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Acta cir. bras ; 34(2): e201900210, 2019. tab, graf
Artigo em Inglês | LILACS | ID: biblio-989058


Abstract Purpose: To analyze aspects of the biomodulating effect of light in biological tissues, bone cells from surgical explants of the femur of rats were irradiated with low intensity laser. Methods: Bone cells were cultured and irradiated with LASER light (GaAlAs). Growth, cell viability, mineralized matrix formation, total protein dosage, immunostimulatory properties, cytochemical analysis, gene expression of bone proteins were examined using live cell imaging and cell counting by colorimetric assay. The gene expression of: alkaline phosphatase (ALP), type 1 collagen, osteocalcin and osteopontin through the real-time polymerase chain reaction. Results: At 8 days, the viability of the irradiated culture was 82.3% and 72.4% in non-irradiated cells. At 18 days, the cellular viability (with laser) was 77.42% and 47.62% without laser. At 8 days, the total protein concentration was 21.622 mg / mol in the irradiated group and 16, 604 mg / mol in the non-irradiated group and at 18 days the concentration was 37.25 mg / mol in the irradiated group and 24, 95 mg / mol in the non-irradiated group. Conclusion: The laser interfered in the histochemical reaction, cell viability, matrix mineralization, and maintained the cellular expression of proteins

Animais , Ratos , Osteoblastos/efeitos da radiação , Diferenciação Celular/efeitos da radiação , Sobrevivência Celular/efeitos da radiação , Terapia com Luz de Baixa Intensidade/métodos , Fatores de Tempo , Células Cultivadas , Ratos Wistar , Relação Dose-Resposta à Radiação
Rev. Asoc. Méd. Argent ; 131(2): 7-26, jun. 2018. tab, ilus
Artigo em Espanhol | LILACS | ID: biblio-973098


Se revisan los nuevos conocimientos sobre la matriz extracelular (MEC), que han permitido descubrir su importante rol en la cicatrización de las heridas cutáneas. Se describen sus características morfofisiológicas y cómo interviene en la curación de las heridas cutáneas. Se presentan cuatro casos clínicos en los que se aplicó este enfoque terapéutico: los sustitutos de piel y la “cura húmeda”.

We review the new knowledge about the extracellular matrix (ECM) that has allowed us to discover its important role in the healing of cutaneous wounds. The morphophysiological characteristics of ECM and its role in the healing of cutaneous wounds are described. Four clinical cases are presented where this therapeutic approach was applied: the skin substitutes and the “moist wound healing”.

Masculino , Feminino , Humanos , Adolescente , Pessoa de Meia-Idade , Idoso , Matriz Extracelular/metabolismo , Matriz Extracelular/genética , Pele/lesões , Cicatrização/fisiologia , Diferenciação Celular , Radiação Eletromagnética , Medicina Regenerativa
An. bras. dermatol ; 93(1): 63-66, Jan.-Feb. 2018. tab, graf
Artigo em Inglês | LILACS | ID: biblio-887148


Abstract: Background: Psoriasis is a chronic inflammatory disorder, characterized by increased keratinocyte proliferation due to abnormal differentiation of basal keratinocytes. The etiology of the disease is unclear, and according to the survey results, it is hypothesized that a combination of genetic and environmental factors prompts an abnormal immune response in patients with psoriasis. CD4+ Th cells play a multifaceted role in both immune defense and pathogenesis of certain diseases such as psoriasis. Nonetheless, the exact contribution of different subpopulations of Th cells in psoriasis is still not clear. Objective: The aim of present study was to determine the mRNA expression level of RORC as potential inducer of Th17 cell differentiation and expression pattern of Th17-signature cytokines (IL-17A and IL-22). Methods: Twenty patients with psoriasis and twenty-one healthy subjects were included in the study. Peripheral blood mononuclear cells (PBMCs) were separated and expression of three genes were determined by quantitative real-time reverse transcriptase PCR (qRT-PCR). Plasma levels of IL-17 and IL-22 were also evaluated by ELISA. Results: RORC, IL-17A and IL-22 gene expression was significantly higher in patients with psoriasis compared with healthy controls (P<0.05). In addition, a marked increase in plasma IL-17A and IL-22 levels was observed in patient group compared to controls (P<0.001). Study limitations: small number of patients. Conclusion: These data suggest that Th17 response may contribute to the pathogenesis of psoriasis.

