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Soares, Christiane Pienna
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Id: biblio-952020
Autor: Gomes-Cornélio, Ana Lívia; Rodrigues, Elisandra Márcia; Mestieri, Leticia Boldrin; Falcoski, Thaís de Oliveira Rodrigues Sanzovo; Soares, Christiane Pienna; Guerreiro-Tanomaru, Juliane Maria; Rossa Junior, Carlos; Tanomaru Filho, Mário.
Título: Cytotoxicity and genotoxicity of calcium silicate-based cements on an osteoblast lineage
Fonte: Braz. oral res. (Online);30(1):e48, 2016. tab, graf.
Idioma: en.
Projeto: Fundação de Amparo à Pesquisa do Estado de São Paulo; . Fundação de Amparo à Pesquisa do Estado de São Paulo; . Fundação de Amparo à Pesquisa do Estado de São Paulo.
Resumo: Abstract Several calcium silicate-based biomaterials have been developed in recent years, in addition to Mineral Trioxide Aggregate (MTA). The aim of this study was to evaluate the cytotoxicity, genotoxicity and apoptosis/necrosis in human osteoblast cells (SAOS-2) of pure calcium silicate-based cements (CSC) and modified formulations: modified calcium silicate-based cements (CSCM) and three resin-based calcium silicate cements (CSCR1) (CSCR 2) (CSCR3). The following tests were performed after 24 hours of cement extract exposure: methyl-thiazolyl tetrazolium (MTT), apoptosis/necrosis assay and comet assay. The negative control (CT-) was performed with untreated cells, and the positive control (CT+) used hydrogen peroxide. The data for MTT and apoptosis were submitted to analysis of variance and Bonferroni's posttest (p < 0.05), and the data for the comet assay analysis, to the Kruskal-Wallis and Dunn tests (p < 0.05). The MTT test showed no significant difference among the materials in 2 mg/mL and 10 mg/mL concentrations. CSCR3 showed lower cell viability at 10 mg/mL. Only CSC showed lower cell viability at 50 mg/mL. CSCR1, CSCR2 and CSCR3 showed a higher percentage of initial apoptosis than the control in the apoptosis test, after 24 hours exposure. The same cements showed no genotoxicity in the concentration of 2 mg/mL, with the comet assay. CSC and CSCR2 were also not genotoxic at 10 mg/mL. All experimental materials showed viability with MTT. CSC and CSCR2 presented a better response to apoptosis and genotoxicity evaluation in the 10 mg/mL concentration, and demonstrated a considerable potential for use as reparative materials.
Descritores: Osteoblastos/efeitos dos fármacos
Silicatos/toxicidade
Compostos de Cálcio/toxicidade
Cimentos Dentários/toxicidade
-Óxidos/toxicidade
Sais de Tetrazólio
Materiais Biocompatíveis/toxicidade
Teste de Materiais
Sobrevivência Celular/efeitos dos fármacos
Células Cultivadas
Reprodutibilidade dos Testes
Análise de Variância
Apoptose/efeitos dos fármacos
Compostos de Alumínio/toxicidade
Ensaio Cometa
Proliferação de Células/efeitos dos fármacos
Combinação de Medicamentos
Formazans
Necrose/induzido quimicamente
Limites: Humanos
Responsável: BR1.1 - BIREME


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Id: biblio-950716
Autor: Hung, Tran Manh; Dang, Nguyen Hai; Dat, Nguyen Tien.
Título: Methanol extract from Vietnamese Caesalpinia sappan induces apoptosis in HeLa cells
Fonte: Biol. Res;47:1-5, 2014. ilus, graf, tab.
Idioma: en.
Projeto: Vietnam National Foundation for Science and Technology Development.
Resumo: BACKGROUND: This study evaluated the cytotoxic activity of extracts from Caesalpinia sappan heartwood against multiple cancer cell lines using an MTT cell viability assay. The cell death though induction of apoptosis was as indicated by DNA fragmentation and caspase-3 enzyme activation. RESULTS: A methanol extract from C. sappan (MECS) showed cytotoxic activity against several of the cancer cell lines. The most potent activity exhibited by the MECS was against HeLa cells with an IC50 value of 26.5 ± 3.2 µg/mL. Treatment of HeLa cells with various MECS concentrations resulted in growth inhibition and induction of apoptosis, as indicated by DNA fragmentation and caspase-3 enzyme activation. CONCLUSION: This study is the first report of the anticancer properties of the heartwood of C. sappan native to Vietnam. Our findings demonstrate that C. sappan heartwood may have beneficial applications in the field of anticancer drug discovery.
