proliferation by the cell cycle arrest in G2/M. Changes in the mRNA expression for CDKN1A (4.9x) and CCNA2 (0.5x) of cell cycle control genes were observed. Cell death occurred due to apoptosis, which may have been induced by increased expression of proapoptotic mRNAs (BAK1, 3.1x; BBC3, 2.4x), and by an increase in 9 and 3/7 active caspases. PLN induced cellular injury by ROS generation and DNA damage. DNA damage induced MDM2 signaling (3.0x) associated with the appearance of the monastral spindle in mitosis. Genes associated with ROS degradation also showed increased mRNA expression (GSR, 2.0x; SOD1, 2.1x). PLN induce endoplasmic reticulum stress with the increase in the mRNA expression of ERN1 (4.5x) and HSPA14 (2.2x). The xenobiotic metabolism showed increased mRNA expression for CYP1A2 (2.2x) and CYP3A4 (3.4x). In addition to 2D culture, PLN treatment also inhibited the growth of 3D culture (spheroids). Conclusion: Thus, the findings of our study show that several gene expression biomarkers (mRNAs) and monastral spindle formation indicated the many pathways of damage induced by PLN treatment that contributes to its antiproliferative effects
Descritores: RNA Mensageiro/efeitos dos fármacos
Morte Celular/efeitos dos fármacos
Técnicas de Cultura de Células
Proliferação de Células/efeitos dos fármacos
Dioxolanos/farmacologia
Antineoplásicos/farmacologia
-Biomarcadores/análise
Expressão Gênica/efeitos dos fármacos
Esferoides Celulares/efeitos dos fármacos
Células Hep G2/efeitos dos fármacos
Limites: Humanos
Responsável: BR30.1 - Biblioteca


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Id: biblio-1254836
Autor: Pérez-Rodriguez, Saumel; Ramírez, Octavio T; Trujillo-Roldán, Mauricio A; Valdez-Cruz, Norma A.
Título: Comparison of protein precipitation methods for sample preparation prior to proteomic analysis of Chinese hamster ovary cell homogenates
Fonte: Electron. j. biotechnol;48:86-94, nov. 2020. tab, graf, ilus.
Idioma: en.
Projeto: Consejo Nacional de Ciencia y Tecnología CONACyT-México; . Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica, Universidad Nacional Autónoma de México, PAPIIT-UNAM; . Institutional Program of the Instituto de Investigaciones Biomédicas-UNAM.
Resumo: BACKGROUND: Chinese hamster ovary (CHO) cells are the workhorse for obtaining recombinant proteins. Proteomic studies of these cells intend to understand cell biology and obtain more productive and robust cell lines for therapeutic protein production in the pharmaceutical industry. Because of the great importance of precipitation methods for the processing of samples in proteomics, the acetone, methanol-chloroform (M/C), and trichloroacetic acid (TCA)-acetone protocols were compared for CHO cells in terms of protein recovery, band pattern resolution, and presence on SDS-PAGE. RESULTS: Higher recovery and similar band profile with cellular homogenates were obtained using acetone precipitation with ultrasonic bath cycles (104.18 ± 2.67%) or NaOH addition (103.12 ± 5.74%), compared to the other two protocols tested. TCA-acetone precipitates were difficult to solubilize, which negatively influenced recovery percentage (77.91 ± 8.79%) and band presence. M/C with ultrasonic homogenization showed an intermediate recovery between the other two protocols (94.22 ± 4.86%) without affecting protein pattern on SDS-PAGE. These precipitation methods affected the recovery of low MW proteins (< 15 kDa). CONCLUSIONS: These results help in the processing of samples of CHO cells for their proteomic study by means of an easily accessible, fast protocol, with an almost complete recovery of cellular proteins and the capture of the original complexity of the cellular composition. Acetone protocol could be incorporated to sample-preparation workflows in a straightforward manner and can probably be applied to other mammalian cell lines as well.
Descritores: Proteínas Recombinantes
Células CHO
Proteômica/métodos
-Acetona
Precipitação Química
Solubilidade
Ácido Tricloroacético
Separação Celular
Clorofórmio
Técnicas de Cultura de Células
Metanol
Eletroforese em Gel de Poliacrilamida
Limites: Animais
Responsável: CL1.1 - Biblioteca Central


