ABSTRACT
The study of biologically active substances-secondary metabolites of plants that exhibit geroprotective properties is an actual and popular direction in medicine to prevent early aging. This work aims to select the cultivation parameters for obtaining in vitro cell cultures of meadowsweet containing the largest amount of biologically active substances (BAS) for their further extraction as candidate substances for geroprotectors. To specify the effectiveness of the selected cell culture cultivation parameters, biomass growth for callus and root cultures, growth index, specific growth rate, and viability for suspension cultures was carried out. The study results made it possible to select the nutrient media for the cultivation of cell cultures of meadowsweet. It has been found that the greater the antioxidant activity of the extracts, the greater the antimicrobial properties it exhibits. In this study, cell cultures in vitro and alcohol extracts from the plant Filipendula ulmaria were considered as raw materials rich in candidate substances for geroprotectors. According to the data obtained, the plant is rich in hydroxybenzoic and salicylic acids, spireoside, avicularin, and hyperoside.
O estudo de substâncias biologicamente ativas - metabólitos secundários de plantas que apresentam propriedades geroprotetoras - é uma tendência atual e popular no campo da medicina para a prevenção do envelhecimento precoce. O objetivo deste trabalho foi selecionar os parâmetros de cultivo para obtenção de culturas celulares in vitro de Ulmária contendo a maior quantidade de substâncias biologicamente ativas (SBA), para sua posterior extração como substâncias candidatas a serem geroprotetoras. Para especificar a eficácia dos parâmetros selecionados de cultivo em cultura de células, foi realizada a análise de crescimento de biomassa para culturas de calos e raízes, índice de crescimento, taxa de crescimento específica e viabilidade para culturas em suspensão. Os resultados do estudo possibilitaram a seleção do meio nutriente para o cultivo de células de Ulmária. Verificou-se que, quanto maior a atividade antioxidante dos extratos, maiores eram as propriedades antimicrobianas exibidas. Neste estudo, culturas celulares in vitro e extratos alcoólicos da planta Filipendula ulmaria foram considerados matérias-primas ricas em substâncias candidatas a serem geroprotetoras. De acordo com os dados obtidos, a planta é rica em ácidos hidroxibenzoico e salicílico, espirosídeo, avicularina e hiperosídeo.
Subject(s)
Plants, Medicinal/genetics , Aging , Aging, Premature , AntioxidantsABSTRACT
Abstract The study of biologically active substances-secondary metabolites of plants that exhibit geroprotective properties is an actual and popular direction in medicine to prevent early aging. This work aims to select the cultivation parameters for obtaining in vitro cell cultures of meadowsweet containing the largest amount of biologically active substances (BAS) for their further extraction as candidate substances for geroprotectors. To specify the effectiveness of the selected cell culture cultivation parameters, biomass growth for callus and root cultures, growth index, specific growth rate, and viability for suspension cultures was carried out. The study results made it possible to select the nutrient media for the cultivation of cell cultures of meadowsweet. It has been found that the greater the antioxidant activity of the extracts, the greater the antimicrobial properties it exhibits. In this study, cell cultures in vitro and alcohol extracts from the plant Filipendula ulmaria were considered as raw materials rich in candidate substances for geroprotectors. According to the data obtained, the plant is rich in hydroxybenzoic and salicylic acids, spireoside, avicularin, and hyperoside.
Resumo O estudo de substâncias biologicamente ativas metabólitos secundários de plantas que apresentam propriedades geroprotetoras é uma tendência atual e popular no campo da medicina para a prevenção do envelhecimento precoce. O objetivo deste trabalho foi selecionar os parâmetros de cultivo para obtenção de culturas celulares in vitro de Ulmária contendo a maior quantidade de substâncias biologicamente ativas (SBA), para sua posterior extração como substâncias candidatas a serem geroprotetoras. Para especificar a eficácia dos parâmetros selecionados de cultivo em cultura de células, foi realizada a análise de crescimento de biomassa para culturas de calos e raízes, índice de crescimento, taxa de crescimento específica e viabilidade para culturas em suspensão. Os resultados do estudo possibilitaram a seleção do meio nutriente para o cultivo de células de Ulmária. Verificou-se que, quanto maior a atividade antioxidante dos extratos, maiores eram as propriedades antimicrobianas exibidas. Neste estudo, culturas celulares in vitro e extratos alcoólicos da planta Filipendula ulmaria foram considerados matérias-primas ricas em substâncias candidatas a serem geroprotetoras. De acordo com os dados obtidos, a planta é rica em ácidos hidroxibenzoico e salicílico, espirosídeo, avicularina e hiperosídeo.