Humanos , Masculino , Feminino , Adolescente , Adulto , Pessoa de Meia-Idade , Idoso , Adulto Jovem , Psoríase/metabolismo , Queratinócitos/fisiologia , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/fisiologia , Células Th17/metabolismo , Psoríase/etiologia , Índice de Gravidade de Doença , RNA Mensageiro/metabolismo , Estudos de Casos e Controles , Expressão Gênica , Queratinócitos/citologia , Diferenciação Celular , Interleucinas/sangue , Interleucina-17/sangue , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Células Th17/imunologia
Mem. Inst. Oswaldo Cruz ; 113(6): e180102, 2018. tab, graf
Artigo em Inglês | LILACS | ID: biblio-955111


BACKGROUND Scedosporium/Lomentospora species are opportunistic mould pathogens, presenting notable antifungal resistance. OBJECTIVES/METHODS We analysed the conidia and germinated conidia of S. apiospermum (Sap), S. aurantiacum (Sau), S. minutisporum (Smi) and L. prolificans (Lpr) by scanning electron microscopy and exposition of surface molecules by fluorescence microscopy. FINDINGS Conidia of Sap, Smi and Sau had oval, ellipsoidal and cylindrical shape, respectively, with several irregularities surrounding all surface areas, whereas Lpr conidia were rounded with a smooth surface. The germination of Sap occurred at the conidial bottom, while Smi and Sau germination primarily occurred at the centre of the conidial cell, and Lpr germination initiated at any part of the conidial surface. The staining of N-acetylglucosamine-containing molecules by fluorescein-labelled WGA primarily occurred during the germination of all studied fungi and in the conidial scars, which is the primary location of germination. Calcofluor white, which recognises the polysaccharide chitin, strongly stained the conidial cells and, to a lesser extent, the germination. Both mannose-rich glycoconjugates (evidenced by fluoresceinated-ConA) and cell wall externally located polypeptides presented distinct surface locations and expression according to both morphotypes and fungal species. In contrast, sialic acid and galactose-containing structures were not detected at fungal surfaces. MAIN CONCLUSIONS The present study demonstrated the differential production/exposition of surface molecules on distinct morphotypes of Scedosporium/Lomentospora species.

Humanos , Esporos Fúngicos/fisiologia , Membrana Celular/ultraestrutura , Scedosporium/crescimento & desenvolvimento , Microscopia Eletrônica de Varredura , Diferenciação Celular , Microscopia de Fluorescência
Braz. j. med. biol. res ; 51(5): e7183, 2018. graf
Artigo em Inglês | LILACS | ID: biblio-889088


Human pluripotent stem cells (hPSCs)/OP9 coculture system is a widely used hematopoietic differentiation approach. The limited understanding of this process leads to its low efficiency. Thus, we used single-cell qPCR to reveal the gene expression profiles of individual CD34+ cells from different stages of differentiation. According to the dynamic gene expression of hematopoietic transcription factors, we overexpressed specific hematopoietic transcription factors (Gata2, Lmo2, Etv2, ERG, and SCL) at an early stage of hematopoietic differentiation. After overexpression, we generated more CD34+ cells with normal expression level of CD43 and CD31, which are used to define various hematopoietic progenitors. Furthermore, these CD34+ cells possessed normal differentiation potency in colony-forming unit assays and normal gene expression profiles. In this study, we demonstrated that single-cell qPCR can provide guidance for optimization of hematopoietic differentiation and transient overexpression of selected hematopoietic transcription factors can enhance hematopoietic differentiation.

Humanos , Células-Tronco Hematopoéticas/citologia , Diferenciação Celular , Técnicas de Cocultura/métodos , Células-Tronco Pluripotentes/citologia , Fenótipo , Expressão Gênica , Reação em Cadeia da Polimerase , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Célula Única/métodos , Citometria de Fluxo
Repert. med. cir ; 27(3): 145-154, 2018.
Artigo em Inglês, Espanhol | LILACS, COLNAL | ID: biblio-981916


Desde su descripción inicial, hace ya más de 40 años, las células madre mesenquimales (MSC) fueron reconocidas como una importante alternativa para el manejo de enfermedades caracterizadas por la pérdida aguda o crónica de tejido, gracias a su capacidad de proliferación y diferenciación, lo cual les permitiría sustituir las células perdidas y de esta forma recuperar la estructura y función. Cada vez es más abundante la evidencia que sugiere el potencial de estas células para el manejo de un amplio grupo de enfermedades, al menos en modelos experimentales preclínicos. No obstante, esta capacidad no ha podido refrendarse contundente y consistentemente en ensayos clínicos. Con la presente revisión, se pretende presentar una visión del estado actual del desarrollo conceptual en torno a las capacidades terapéuticas de las MSC y un análisis crítico de algunos de los factores, que han impedido que estas sean una opción terapéutica usable en la práctica clínica diaria