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Id: biblio-1249655
Autor: Campos, Marcelo Ferraz; Silva, Mariane de Barros Ribeiro da; Pinhal, Maria Aparecida Silva; Salati, Thiago; Rodrigues, Luciano Miller Reis; Melo, Carina Mucciolo.
Título: Cell therapy in the treatment of intervertebral disc degeneration / Terapia celular no tratamento da degeneração de disco intervertebral / Terapia celular en el tratamiento de la degeneración de disco intervertebral
Fonte: Coluna/Columna;20(2):101-104, Apr.-June 2021. graf.
Idioma: en.
Resumo: ABSTRACT Approximately 80% of the world population experiences some type of back pain at some point in their life, and in 10% of this population the pain causes chronic disability resulting in a high cost for the treatment of these patients, in addition to compromising their work and social interaction abilities. Current treatment strategies include the surgical procedure for degenerated intervertebral disc resection, the nerve root block and physiotherapy. However, such treatments only relieve symptoms and do not prevent the degeneration of intervertebral discs. Therefore, new therapeutic strategies have emerged and include manipulating cells to recover the degenerated disc. This article will discuss the possible cell therapy alternatives used in the disc regeneration process, featuring a descriptive study of translational medicine that involves clinical aspects of new treatment alternatives and knowledge of basic research areas, such as cellular and molecular biology. Level of evidence V; Expert Opinion.

RESUMO Aproximadamente 80% da população mundial sofre algum tipo de dor nas costas em alguma fase de vida, sendo que em 10% dessa população, as dores acarretam incapacidade crônica, deflagrando alto custo de tratamento desses pacientes, além de comprometer as habilidades de trabalho e convívio social desses indivíduos. As estratégias de tratamento atuais incluem o procedimento cirúrgico por ressecção do disco intervertebral degenerado, bloqueio de raízes nervosas e fisioterapia. Entretanto, tais tratamentos apenas aliviam os sintomas e não impedem que ocorra a degeneração de discos intervertebrais. Portanto, novas estratégias terapêuticas têm surgido e incluem a manipulação de células com o objetivo de recuperar o disco degenerado. No presente artigo, serão discutidas as diferentes possibilidades alternativas de terapias celulares no processo de regeneração discal, caracterizando um estudo descritivo da medicina translacional que envolve aspectos clínicos de novas alternativas de tratamento e o conhecimento de áreas básicas de pesquisa como biologia celular e molecular. Nível de evidência V; Opinião do Especialista.

RESUMEN Aproximadamente 80% de la población mundial sufre algún tipo de dolor de espalda en alguna etapa de la vida, y en 10% de esa población, los dolores causan incapacidad crónica, deflagrando alto costo de tratamiento de esos pacientes, además de comprometer las habilidades laborales y convivencia social de esos individuos. Las estrategias de tratamiento actuales incluyen el procedimiento quirúrgico para la resección del disco intervertebral degenerado, bloqueo de las raíces nerviosas y fisioterapia. Entretanto, tales tratamientos solo alivian los síntomas y no impiden que ocurra la degeneración de discos intervertebrales. Por lo tanto, han surgido nuevas estrategias terapéuticas e incluyen la manipulación de células con el objetivo de recuperar el disco degenerado. En el presente artículo se discutirán las diferentes posibilidades alternativas de las terapias celulares en el proceso de regeneración discal, caracterizando un estudio descriptivo de la medicina traslacional que involucra aspectos clínicos de nuevas alternativas de tratamiento y conocimiento de áreas básicas de investigación como biología celular y molecular. Nivel de evidencia V; Opinión del especialista.
Descritores: Técnicas de Cultura de Células
Terapia Baseada em Transplante de Células e Tecidos
Disco Intervertebral
Limites: Humanos
Tipo de Publ: Artigo Clássico
Responsável: BR15.3 - Biblioteca Emília Bustamante