Descritores: Extratos Vegetais/farmacologia
Apoptose
Caesalpinia/química
Feixe Vascular de Plantas/metabolismo
Antineoplásicos Fitogênicos/farmacologia
-Sais de Tetrazólio
Vietnã
Ensaios de Seleção de Medicamentos Antitumorais/métodos
Células HeLa
Sobrevivência Celular
Concentração Inibidora 50
Citotoxinas/farmacologia
Linhagem Celular Tumoral
Proliferação de Células/efeitos dos fármacos
Metanol
Ativação Enzimática/efeitos dos fármacos
Caspase 3/metabolismo
Fragmentação do DNA
Formazans
Inibidores do Crescimento/farmacologia
Indicadores e Reagentes
Camundongos Endogâmicos C57BL
Antineoplásicos Fitogênicos/isolamento & purificação
Limites: Humanos
Animais
Feminino
Camundongos
Tipo de Publ: Research Support, Non-U.S. Gov't
Responsável: CL1.1 - Biblioteca Central


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Id: biblio-1040072
Autor: Li, Xue-Chao; Huang, Chuan-Xi; Wu, Shi-Kui; Yu, Lan; Zhou, Guang-Jian; Chen, Li-Jun.
Título: Biological roles of filamin a in prostate cancer cells
Fonte: Int. braz. j. urol;45(5):916-924, Sept.-Dec. 2019. graf.
Idioma: en.
Projeto: National Key Research and Development Program of China.
Resumo: ABSTRACT Objective This study aims to investigate the association of filamin A with the function and morphology of prostate cancer (PCa) cells, and explore the role of filamin A in the development of PCa, in order to analyze its significance in the evolvement of PCa. Materials and Methods A stably transfected cell line, in which filamin A expression was suppressed by RNA interference, was first established. Then, the effects of the suppression of filamin A gene expression on the biological characteristics of human PCa LNCaP cells were observed through cell morphology, in vitro cell growth curve, soft agar cloning assay, and scratch test. Results A cell line model with a low expression of filamin A was successfully constructed on the basis of LNCaP cells. The morphology of cells transfected with plasmid pSilencer-filamin A was the following: Cells were loosely arranged, had less connection with each other, had fewer tentacles, and presented a fibrous look. The growth rate of LNCap cells was faster than cells transfected with plasmid pSilencer-filamin A (P <0.05). The clones of LNCap cells in the soft agar cloning assay was significantly fewer than that of cells stably transfected with plasmid pSilencer-filamin A (P <0.05). Cells stably transfected with plasmid pSilencer-filamin A presented with a stronger healing and migration ability compared to LNCap cells (healing rate was 32.2% and 12.1%, respectively; P <0.05). Conclusion The expression of the filamin A gene inhibited the malignant development of LNCap cells. Therefore, the filamin A gene may be a tumor suppressor gene.
Descritores: Neoplasias da Próstata/patologia
Filaminas/análise
Filaminas/fisiologia
-Plasmídeos
Neoplasias da Próstata/genética
Sais de Tetrazólio
Fatores de Tempo
Cicatrização/fisiologia
Transfecção/métodos
Células Cultivadas
Western Blotting
Colorimetria/métodos
Linhagem Celular Tumoral
Proliferação de Células
Filaminas/genética
Formazans
Limites: Humanos
Masculino
Responsável: BR1.1 - BIREME


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Id: biblio-950783
Autor: NK, Asha Tukappa; Londonkar, Ramesh L; Nayaka, Hanumantappa B; CB, Sanjeev Kumar.
Título: Cytotoxicity and hepatoprotective attributes of methanolic extract of Rumex vesicarius L
Fonte: Biol. Res;48:1-9, 2015. ilus, graf, tab.
Idioma: en.