Since they were initially identified more than 40 years ago, mesenchymal stem cells (MSCs) were recognized as an important therapeutic alternative for diseases characterized by acute or chronic loss of tissue, thanks to their proliferation and differentiation ability, which would allow the replacement of lost cells and their structural and functional recuperation. Evidence suggesting the therapeutic potential of these cells for a vast group of diseases increases day by day, at least in preclinical experimental models. However, clinical trials have been unable to obtain consistent and categorical results to demonstrate this capacity. This review aims to provide, an overview on the current status of the conceptual development on the therapeutic properties of MSCs, and a critical analysis of some factors which have hindered the application of this therapeutic option in daily clinical practice.

Humanos , Células-Tronco , Diferenciação Celular , Células-Tronco Adultas
Univ. med ; 59(3)2018. ilus
Artigo em Espanhol | LILACS, COLNAL | ID: biblio-995004


El desarrollo neurológico humano requiere una serie de pasos que permitan orientar, regular y diferenciar los diversos componentes cerebrales, para así garantizar, de una manera bastante precisa, la correcta organización y funcionamiento de las estructuras neuronales. La neurogénesis está clásicamente dividida en cuatro etapas consecutivas: proliferación, migración, diferenciación y maduración. En los humanos, estas ocurren desde la tercera semana de gestación hasta la vida adulta y precisan de un complejo grupo de paquetes genéticos, así como de algunos factores asociados, que se han ido descubriendo gracias a los avances en la biología molecular. El artículo es una revisión acerca del desarrollo neuroembriológico humano y los componentes genéticos más relevantes encontrados en la literatura.

The human neuronal development requires a number of concrete steps which lead to orientation, regulation and differentiation of several brain components. They must be done to guarantee, in a very precise way, the correct organization and functioning of the neuronal structures. Neurogenesis is commonly divided into four consecutive stages: proliferation, migration, differentiation and maturation. In humans, those stages take place since the third week of prenatal Iife until the adult Iife. They also require a complex group of genetic packs and associated molecular factors, most of which have been recen tly discovered by the molecular biology technology. A review was made about the human neuronal and embryological development and the most relevant genetic components described by the literature so far.

Movimento Celular/genética , Neurogênese/genética , Desenvolvimento Embrionário e Fetal , Diferenciação Celular/genética
São Paulo; s.n; 20180000. 87 p.
Tese em Português | LILACS, BBO - Odontologia | ID: biblio-970272


O carcinoma epidermoide é a neoplasia maligna mais comum em boca e está entre as principais causas de morbidade e mortalidade em todo o mundo, devido a seu comportamento agressivo, evoluindo à metástase loco regional e a distância. O microambiente tumoral contém numerosos tipos celulares e muita atenção tem sido dada na literatura científica sobre a participação das células inflamatórias no desenvolvimento e progressão do câncer, pois as células neoplásicas são capazes de subverter a resposta imune. Os linfócitos T são o componente central na imunidade antitumoral, através da produção de citocinas por células e morte celular. Pouco se sabe sobre como substratos derivados de células neoplásicas influenciam as células no microambiente tumoral. Assim, o presente estudo propôs analisar a influência do meio condicionado derivado de células de carcinoma epidermoide de língua (SCC4 e MC SCC9) sobre linfoblastos (CEM) e células mononucleares do sangue periférico (PBMC-A e PBMC-B) para compreender melhor seu papel na imunidade anti-tumoral, imunoedição e evasão imune. Após estimulação com meio condicionado, os linfoblastos e as PBMCs foram submetidas ao ensaio de viabilidade celular, de citometria de fluxo e RT-qPCR para analisar a expressão de genes de apoptose e citocinas. O meio condicionado também foi coletado e avaliado por ELISA para verificar as citocinas secretadas pelas SCCs, bem como pela CEM e PBMC. Ambos meios condicionados foram capazes de reduzir a viabilidade da CEM e das PBMCs. A expressão de BCL2 e BAK não foi afetada na CEM, enquanto que MC SCC4 aumentou a expressão de BAK na PBMC-B. Os MCs das SCCs apresentaram expressão reduzida de IL-1?, IL-10 e INF-?. A IL-6 e IL-8 são expressas em níveis um pouco maiores pela SCC4 e superexpressas pela SCC9. A linhagem CEM não apresentou expressão de RNAm de IL-6, enquanto que a PBMC-B apresentou redução da expressão de IL-6 quando cultivada com ambos meios, sendo significativa com o meio MC SCC9. A expressão de RNAm de IL-8 reduziu na CEM e aumentou na PBMC-B com ambos os meios. A diferenciação para células CD4+ aumentou com ambos os MCs nas duas linhagens, reduzindo células CD34+. O MC SCC4 não alterou o número de linfócitos T CD4+/FOXP3+ da CEM e PBMC-B. O MC SCC9 induziu aumento da população CD4+/CD8+ na PBMC-B e amos os MCs induziram aumento da população CD8+/FOXP3+ da PBMC-B. Os resultados sugerem que os produtos derivados de carcinoma epidermoide de língua podem variar nas linhagens celulares, reduzindo a viabilidade, alterando a expressão de citocinas e aumentando as células CD4+ nas duas linhagens e aumentando o perfil CD8+/FOXP3+ e CD4+/CD8+ nas PBMCs.