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Id: biblio-990004
Autor: Liu, Lu-Lu; Zhang, Bin-Bin; Gao, Quan-Wen; Bing, Li; Sha, Huang; Bin, Yao.
Título: Morphological observation of bone marrow mesenchymal stem cells under matrigel three dimensional culture conditions / Observación morfológica de células madre mesenquimales de médula ósea en condiciones de cultivo tridimensional con matrigel
Fonte: Int. j. morphol;37(1):54-58, 2019. graf.
Idioma: en.
Projeto: Beijing Natural Science Foundation of China.
Resumo: SUMMARY: Matrigel is a basement membrane matrix extracted from the EHS mouse tumor containing extracellular matrix protein, its main components are laminin, type IV collagen, nestin, heparin sulfate, growth factor and matrix metalloproteinase.At room temperature, Matrigel polymerized to form a three dimensional matrix with biological activity. It can simulate the structure, composition, physical properties and functions of the cell basement membrane in vivo, which is beneficial to the culture and differentiation of the cells in vitro, and can be used for the study of cell morphology, biochemical function, migration, infection and gene expression. In this study, Matrigel three-dimensional culture model of bone marrow mesenchymal stem cells(BMSCs) was established, and its morphology, proliferation and survival were observed. BMSCs were isolated and cultured with whole bone marrow adherence method. The Second generation BMSCs with good growth condition were selected and mixed with Matrigel to form cell gel complexes. The morphology and proliferation of mesenchymal stem cells were observed by phase contrast microscope and HE staining,Live/Dead staining was used to evaluate the cell activity.Phase contrast microscopy showed that BMSCs were reticulated in Matrigel and proliferated well, After 7 days, the matrix gel gradually became soft and collapsed, a few cell reticular crosslinking growth was seen at 14 days; HE staining showed that the cytoplasm of the cells was larger on the fourth day and the cells were elongated and cross-linked on the seventh day; Live/dead staining showed that most cells showed green fluorescence with the prolongation of culture time, on the first, 4 and 7 days, the activity of bone marrow mesenchymal stem cells in Matrigel gradually increased, and the percentages were 92.57 %, 95.54 % and 97.37 %, respectively. Matrigel three-dimensional culture system can maintain the morphology, function and proliferation ability of bone marrow mesenchymal stem cells.

RESUMEN: Matrigel es una matriz de membrana basal extraída del tumor de ratón EHS que contiene proteína de matriz extracelular. Los componentes principales son laminina, el colágeno tipo IV, nestina, sulfato de heparina, factor de crecimiento y metaloproteinasa de matriz. A temperatura ambiente, Matrigel se polimerizó para formar una matriz tridimensional. Es posible simular la estructura, la composición, las propiedades físicas y las funciones de la membrana basal celular in vivo, lo que es beneficioso para el cultivo y la diferenciación de las células in vitro, y se puede utilizar para el estudio de la morfología celular, la función bioquímica, la migración, infección y expresión génica. En este estudio, se estableció el modelo de cultivo tridimensional Matrigel de células madre mesenquimales de médula ósea (BMSC), y se observó su morfología, proliferación y supervivencia. Las BMSC fueron aisladas y cultivadas con el método de adherencia de la médula ósea completa. Se seleccionaron las BMSC de segunda generación con buenas condiciones de crecimiento y se mezclaron con Matrigel para formar complejos de gel de células. La morfología y la proliferación de las células madre mesenquimales se observaron con microscopio de contraste de fase y se tiñó con Hematoxilina-Eosina (HE); para evaluar la actividad celular se usó la tinción Live/Dead. La microscopía de contraste mostró que las BMSC se reticularon en Matrigel y proliferaron bien. Después de 7 días, se observó que el gel de matriz gradualmente se volvió blando y colapsó, y se visualizó un cruce transversal de algunas células reticulares a los 14 días. La tinción mostró que la mayoría de las células mostraron una fluorescencia verde con la prolongación del tiempo de cultivo; en los primeros 4 y 7 días, la actividad de las células madre mesenquimales de la médula ósea en Matrigel aumentó gradualmente y los porcentajes fueron de 92,57 %, 95,54 % y 97,37 %, respectivamente. El sistema de cultivo tridimensional de Matrigel puede mantener la morfología, la función y la capacidad de proliferación de las células madre mesenquimales de la médula ósea.
Descritores: Proteoglicanas/química
Colágeno/química
Laminina/química
Técnicas de Cultura de Células/métodos
Células-Tronco Mesenquimais/citologia
-Engenharia Tecidual
Combinação de Medicamentos
Limites: Animais
Cães
Responsável: CL1.1 - Biblioteca Central