Resumo: BACKGROUND: To evaluate the hepatoprotective potential and invitro cytotoxicity studies of whole plant methanol extract of Rumex vesicarius L. Methanol extract at a dose of 100 mg/kg bw and 200 mg/kg bw were assessed for its hepatoprotective potential against CCl4-induced hepatotoxicity by monitoring activity levels of SGOT (Serum glutamic oxaloacetic transaminase), SGPT (Serum glutamic pyruvic transaminase), ALP (Alkaline phosphatase), TP (Total protein), TB (Total bilirubin) and SOD (Superoxide dismutase), CAT (Catalase), MDA (Malondialdehyde). The cytotoxicity of the same extract on HepG2 cell lines were also assessed using MTT assay method at the concentration of 62.5, 125, 250, 500 µg/ml. RESULTS: Pretreatment of animals with whole plant methanol extracts of Rumex vesicarius L. significantly reduced the liver damage and the symptoms of liver injury by restoration of architecture of liver. The biochemical parameters in serum also improved in treated groups compared to the control and standard (silymarin) groups. Histopathological investigation further corroborated these biochemical observations. The cytotoxicity results indicated that the plant extract which were inhibitory to the proliferation of HepG2 cell line with IC50 value of 563.33 ± 0.8 Mg/ml were not cytotoxic and appears to be safe. CONCLUSIONS: Rumex vesicarius L. whole plant methanol extract exhibit hepatoprotective activity. However the cytotoxicity in HepG2 is inexplicable and warrants further study.
Descritores: Extratos Vegetais/farmacologia
Citotoxinas/farmacologia
Rumex/química
Proliferação de Células/efeitos dos fármacos
Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico
Fitoterapia/métodos
-Aspartato Aminotransferases/metabolismo
Silimarina/farmacologia
Superóxido Dismutase/metabolismo
Sais de Tetrazólio
Bilirrubina/metabolismo
Tetracloreto de Carbono
Catalase/metabolismo
Anticarcinógenos/farmacologia
Ratos Wistar
Alanina Transaminase/metabolismo
Metanol
Ingestão de Líquidos/efeitos dos fármacos
Ingestão de Alimentos/efeitos dos fármacos
Fosfatase Alcalina/metabolismo
Células Hep G2
Doença Hepática Induzida por Substâncias e Drogas/metabolismo
Doença Hepática Induzida por Substâncias e Drogas/patologia
Formazans
Fígado/efeitos dos fármacos
Fígado/metabolismo
Malondialdeído/metabolismo
Antioxidantes/metabolismo
Antioxidantes/farmacologia
Limites: Humanos
Animais
Masculino
Ratos
Tipo de Publ: Research Support, Non-U.S. Gov't
Responsável: CL1.1 - Biblioteca Central


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Id: biblio-950822
Autor: Aftab, Usman; Zechel, David L; Sajid, Imran.
Título: Antitumor compounds from Streptomyces sp. KML-2, isolated from Khewra salt mines, Pakistan
Fonte: Biol. Res;48:1-10, 2015. ilus, graf, tab.
Idioma: en.
Resumo: BACKGROUND: Actinomycetes are gram positive bacteria with high G + C content in their DNA and are capable of producing variety of secondary metabolites. Many of these metabolites possess different biological activities and have the potential to be developed as therapeutic agents. The aim of the present study was to screen actinomycetes inhabiting halophilic environment such as Khewra salt mines present in Pakistan for cytotoxic and antitumor compounds. RESULTS: An actiomycetes strain designated as Streptomyces sp. KML-2 was isolated from a saline soil of Khewra salt mines, Pakistan. The strain Streptomyces sp. KML-2 showed 84 % cytotoxic activity against larvae of Artemiasalina. In the screening phase, the strain exhibited significant antitumor activity with IC50 values of 12, 48 and 56 µg/ml against Hela, MDBK and Vero cell lines, respectively. After that extract from 20 l fermentation was used to purify secondary metabolites by several chromatographic techniques. Structure elucidation of isolated compounds revealed that it is highly stable producer of Chromomycin SA (1) and 1-(1H-indol-3-yl)-propane-1,2,3-triol (2). Both of the isolated compounds showed significant antitumor activity against Hela and MCF-7 cancer cell lines (IC50 values 8.9 and 7.8 µg/ml against Hela; 12.6 and 0.97 µg/ml against MCF-7, respectively). The 16S rRNA gene sequence (1437 bp) of the strain confirm its identity (99 %) with Streptomyces griseus. CONCLUSIONS: From this research work we were successful in isolating two potent antitumor compounds, Chromomycin SA and 1-(1H-indol-3-yl)-propane-1,2,3-triol from Streptomyces KML-2 strain, isolated from Khewra salt mine. As such this is the second report which confirms that S. griseus can produce Chromomycin SA without introducing any mutagenesis in its biosynthesizing gene cluster and isolated indole derivative is being reported first time from any member of actinomycetes group with having novel antitumor activity against Hela and MCF-7 cells Nucleotide sequences: Nucleotide sequence data reported are available in the GenBank database under the accession number: GenBank KJ009562.