Carcinoma de Células Escamosas , Diferenciação Celular , Citocinas
Int. j. morphol ; 35(4): 1597-1606, Dec. 2017. tab, graf
Artigo em Espanhol | LILACS | ID: biblio-893174


RESUMEN: Las células madre de la línea germinal masculina son factores clave para la espermatogénesis masculina y la fertilidad. Las células sustentaculares (células de Sertoli) como células somáticas juegan un papel fundamental en la creación de un microambiente esencial para la auto-renovación y diferenciación de las células de la línea germinal masculina. Las células madre mesenquimales son reconocidas como células auto-renovables y multipotentes capaces de diferenciarse en múltiples tipos de células. La generación de células germinales masculinas a partir de células madre mesenquimales puede proporcionar un método terapéutico para tratar la infertilidad masculina. En este estudio, las células mesenquimales derivadas de la médula ósea (BMMSCs) se recuperaron de la médula ósea de ratones de 6-8 semanas de edad del Instituto de Investigación Médico Naval (NMRI). En el estudio se aislaron las células sustentaculares y se enrriquecieron usando placas revestidas con lectina. Se obtuvo el medio de condición celular después de diferentes intervalos de tiempo. Posteriormente se cultivaron las BMMSC con diferentes concentraciones de SCCM y medio de Eagle modificado por Dulbecco (DMEM) en diversos momentos. Se evaluaron marcadores específicos de células de línea germinal usando la reacción en cadena de polimerasa transcriptasa inversa (RT-PCR) e inmunocitoquímica. Los resultados mostraron que las BMMSCs cultivadas con SCCM durante 48h exhibieron transcritos específicos de línea germinal (Mvh, Iid4, piwil2) (p <0,05) y marcadores (Mvh, Scp3). Nuestros resultados indican que el cultivo de BMMSCs con SCCM puede conducir a la diferenciación efectiva de BMMSCs en células germinales y proporcionar una estrategia de tratamiento para la infertilidad masculina.

SUMMARY: Male germ line stem cells are key factors for male spermatogenesis and fertility. Sustentacular cells (Sertoli cells) as somatic cells play a pivotal role in creating essential microenvironment for the self-renewal and differentiation of the male germ line cells. Mesenchymal stem cells are recognized as self-renewing and multipotent cells able to differentiate into multiple cell types. The generation of male germ cells from mesenchymal stem cells may provide a therapeutic method to treat male infertility. In this study, Bone marrow derived mesenchymal cells (BMMSCs) were retrieved from the bone marrow of 6-8-week old Naval Medical Research Institute (NMRI) mice. Sustentacular cells (Sertoli cells) were isolated and made rich using lectin coated plates. Sustentacular cell condition medium (SCCM) was collected after different time intervals. Then the BMMSCs were cultured with different concentration of SCCM and Dulbecco's Modified Eagle's medium (DMEM) at various times. Specific markers of Germ line cells were evaluated by using Reverse transcriptase polymerase chain reaction (RT-PCR) and immunocytochemistry. The results showed that BMMSCs cultured with SCCM for 48h exhibited germ line specific transcripts (Mvh, Iid4, piwil2) (p< 0.05) and markers (Mvh, Scp3). Our findings represent that culturing BMMSCs with SCCM may lead to effective differentiation of BMMSCs into germline cells and provide a treatment strategy for male infertility.