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Id: lil-794669
Autor: Zhu, Yong-tong; Pang, Shi-yu; Luo, Yang; Chen, Wei; Bao, Ji-ming; Tan, Wan-long.
Título: A modified method by differential adhesion for enrichment of bladder cancer stem cells
Fonte: Int. braz. j. urol;42(4):817-824, July-Aug. 2016. tab, graf.
Idioma: en.
Projeto: Education Department in Guangdong Province; . Southern Medical University.
Resumo: ABSTRACT Purpose: In a previous study the vaccine was effective against bladder cancer in a mouse model. However, a small portion of tumors regrew because the vaccine could not eliminate bladder cancer stem cells (CSCs). In this study, we showed a modified method for the isolation of bladder CSCs using a combination of differential adhesion method and serum-free culture medium (SFM) method. Materials and Methods: Trypsin-resistant cells and trypsin-sensitive cells were isolated from MB49, EJ and 5637 cells by a combination of differential adhesion method and SFM method. The CSCs characterizations of trypsin-resistant cells were verified by the flow cytometry, the western blotting, the quantitative polymerase chain reaction, the resistance to chemotherapy assay, the transwell assay, and the tumor xenograft formation assay. Results: Trypsin-resistant cells were isolated and identified in CSCs characters, with high expression of CSCs markers, higher resistance to chemotherapy, greater migration in vitro, and stronger tumorigenicity in vivo. Conclusion: Trypsin-resistant cells displayed specific CSCs properties. Our study showed trypsin-resistant cells were isolated successfully with a modified method using a combination of differential adhesion method and SFM method.
Descritores: Células-Tronco Neoplásicas/citologia
Neoplasias da Bexiga Urinária/patologia
Tripsina/farmacologia
Adesão Celular/efeitos dos fármacos
Separação Celular/métodos
Técnicas de Cultura de Células/métodos
-Células-Tronco Neoplásicas/química
Biomarcadores Tumorais
Diferenciação Celular
Meios de Cultura Livres de Soro
Vacinas Anticâncer/imunologia
Linhagem Celular Tumoral
Reação em Cadeia da Polimerase em Tempo Real
Citometria de Fluxo
Camundongos Nus
Limites: Animais
Camundongos
Responsável: BR1.1 - BIREME