Descritores: Microbiologia do Solo
Streptomyces/química
Antineoplásicos/farmacologia
-Paquistão
Filogenia
Artemia/classificação
Artemia/efeitos dos fármacos
Sais
Solo/química
Streptomyces/isolamento & purificação
Streptomyces/ultraestrutura
Streptomyces griseus/classificação
Sais de Tetrazólio
Células Vero
RNA Ribossômico 16S/genética
Cromomicinas/classificação
Cromomicinas/farmacologia
Células HeLa
Microscopia Eletrônica de Varredura
Linhagem Celular
Chlorocebus aethiops
Cromatografia/métodos
Análise de Sequência de RNA
Concentração Inibidora 50
Células MCF-7
Formazans
Glicerol/análogos & derivados
Glicerol/farmacologia
Larva/efeitos dos fármacos
Mineração
Antibacterianos/isolamento & purificação
Antibacterianos/farmacologia
Antineoplásicos/isolamento & purificação
Limites: Humanos
Animais
Bovinos
Tipo de Publ: Research Support, Non-U.S. Gov't
Responsável: CL1.1 - Biblioteca Central


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Id: biblio-950834
Autor: Orozco-Ibarra, Marisol; Muñoz-Sánchez, Jorge; Zavala-Medina, Martín E; Pineda, Benjamín; Magaña-Maldonado, Roxana; Vázquez-Contreras, Edgar; Maldonado, Perla D; Pedraza-Chaverri, José; Chánez-Cárdenas, María Elena.
Título: Aged garlic extract and S-allylcysteine prevent apoptotic cell death in a chemical hypoxia model
Fonte: Biol. Res;49:1-10, 2016. ilus, graf.
Idioma: en.
Projeto: PAPIIT; . CONACYT; . Armstrong Foundation in México; . National Council of Science and Technology; . National Autonomous University of Mexico.
Resumo: BACKGROUND: Aged garlic extract (AGE) and its main constituent S-allylcysteine (SAC) are natural antioxidants with protective effects against cerebral ischemia or cancer, events that involve hypoxia stress. Cobalt chloride (CoCl2) has been used to mimic hypoxic conditions through the stabilization of the α subunit of hypoxia inducible factor (HIF-1α) and up-regulation of HIF-1α-dependent genes as well as activation of hypoxic conditions such as reactive oxygen species (ROS) generation, loss of mitochondrial membrane potential and apoptosis. The present study was designed to assess the effect of AGE and SAC on the CoCl2-chemical hypoxia model in PC12 cells. RESULTS: We found that CoCl2 induced the stabilization of HIF-1α and its nuclear localization. CoCl2 produced ROS and apoptotic cell death that depended on hypoxia extent. The treatment with AGE and SAC decreased ROS and protected against CoCl2-induced apoptotic cell death which depended on the CoCl2 concentration and incubation time. SAC or AGE decreased the number of cells in the early and late stages of apoptosis. Interestingly, this protective effect was associated with attenuation in HIF-1α stabilization, activity not previously reported for AGE and SAC. CONCLUSIONS: Obtained results show that AGE and SAC decreased apoptotic CoCl2-induced cell death. This protection occurs by affecting the activity of HIF-1α and supports the use of these natural compounds as a therapeutic alternative for hypoxic conditions
Descritores: Extratos Vegetais/farmacologia
Apoptose/efeitos dos fármacos
Cisteína/análogos & derivados
Fatores de Transcrição Hélice-Alça-Hélice Básicos/efeitos dos fármacos
Alho/química
Antioxidantes/farmacologia
-Sais de Tetrazólio
Hipóxia Celular/efeitos dos fármacos
Sobrevivência Celular/efeitos dos fármacos
Células Cultivadas
Análise de Variância
Células PC12
Espécies Reativas de Oxigênio/análise
Cobalto
Cisteína/farmacologia
Citometria de Fluxo
Formazans
Limites: Animais
Ratos
Tipo de Publ: Research Support, Non-U.S. Gov't
Responsável: CL1.1 - Biblioteca Central


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Id: biblio-950848
Autor: Li, JinLong; Li, Hui; Zhang, ZhaoLi; Wang, NianYue; Zhang, Yong Chen.