Animais , Masculino , Camundongos , Células de Sertoli/citologia , Células-Tronco Mesenquimais/citologia , Células de Sertoli/ultraestrutura , Testículo/citologia , Medula Óssea , Imuno-Histoquímica , Diferenciação Celular , Meios de Cultivo Condicionados , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Citometria de Fluxo
J. appl. oral sci ; 25(6): 631-640, Nov.-Dec. 2017. tab, graf
Artigo em Inglês | LILACS | ID: biblio-893662


Abstract Objectives: The primary purpose of this study was to examine the effects of triethylene glycol dimethacrylate (TEGDMA) on odontoclastic differentiation in the dental pulp tissue. Material and Methods: The effects of different TEGDMA dosages on the odontoclastic differentiation capability of dental pulp cells were analyzed in vitro using the following methodologies: i) flow cytometry and tartrate-resistant acid phosphatase (TRAP) staining; ii) apoptotic effects using Annexin V staining; iii) mRNA expression of osteoprotegerin (OPG) and receptor activator of nuclear factor (NF)-kB ligand (RANKL) genes by quantitative Real-time PCR (qRT-PCR); and iv) OPG and RANKL protein expression by enzyme-linked immunosorbent assay (ELISA). Results: TEGDMA caused relatively less odontoclastic differentiation in comparison with the control group; however, odontoclastic differentiation augmented with increasing doses of TEGDMA (p<0.05). The mRNA and protein expression of OPG was lower in TEGDMA treated pulp cells than in the control group (p<0.05). While the mRNA expression of RANKL remained unchanged compared to the control group (p>0.05), its protein expression was higher than the control group (p<0.05). In addition, TEGDMA increased the apoptosis of dental pulp cells dose dependently. Conclusions: TEGDMA reduced the odontoclastic differentiation ability of human dental pulp cells. However, odontoclastic differentiation ratios increased proportionally with the increasing dose of TEGDMA.

Humanos , Polietilenoglicóis/farmacologia , Ácidos Polimetacrílicos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Polpa Dentária/efeitos dos fármacos , Fosfatase Ácida Resistente a Tartarato/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Receptores de Lipopolissacarídeos/metabolismo , Polpa Dentária/citologia , Ligante RANK/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Citometria de Fluxo
J. appl. oral sci ; 25(6): 708-715, Nov.-Dec. 2017. graf
Artigo em Inglês | LILACS | ID: biblio-893672


Abstract Research on cancer stem cells (CSCs) has greatly increased in the field of medicine and pathology; however, some conceptual misunderstandings are still present among the public as well as within the general scientific community that is not yet familiar with the subject. The very first problem is the misinterpretation of CSCs as a synonym of their normal counterparts, the well-known stem cells (SCs). Particularly in Dentistry, another common mistake is the misinterpretation of oral CSCs as normal tooth-derived SCs. The present review aims to clarify important concepts related to normal SCs and CSCs, as well as discuss the relevance of CSCs to the development, metastasis and therapy resistance of oral squamous cell carcinoma.

Humanos , Células-Tronco Neoplásicas/patologia , Neoplasias Bucais/patologia , Carcinoma de Células Escamosas/patologia , Diferenciação Celular , Transformação Celular Neoplásica , Transição Epitelial-Mesenquimal
Acta neurol. colomb ; 33(4): 299-306, oct.-dic. 2017. tab, graf
Artigo em Espanhol | LILACS | ID: biblio-886462


RESUMEN INTRODUCCIÓN: Las células madre mesenquimales son células con la capacidad de autorrenovarse y diferenciarse a linajes mesenquimales e inclusive a células de origen no mesenquimal, como las del tejido nervioso. Teniendo en cuenta la idoneidad de estas células para diferenciarse en neuronas, han sido utilizadas con propósitos terapéuticos debido a que son capaces de restaurar neuronas que se deterioran en diversas enfermedades neurodegenerativas. OBJETIVO: Informar y comparar los métodos más utilizados para inducir a las células madre mesenquimales a diferenciarse en neuronas, además de mencionar las ventajas y desventajas de cada una de estas. METODOLOGÍA: La primera metodología para inducir a la diferenciación neural fue utilizada en 1999 y a partir de ese momento, se han empleado compuestos químicos, factores de crecimiento, compuestos sintéticos, entre otros métodos para diferenciar este tipo de células a linaje neuronal. El problema radica en que algunas de estas metodologías son tóxicas para las células, costosos o presentan otro tipo de efecto colateral. CONCLUSIÓN: La elección de uno de estos métodos depende de los intereses y las condiciones con las que cuente cada investigador. Además, es indispensable conocer las falencias que tenemos en este campo de la investigación con el propósito de continuar con la búsqueda de alternativas que no tengan desventajas, si no por el contario, reúna todas las ventajas de los métodos aquí mencionados.