  9 / 495 LILACS  
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Id: biblio-838966
Autor: Ramachandran, Gokula Krishnan; Yeow, Chen Hua.
Título: Proton NMR characterization of intact primary and metastatic melanoma cells in 2D & 3D cultures
Fonte: Biol. Res;50:12, 2017. tab, graf.
Idioma: en.
Projeto: MOE Academic research fund.
Resumo: OBJECTIVE: To characterize the differences between the primary and metastatic melanoma cell lines grown in 2D cultures and 3D cultures. METHODS: Primary melanoma cells (WM115) and metastatic melanoma cells (WM266) extracted from a single donor was cultured in 2D as well as 3D cultures. These cells were characterized using proton NMR spectrometry, and the qualitative chemical shifts markers were identified and discussed. RESULTS: In monolayer culture (2D), we observed one qualitative chemical shift marker for primary melanoma cells. In spheroid cultures (3D), we observed nine significant chemical shifts, of which eight markers were specific for primary melanoma spheroids, whereas the other one marker was specific to metastatic melanoma spheroids. This study suggests that the glucose accumulation and phospholipid composition vary significantly between the primary and metastatic cells lines that are obtained from a single donor and also with the cell culturing methods. 14 qualitative chemical shift markers were obtained in the comparison between monolayer culture and spheroids cultures irrespective of the differences in the cell lines. Among which 4 were unique to monolayer cultures whereas 10 chemical shifts were unique to the spheroid cultures. This study also shows that the method of cell culture would drastically affect the phospholipid composition of the cells and also depicts that the cells in spheroid culture closely resembles the cells in vivo. CONCLUSION: This study shows the high specificity of proton NMR spectrometry in characterizing cancer cell lines and also shows the variations in the glucose accumulation and phospholipid composition between the primary and metastatic melanoma cell lines from the same donor. Differences in the cell culture method does plays an important role in phospholipid composition of the cells.
Descritores: Espectroscopia de Ressonância Magnética/métodos
Técnicas de Cultura de Células/métodos
Melanoma/patologia
Melanoma/secundário
-Fosfolipídeos/análise
Fosfolipídeos/metabolismo
Fatores de Tempo
Biomarcadores Tumorais
Análise de Variância
Esferoides Celulares
Linhagem Celular Tumoral
Glucose/análise
Glucose/metabolismo
Melanoma/metabolismo
Limites: Humanos
Responsável: CL1.1 - Biblioteca Central


  10 / 495 LILACS  
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Id: biblio-890754
Autor: Zuliani, Carolina Coli; Bombini, Mariana Freschi; Andrade, Kleber Cursino de; Mamoni, Ronei; Pereira, Ana Helena; Coimbra, Ibsen Bellini.
Título: Micromass cultures are effective for differentiation of human amniotic fluid stem cells into chondrocytes
Fonte: Clinics;73:e268, 2018. tab, graf.
Idioma: en.
Projeto: CNPq.
Resumo: OBJECTIVES: Articular cartilage is vulnerable to injuries and undergoes an irreversible degenerative process. The use of amniotic fluid mesenchymal stromal stem cells for the reconstruction of articular cartilage is a promising therapeutic alternative. The aim of this study was to investigate the chondrogenic potential of amniotic fluid mesenchymal stromal stem cells from human amniotic fluid from second trimester pregnant women in a micromass system (high-density cell culture) with TGF-β3 for 21 days. METHODS: Micromass was performed using amniotic fluid mesenchymal stromal stem cells previously cultured in a monolayer. Chondrocytes from adult human normal cartilage were used as controls. After 21 days, chondrogenic potential was determined by measuring the expression of genes, such as SOX-9, type II collagen and aggrecan, in newly differentiated cells by real-time PCR (qRT-PCR). The production of type II collagen protein was observed by western blotting. Immunohistochemistry analysis was also performed to detect collagen type II and aggrecan. This study was approved by the local ethics committee. RESULTS: SOX-9, aggrecan and type II collagen were expressed in newly differentiated chondrocytes. The expression of SOX-9 was significantly higher in newly differentiated chondrocytes than in adult cartilage. Collagen type II protein was also detected. CONCLUSION: We demonstrate that stem cells from human amniotic fluid are a suitable source for chondrogenesis when cultured in a micromass system. amniotic fluid mesenchymal stromal stem cells are an extremely viable source for clinical applications, and our results suggest the possibility of using human amniotic fluid as a source of mesenchymal stem cells.
Descritores: Técnicas de Cultura de Células/métodos
Condrócitos/citologia
Condrogênese
Células-Tronco Mesenquimais/citologia
-Expressão Gênica
Diferenciação Celular
Colágeno Tipo II/análise
Agrecanas/metabolismo
Fator de Crescimento Transformador beta3/metabolismo
Fatores de Transcrição SOX9/metabolismo
Líquido Amniótico
Limites: Humanos
Gravidez
Tipo de Publ: Research Support, Non-U.S. Gov't
Responsável: BR1.1 - BIREME



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