Título: The anti-cancerous activity of recombinant trichosanthin on prostate cancer cell PC3
Fonte: Biol. Res;49:1-6, 2016. ilus, graf, tab.
Idioma: en.
Projeto: Bureau of Health.
Resumo: CONTEXT: Trichosanthin produced in the root tube of Trichosanthes kirilowii shows anti-tumor activity on a series of cancer cells including Hela, MCF-7, HL-60. But there is little information about its effect on the carcinogenesis of prostate cancer. OBJECTIVE: This work was designed to study the role of trichosanthin on prostate cancer cells PC3. MATERIALS AND METHODS: Trichosanthin was expressed in BL21 strain and purified by affinity chromatography. MTT assay was designed to determine the effect of trichosanthin on growth of PC3 cells at doses of 10, 20, 40, 60, 80, and 120 µg/ml.Then the effect of 50 µg/ml rTCS alone or combined with 2 µM IL-2 on PC3 cell proliferation was analyzed. And the mechanism of rTCS was studied by western blot. After that the in vivo effect of rTCS combined with IL-2 was explored in mice bearing PC3 xenograft tumor. RESULTS: Trichosanthin was successfully expressed in BL21 and purified by 100 mM imidazole. It was shown to inhibit proliferation of PC3 cells in a dose-dependent manner with IC50 50.6 µg/ml. When combined with cytokine IL-2, a significant synergic effect was obtained. The inhibition rate on PC3 was around 50 % in combination group while only 35.5 % in single rTCS group at 50 µg/ml. Further, the expression of full length caspase-8 and Bcl-2 decreased significantly while cleaved caspase-8 and Bax were up-regulated, which suggest that caspase-8-mediated apoptosis pathway may be activated by rTCS in PC3 cells. Moreover, our data demonstrated that tumor volume and tumor weight were significantly reduced in rTCS-treated or rTCS/IL-2-treated nude mice bearing PC3 xenograft tumor compared with control. And significant difference was also found between rTCS and rTCS/IL-2 group. CONCLUSIONS: This study demonstrates that rTCS is a potential agent with high in vitro and in vivo anti-tumor activity on PC3 cells. And rTCS combined with IL-2 is a promising strategy in treating patients with prostate cancer in future.
Descritores: Neoplasias da Próstata/tratamento farmacológico
Tricosantina/farmacologia
Antineoplásicos Fitogênicos/farmacologia
-Neoplasias da Próstata/patologia
Sais de Tetrazólio
Fatores de Tempo
Proteínas Recombinantes/farmacologia
Western Blotting
Reprodutibilidade dos Testes
Apoptose/efeitos dos fármacos
Linhagem Celular Tumoral
Carga Tumoral
Proliferação de Células/efeitos dos fármacos
Formazans
Limites: Animais
Masculino
Feminino
Camundongos
Tipo de Publ: Research Support, Non-U.S. Gov't
Estudo de Avaliação
Responsável: CL1.1 - Biblioteca Central


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Id: biblio-950875
Autor: Cheng, Ming-Jun; Cao, Yun-Gui.
Título: TMPYP4 exerted antitumor effects in human cervical cancer cells through activation of p38 mitogen-activated protein kinase
Fonte: Biol. Res;50:24, 2017. graf.
Idioma: en.
Projeto: National Natural Science Foundation of China; . Chongqing Science and Technology.
Resumo: BACKGROUND: The aim of the present study was to investigate the potential effects of the 5,10,15,20-tetrakis (1-methylpyridinium-4-yl) porphyrin (TMPyP4) on the proliferation and apoptosis of human cervical cancer cells and the underlying mechanisms by which TMPyP4 exerted its actions. RESULTS: After human cervical cancer cells were treated with different doses of TMPyP4, cell viability was determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) method, the apoptosis was observed by flow cytometry (FCM), and the expression of p38 mitogen-activated protein kinase (MAPK), phosphated p38 MAPK (p-p38 MAPK), capase-3, MAPKAPK2 (MK-2) and poly ADP-ribose polymerase (PARP) was measured by Western blot analysis. The analysis revealed that TMPyP4 potently suppressed cell viability and induced the apoptosis of human cervical cancer cells in a dose-dependent manner. In addition, the up-regulation of p-p38 MAPK expression levels was detected in TMPyP4-treated human cervical cancer cells. However, followed by the block of p38 MAPK signaling pathway using the inhibitor SB203580, the effects of TMPyP4 on proliferation and apoptosis of human cervical cancer cells were significantly changed. CONCLUSIONS: It was indicated that TMPyP4-inhibited proliferation and -induced apoptosis in human cervical cancer cells was accompanied by activating the p38 MAPK signaling pathway. Taken together, our study demonstrates that TMPyP4 may represent a potential therapeutic method for the treatment of cervical carcinoma.