SUMMARY INTRODUCTION: Mesenchymal stem cells are cells with the ability to self-renew and differentiate into mesen-chymal lineages and even cells of non-mesenchymal origin, such as those of nerve tissue. Taking into account the suitability of these cells to differentiate into neurons, they have been used for therapeutic purposes since they are able to restore neurons that deteriorate in various neurodegenerative diseases. OBJECTIVE: To inform and to compare the methods most used to induce mesenchymal stem cells to differentiate into neurons and to mention the advantages and disadvantages of each of these. DEVELOPMENT: The first methodology to induce neural differentiation was used in 1999 and since then, chemical compounds, growth factors, synthetic compounds have been used, among other methods to differentiate this type of cells into neuronal lineage. The problem is that some of these methodologies are toxic to cells, expensive or have other side effects. CONCLUSION: The choice of one of these methods depends on the interests and the conditions that each researcher has. In addition, it is indispensable to know the shortcomings that we have in this field of research with the purpose of continuing with the search for alternatives that do not have disadvantages, if not by the contrary, gather all the advantages of the methods mentioned here.

Células-Tronco , Diferenciação Celular , Doenças Neurodegenerativas
Acta cir. bras ; 32(11): 984-994, Nov. 2017. tab, graf
Artigo em Inglês | LILACS | ID: biblio-886180


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

Humanos , Animais , Células-Tronco/citologia , Queratinócitos/citologia , Diferenciação Celular/fisiologia , Citometria de Fluxo/métodos , Pele/citologia , Biomarcadores/análise , Células Cultivadas , Protocolos Clínicos , Técnicas de Cultura de Células
J. appl. oral sci ; 25(5): 515-522, Sept.-Oct. 2017. graf
Artigo em Inglês | LILACS | ID: biblio-893656


Abstract Hypersensitivity, local irritative and cytotoxic effects are known for the chemical components of Syzygium aromaticum and Cinnamomum zeylanicum contained in dental materials. However, there is no intimate data in dentistry using the whole extracts of these plants and introducing new ones. Salvia triloba is a well-known anti-inflammatory plant that correspondingly could be used in several dental traumas. Objectives: We aimed to show and compare the effect of S. aromaticum, C. zeylanicum, and S. triloba extracts on dental pulp stem cells (DPSCs) proliferation, differentiation, and immune responses. Material and Methods: Using xCELLigence, a real time monitoring system, we obtained a growth curve of DPSCs with different concentrations of the Extracts. A dose of 10 μg/mL was the most efficient concentration for vitality. Osteogenic differentiation and anti-inflammatory activities were determined by using an ELISA Kit to detect early and late markers of differentiation. Results: The level of osteonectin (ON, early osteogenic marker) decreased, which indicated that the osteogenic differentiation may be accelerated with addition of extracts. However, the level of osteocalcin (OCN, late osteogenic marker and sign of calcium granulation) differed among the extracts, in which S. aromaticum presented the highest value, followed by S. triloba and C. zeylanicum. Surprisingly, the determined calcium granules were reduced in S. aromaticum and S. triloba. In response to tumor necrosis factor alpha (TNF-α), S. triloba-treated DPSCs showed the most reduced level of IL-6 cytokine level. We suggest C. zeylanicum as a promising osteogenic inducer and S. triloba as a potent anti-inflammatory agent, which could be used safely in biocomposite or scaffold fabrications for dentistry. Conclusions: Because calcium granule formation and cell viability play a critical role in hard tissue formation, S. aromaticum in dentistry should be strictly controlled, and the mechanism leading to reduced calcium granule formation should be identified.

Humanos , Adolescente , Adulto Jovem , Anti-Inflamatórios/farmacologia , Cinnamomum zeylanicum/química , Polpa Dentária/citologia , Medicamentos de Ervas Chinesas/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Extratos Vegetais/farmacologia , Syzygium/química , Análise de Variância , Antígenos de Diferenciação/análise , Cálcio/análise , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Citocinas/análise , Polpa Dentária/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Osteocalcina/análise , Osteogênese/efeitos dos fármacos , Osteonectina/análise , Reprodutibilidade dos Testes , Fatores de Tempo
J. appl. oral sci ; 25(3): 299-309, May-June 2017. graf
Artigo em Inglês | LILACS | ID: biblio-893619