ABSTRACT
Abstract At present, tissue engineering is transforming the area of cardiovascular regenerative medicine, which combines the principles and methods of materials engineering and biological sciences, interacting with biochemical and physicochemical factors, for the understanding of their structure-function relationship. Thus, the course of diseases is reoriented by implementing methods and procedures involved in the regeneration of organs and tissues by means of the interaction with biocompatible matrices, pre-treated organs or stem cell management, among others, thus recovering the functionality in the system affected by acquired pathologies, alterations or congenital defects. Consequently, these procedures are increasingly becoming one the most promising treatment alternative for patients who suffer from any type of functional deficit. Known that all these possibilities make cell cultures a promising study environment to be used in biomedical applications, especially in tissue engineering and regenerative medicine, this manuscript presents a general reviews of established cell lines or primary tissue lines and how cell cultures serve as a model before experimental work on laboratory animals and human subjects which makes it a valuable tool for broad models of study in the research on cardiology.
Resumen En la actualidad, la ingeniería de tejidos está transformando el área de la medicina regenerativa cardiovascular, combinando los principios y métodos de la ingeniería de materiales y las ciencias biológicas, interactuando entre factores bioquímicos y fisicoquímicos, para la comprensión de su relación estructura-función. Así, el curso de las enfermedades se viene a reorientar mediante la implementación de métodos y procedimientos implicados en la regeneración de órganos y tejidos a través de la interacción con matrices biocompatibles, órganos pretratados o manejo de células madre, entre otros, recuperando así la funcionalidad en el sistema afectado por enfermedades adquiridas y alteraciones o defectos congénitos. En consecuencia, estos procedimientos se están convirtiendo en una de las alternativas de tratamiento cada vez más prometedoras para los pacientes que sufren de algún tipo de alteración funcional. Considerando que todas estas posibilidades hacen de los cultivos celulares un entorno de estudio prometedor para ser utilizado en aplicaciones biomédicas, especialmente en ingeniería de tejidos y medicina regenerativa, este manuscrito presenta una revisión general de las líneas celulares establecidas o líneas de tejido primario y cómo los cultivos celulares sirven como modelo de evaluación antes del trabajo experimental en animales de laboratorio y sujetos humanos, lo cual los convierte en una herramienta valiosa para amplios modelos de estudio en la investigación en cardiología.
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Abstract Thevetia peruviana is a medicinal plant that has valuable secondary terpenoid-type metabolites and phenolic compounds. Some flavonoid compounds of pharmaceutical interest stand out in the latter group. The concentration of these bioactive compounds in natural conditions is limited by environmental; therefore, it has been considered necessary to make in vitro plant cell suspension cultures that admit the use of elicitors to increase the content of active principles. Accordingly, in this study, for the optimization of flavonoid production in cell suspension culture of T. peruviana, different parameters related to elicitation with methyl jasmonate (MeJa), and salicylic acid (SA) were evaluated, at stirred flask scale. Firstly, 3 μM MeJa and 300 μM SA were added separately in cell cultures of T. peruviana, to assess their potential effects. Secondly, several experimental conditions were evaluated, for optimization purpose. In the first part, MeJa and SA increased the total flavonoid content, in 1.07 and 1.3 times, respectively, compared to the control culture; in the second part, total flavonoid content produced in MeJa mediated cell suspension cultures were 4.14 mg QE/g DW (milligrams of quercetin equivalent per gram of dry biomass) with: concentration 0.3 μM, addition time day 5 and harvest time 90 h. On the other hand, total flavonoid content produced in SA mediated cell suspension cultures were 3.75 mg QE/g DW with: concentration 100 μM, addition time day 0 and harvest time 96 h. Elicitation of cell suspension cultures of T. peruviana with MeJa and SA under their ideal parameter values increased flavonoid content.