Descritores: Porfirinas/farmacologia
Neoplasias do Colo do Útero/tratamento farmacológico
Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
Antineoplásicos/farmacologia
-Sais de Tetrazólio
Células HeLa/efeitos dos fármacos
Sobrevivência Celular/efeitos dos fármacos
Células Cultivadas
Western Blotting
Reprodutibilidade dos Testes
Apoptose/efeitos dos fármacos
Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores
Proliferação de Células/efeitos dos fármacos
Ativação Enzimática/efeitos dos fármacos
Caspase 3/análise
Formazans
Limites: Humanos
Feminino
Tipo de Publ: Estudo de Avaliação
Responsável: CL1.1 - Biblioteca Central


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Id: lil-787334
Autor: Covre, Joyce L; Cristovam, Priscila C; Loureiro, Renata R; Hazarbassanov, Rossen M; Campos, Mauro; Sato, Élcio H; Gomes, José Álvaro P.
Título: The effects of riboflavin and ultraviolet light on keratocytes cultured in vitro / Avaliação do efeito da riboflavina e luz ultravioleta sobre os ceratócitos in vitro
Fonte: Arq. bras. oftalmol;79(3):180-185graf.
Idioma: en.
Resumo: ABSTRACT Purpose: To culture quiescent human keratocytes and evaluate the effects of ultraviolet light and riboflavin on human corneal keratocytes in vitro. Methods: Keratocytes were obtained from remaining corneoscleral ring donor corneas previously used in corneal transplant surgeries and cultured in DMEM/F12 with 2% FBS until confluence. Characterization of cultured cells was performed by immunofluorescence analysis for anti-cytokeratin-3, anti-Thy-1, anti-α-smooth muscle actin, and anti-lumican. Immunofluorescence was performed before and after treatment of cultured cells with either ultraviolet light or riboflavin. Corneal stromal cells were covered with collagen (200 µL or 500 µL) and 0.1% riboflavin, and then exposed to ultraviolet light at 370 nm for 30 minutes. After 24 hours, cytotoxicity was determined using MTT colorimetric assays, whereas cell viability was assessed using Hoechst 33342 and propidium iodide. Results: Cell cultures achieved confluence in approximately 20 days. Expression of the lumican was high, whereas no expression of CK3, Thy-1, and α-SMA was observed. After crosslinking, MTT colorimetric assays demonstrated a low toxicity rate, whereas Hoechst 33342/propidium iodide staining demonstrated a low rate of apoptosis and necrosis, respectively, in all collagen-treatment groups. Conclusion: Keratocytes can be successfully cultured in vitro and characterized by immunofluorescence using lumican. MTT colorimetric assays, and Hoechst 33342, and propidium iodide staining demonstrated a higher rate of cell death in cells cultured without collagen, indicating collagen protects keratocytes from the cytotoxic effects of ultraviolet light.

RESUMO Objetivo: Avaliar o efeito da aplicação da luz ultravioleta e riboflavina sobre ceratócitos da córnea humana in vitro. Métodos: Os ceratócitos foram obtidos a partir das rimas corneoesclerais remanescentes da trepanação de córneas previamente utilizadas em cirurgias de transplante de córnea e cultivadas em meio DMEM/F12 com 2% de FBS até atingir confluência. As culturas de células foram caracterizadas por imunofluorescência com os anticorpos K3 (marcador de células epiteliais), Thy-1 (marcador de fibroblasto) SMA (marcador de miofibroblasto) e Lumican (marcador de ceratócitos). Imunofluorescência também foi feita após o tratamento. As células do estroma da córnea foram cobertas com colágeno (200 µL e 500 µL) e 0,1% de riboflavina e exposta a luz UVA a 370 nm por 30 minutos. Após 24 horas, citotoxicidade foi determinada por ensaio de MTT e a viabilidade celular foi feita por Hoechst 33342/Iodeto de propideo. Resultados: As culturas de células atingiram confluência em aproximadamente 20 dias. Imunofluorescência apontou alta expressão para o marcador de ceratócitos (Lumican) e expressão negativa par os marcadores de células epiteliais (K3), fibroblasto (Thy-1) e miofibroblasto (α-SMA). Após o cross linking a análise de MTT mostrou baixa taxa de toxicidade e com a coloração de Hoechst 33342/Iodeto de propideo baixa taxa de apoptose e necrose respectivamente em todos os grupos que continham colágeno. Conclusão: As culturas de ceratócitos foram obtidas e caracterizadas por imunofluorescência através do marcador Lumican com sucesso. O ensaio de MTT e a coloração por Hoechst 33342 e iodeto de propídio, apresentaram maior índice de morte celular nos grupos que não continham colágeno, provando que protege as células contra os efeitos da luz UVA.