Abstract Objective To assess the effect of fibronectin (Fn) and porcine type I collagen (PCOL) on odontoblast-like cells in vitro. Material and Methods Rat odontoblast-like cells (MDPC-23 cells) were inoculated and cultured on Fn-coated or type I collagen-coated substrates. Proliferation assay, alkaline phosphatase activity (ALP activity), mRNA expression of hard tissue-forming markers, and Alizarin red staining were investigated over a period of 10 days. Results Cells maintained a high proliferation activity on Fn and PCOL even at a low seeding concentration (0.5×104/mL) as demonstrated by CCK-8 assay. The proliferation activity of cells on Fn increases in a concentration-dependent manner while it reached a plateau after 10 µg/mL. Cells adopted long, thin and spindle shape on Fn(10-50) and PCOL. Parallel actin filaments were observed in MDPC-23 cells cultured on Fn and PCOL. ALP activity was markedly up-regulated on Fn and PCOL-coated surfaces. Importantly, gene expression of BSP (Fn10: 2.44±0.32; Fn20: 3.05±0.01; Fn30: 2.90±0.21; Fn40: 2.74±0.30; Fn50: 2.64±0.12; PCOL: 2.20±0.03) and OCN (Fn10: 2.52±0.23; Fn20: 2.28±0.24; Fn30: 2.34±0.21; Fn40: 2.34±0.25; Fn50: 2.20±0.22; PCOL: 1.56±0.16) was significantly enhanced on Fn and PCOL substrates as compared with control; moreover, expression of integrin beta 1 (ITGB1), an ubiquitous cell surface receptor was augmented in Fn(10-50) and PCOL groups simultaneously. In accordance with the ALP activity and gene expression data, calcific deposition in cells grown on Fn(10-50) and PCOL was observed as well. Conclusion Despite the limitation of this study, the findings indicate that a surface coating of Fn enhances the proliferation, differentiation and mineralization of odontoblast-like cells by activation of integrin beta 1 (ITG B1). The promoting effects of Fn on MDPC-23 cells were achieved at a comparatively lower coating concentration than type I collagen (300 µg/mL). Specifically, it is suggested that the optimum coating concentration of Fn to be 10 µg/mL.

Humanos , Animais , Ratos , Diferenciação Celular/efeitos dos fármacos , Fibronectinas/farmacologia , Proliferação de Células/efeitos dos fármacos , Odontoblastos/efeitos dos fármacos , Fatores de Tempo , Expressão Gênica , Células Cultivadas , Reprodutibilidade dos Testes , Imunofluorescência , Antraquinonas , Integrina beta1/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Colágeno Tipo I/farmacologia , Fosfatase Alcalina/análise
Int. j. morphol ; 35(2): 413-419, June 2017. ilus
Artigo em Inglês | LILACS | ID: biblio-892996


Surgical techniques for treatment of sensory neural hearing loss (SNHL) have unpredictable outcomes and in recent years cell therapy investigated for treatment of SNHL. Olfactory epithelium proceed neurogenesis during life time and provide an easily accessible source of neural stem cells. So the aim of this study was isolating neural stem cells from olfactory epithelium of rat and differentiation of these cells into hair cells of inner ear in vitro. The epithelium tissue of olfactory mucosa of rats were removed and digested by collagenase H. The digested tissue was cultured in flasks in suspension forms to create spheres. Spheres were passaged and from passage 2 spheres selected for differentiation. At this stage cells of spheres isolated from each other and placed in flask containing defined differentiation medium. Cells at this stage cultured in adhesive form. Immunohistochemistry and RT-PCR were used for neural stem cells and hair cells identification. Spheres formed from olfactory epithelium culture and immunohistochemistry revealed that cells of spheres from passage one and two expressed the neural stem cells markers. After culture of isolated cells in differentiation medium, the morphology of cells begun to change. The cells presented neural cells projections and after 10 days the projections elongated more and interact to each other in multi layers. RT-PCR and immunohistochemistry revealed that differentiated cells expressed hair cells specific genes. In this study we showed that neural stem cells of olfactory epithelium can differentiate into hair cells of inner ear and therefore can be used for treatment of SNHL.