Descritores: Riboflavina/farmacologia
Raios Ultravioleta
Fármacos Fotossensibilizantes/farmacologia
Ceratócitos da Córnea/efeitos dos fármacos
Ceratócitos da Córnea/efeitos da radiação
-Sobrevivência Celular/efeitos dos fármacos
Sobrevivência Celular/efeitos da radiação
Células Cultivadas
Análise de Variância
Imunofluorescência
Colágeno/farmacologia
Apoptose/efeitos dos fármacos
Apoptose/efeitos da radiação
Substância Própria/citologia
Reagentes para Ligações Cruzadas/farmacologia
Fibroblastos/efeitos dos fármacos
Fibroblastos/efeitos da radiação
Formazans
Necrose
Limites: Humanos
Tipo de Publ: Estudo de Avaliação
Responsável: BR1.1 - BIREME


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Id: biblio-1089334
Autor: Gutiérrez-Barbosa, Hernando; Castañeda, Nadia Y; Castellanos, Jaime E.
Título: Differential replicative fitness of the four dengue virus serotypes circulating in Colombia in human liver Huh7 cells
Fonte: Braz. j. infect. dis;24(1):13-24, Feb. 2020. tab, graf.
Idioma: en.
Resumo: ABSTRACT Dengue has been a significant public health problem in Colombia since the simultaneous circulation of the four dengue virus serotypes. The replicative fitness of dengue is a biological feature important for virus evolution and contributes to elucidating the behavior of virus populations and viral pathogenesis. However, it has not yet been studied in Colombian isolates. This study aimed to compare the replicative fitness of the four dengue virus serotypes and understand the association between the serotypes, their in vitro infection ability, and their replication in target cells. We used three isolates of each DENV serotype to infect Huh-7 cells at an MOI of 0.5. The percentage of infected cells was evaluated by flow cytometry, cell viability was evaluated by MTT assay, and the pathogenicity index was calculated as a ratio of both parameters. The replicative fitness was measured by the number of viral genome copies produced using quantitative PCR and the production of infectious viral progeny was measured by plaque assay. We showed that Huh-7 cells were susceptible to infection with all the different strain isolates. Nevertheless, the biological characteristics, such as infectious ability and cell viability, were strain-dependent. We also found different degrees of pathogenicity between strains of the four serotypes, representative of the heterogeneity displayed in the circulating population. When we analyzed the replicative fitness using the mean values obtained from RT-qPCR and plaque assay for the different strains, we found serotype-dependent behavior. The highest mean values of replicative fitness were obtained for DENV-1 (log 4.9 PFU/ml) and DENV-4 (log 5.28 PFU/ml), followed by DENV-2 (log 3.9 PFU/ml) and DENV-3 (log 4.31 PFU/ml). The internal heterogeneity of the replicative fitness within each serotype could explain the simultaneous circulation of the four DENV serotypes in Colombia.
Descritores: Replicação Viral/genética
Vírus da Dengue/genética
Vírus da Dengue/patogenicidade
Sorogrupo
-Ensaio de Placa Viral
Valores de Referência
Sais de Tetrazólio
Fatores de Tempo
RNA Viral/genética
Linhagem Celular
Sobrevivência Celular
Células Cultivadas
Colômbia
Reação em Cadeia da Polimerase Via Transcriptase Reversa
Citometria de Fluxo
Formazans
Fígado/citologia
Limites: Humanos
Responsável: BR1.1 - BIREME



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