Las técnicas quirúrgicas para el tratamiento de la pérdida auditiva neural sensorial (PANS) tienen resultados impredecibles y en los últimos años la terapia celular ha sido investigada para su tratamiento. El epitelio olfatorio se forma durante la neurogénesis y proporciona una fuente fácilmente accesible de células madre neurales. El objetivo de este estudio fue aislar las células madre neurales del epitelio olfativo de la rata y la diferenciación de estas células en vestibulocitos del oído interno in vitro. Se retiró el tejido del epitelio de la mucosa olfatoria de ratas y fue digerido con colagenasa H. El tejido se cultivó en forma de suspensión para crear esferas. Se seleccionaron dos esferas para la diferenciación. En esta fase, las células de esferas fueron aisladas unas de otras y colocadas en un medio de diferenciación definido. Células en esta etapa fueron cultivadas en forma adhesiva. Inmunohistoquímica y RT-PCR se utilizó para las células madre neurales y la identificación de células ciliadas. Las esferas formadas a partir del cultivo del epitelio olfatorio y la inmunohistoquímica revelaron que las células de esferas en etapas uno y dos expresaban los marcadores de células madre neurales. Se observaron cambios en la morfología de las células después del cultivo de células aisladas. RT-PCR e inmunohistoquímica revelaron que las células diferenciadas expresaron células específicas de gen de vestibulocitos. Se observó que las células madre neuronales de epitelio olfatorio puede diferenciarse en células en forma de cabello del oído interno y por lo tanto puede ser utilizado para el tratamiento de PANS.

Animais , Ratos , Perda Auditiva Neurossensorial/cirurgia , Células-Tronco Neurais/transplante , Mucosa Olfatória/citologia , Diferenciação Celular , Imuno-Histoquímica , Reação em Cadeia da Polimerase , Ratos Sprague-Dawley
Braz. j. microbiol ; 48(1): 125-131, Jan.-Mar. 2017. graf
Artigo em Inglês | LILACS | ID: biblio-839349


Abstract Small ruminant lentiviruses isolated from peripheral blood leukocytes and target organs can be propagated in vitro in fibroblasts derived from goat synovial membrane cells. These cells are obtained from tissues collected from embryos or fetuses and are necessary for the establishment of the fibroblast primary culture. A new alternative type of host cells, derived from goat umbilical cord, was isolated and characterized phenotypically with its main purpose being to obtain cell monolayers that could be used for the diagnosis and isolation of small ruminant lentiviruses in cell culture. To accomplish this goal, cells were isolated from umbilical cords; characterized phenotypically by flow cytometry analysis; differentiate into osteogenic, chondrogenic and adipogenic lineage; and submitted to viral challenge. The proliferation of goat umbilical cord cells was fast and cell monolayers formed after 15 days. These cells exhibited morphology, immunophenotype, growth characteristics, and lineage differentiation potential similar to mesenchymal stem cells of other origins. The goat umbilical cord derived cells stained positive for vimentin and CD90, but negative for cytokeratin, CD34 and CD105 markers. Syncytia and cell lysis were observed in cell monolayers infected by CAEV-Cork and MVV-K1514, showing that the cells are permissive to small ruminant lentivirus infection in vitro. These data demonstrate the proliferative competence of cells derived from goat umbilical cords and provide a sound basis for future research to standardize this cell lineage.

Animais , Cordão Umbilical/citologia , Lentivirus/fisiologia , Células-Tronco Mesenquimais/virologia , Osteogênese , Replicação Viral , Técnicas In Vitro , Cabras , Biomarcadores , Diferenciação Celular , Células Cultivadas , Imunofenotipagem , Técnicas de Cultura de Células , Condrogênese , Efeito Citopatogênico Viral , Adipogenia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/patologia
Electron. j. biotechnol ; 26: 40-45, Mar. 2017. ilus, graf
Artigo em Inglês | LILACS | ID: biblio-1009000


Background: Ethylene plays an important role in the regulation of floral organ development in soybean, and 1-aminocyclopropane-1-carboxylate synthase (ACS) is a rate-limiting enzyme for ethylene biosynthesis. However, whether ACS also regulates floral organ differentiation in soybean remains unknown. To address this, we constructed an RNAi vector to inhibit ACS expression in cotyledonary nodes. Linear DNA cassettes of RNAi-ACS obtained by PCR were used to transform soybean cotyledonary nodes. Results: In total, 131 of 139 transiently transformed plants acquired herbicide resistance and displayed GUS activities in the new buds. In comparison to untransformed seedling controls, a greater number of flower buds were differentiated at the cotyledonary node; GM-ACS1 mRNA expression levels and ethylene emission in the transformed buds were reduced. Conclusion: These results indicate that the cotyledonary node transient transformation system may be suitable for stable transformation and that the inhibition of ACS expression may be an effective strategy for promoting floral organ differentiation in soybean.

Soja/enzimologia , Soja/genética , Interferência de RNA , Liases/metabolismo , Soja/crescimento & desenvolvimento , Transformação Genética , Expressão Gênica , Diferenciação Celular , Reação em Cadeia da Polimerase , Regulação da Expressão Gênica de Plantas , Etilenos/biossíntese , Resistência a Herbicidas , Vetores Genéticos , Glucuronidase