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Introducción: El desarrollo de herramientas para investigar la actividad electrofisiológica cardiaca ha permitido profundizar en el conocimiento sobre los mecanismos subyacentes a las arritmias cardiacas. Los sistemas de mapeo óptico constituyen una tecnología que responde a la necesidad de superar varios obstáculos en la experimentación. Objetivo: Proporcionar una visión general de la importancia del mapeo óptico en cultivos celulares HL-1, en las investigaciones en electrofisiología cardiaca. Métodos: Se realizó una revisión sobre los estudios electrofisiológicos que involucran la línea celular HL-1 utilizando la técnica de mapeo óptico. Conclusiones: Los trabajos se caracterizan por la implementación de la técnica respecto a la tecnología de los equipos de mapeo, a la utilización de diferentes colorantes y al objetivo de la investigación. Están enfocados en el estudio de mecanismos arritmogénicos, procesos de estiramiento mecánico o remodelación del tejido y en el análisis de nuevos biomateriales. Lo anterior, sustenta la relevancia del mapeo óptico en la investigación cardiaca(AU)
Introduction: The development of tools to study cardiac electrophysiological activity has made it possible to broaden knowledge about the mechanisms underlying cardiac arrhythmias. Optical mapping systems constitute a technology that responds to the need to overcome several hurdles in experimentation. Objective: Provide an overview of the importance of optical mapping in HL-1 cell cultures in cardiac electrophysiology research. Methods: A review was conducted of electrophysiological studies involving the HL-1 cell line using the optical mapping technique. Conclusions: The studies are characterized by implementation of the technique with respect to the technology of mapping equipment, the use of different colorants and the purpose of the research. They focus on the study of arrhythmogenic mechanisms, mechanical stretch processes or tissue remodeling as well as the analysis of new biomaterials. The above substantiates the relevance of optical mapping in cardiac research(AU)
Subject(s)
Humans , Male , Female , Electrophysiologic Techniques, Cardiac/methods , Optical Restriction Mapping/methodsABSTRACT
Abstract Thevetia peruviana is an ornamental shrub grown-up in many tropical region of the world. This plant produces secondary metabolites with biological properties of interest for the pharmaceutical industry. The objective was to determine the secondary metabolites profile of callus and cell suspension cultures of T. peruviana and compare them with those from explant (fruit pulp). Extracts in 50% aqueous ethanol and ethyl acetate were prepared. The phytochemical analysis was performed using standard chemical tests and thin layer chromatography. In addition, total phenolic and flavonoids compounds (TPC and TFC), total cardiac glycosides (TCG) and total antioxidant activity (TAA) was determined during the cell suspension growth. Phenolic chemical profile was also analyzed by high performance liquid chromatography (HPLC). Common metabolites (alkaloids, amino acids, antioxidants, cardiac glycosides, leucoanthocyanidins, flavonoids, phenols, sugars and triterpenes) were detected in all samples. The maximum production of extracellular TCG, TPC, TFC and TAA in cells suspensions were at 6-12 days; in contrast, intracellular content was relatively constant during the exponential grown phase (0 to 12-days). HPLC analysis detected one compound with retention time at 11.6 min; this compound was tentatively identified as dihydroquercetin, a flavonoid with anti-cancer properties. These results provide evidence on the utility of the in vitro cell cultures of T. peruviana for valuable pharmaceutical compounds production.
Subject(s)
Cells, Cultured , Thevetia/cytology , Phytochemicals/biosynthesis , Triterpenes , Flavonoids , Chromatography, High Pressure Liquid , Anticarcinogenic Agents , Thevetia/chemistry , Culture Techniques , Phytochemicals/analysis , AntioxidantsABSTRACT
ABSTRACT: Canine transmissible venereal tumor (CTVT) is a transmissible neoplasm, which spreads naturally between dogs through the halogenic transfer of tumor cells, mainly during coitus. It is the oldest known tumoral lineage in nature and reports on gene mutations have been extended. Also, this tumor shares several genetic mutations with some cancers in humans, among them lung carcinomas, melanoma, prostate, breast, among other cancers. Thus, expression of tumor suppressor genes such as TP53, P21, and apoptosis-related genes such as BAX, BCL-2, and BCL-xL, both in vivo and in vitro (primary cell culture) were quantified. In the present study, the comparison of gene expression, the TP53 gene, in most cases, was shown to be high in the majority of tissues (65%) and primary cell culture (100%), while BCL-2, BCL-xL, and BAX presented variation among the animals analyzed. Moreover, in these situations, the results suggested that the apoptotic regulation of these genes did not occur for TP53. The P21 gene was shown to be mostly normal (70%); although, absence (6%) and underexpressions (24%) were also observed. Statistical analysis of the BCL-xL gene demonstrated significant differences between the tissues of the animals when compared to the cell cultures; however, to the other genes, no statistical difference was observed between the groups. Preliminarily, the results suggested the presence of alterations in the gene expressions of the TP53, P21, BAX, BCL-2 and BCL-xL leading to loss of function in these genes, which affect the tumorigenesis of CTVT.
RESUMO: O tumor venéreo transmissível canino (TVTC) se trata de uma neoplasia transmissível, que se propaga naturalmente entre os cães pela transferência halogênica de células tumorais, principalmente, durante o coito. É a mais antiga linhagem tumoral conhecida na natureza e relatos sobre mutações gênicas vêm sendo ampliadas. Além disso, este tumor compartilha uma série de mutações genéticas com alguns cânceres em seres humanos, dentre eles, carcinomas de pulmão, melanoma, próstata, mama, entre outros tipos de câncer. Assim, quantificou-se a expressão de genes supressores de tumores, como TP53, P21 e genes relacionados à apoptose, como BAX, BCL-2 e BCL-xL, tanto in vivo quanto in vitro (cultura celular primária). No presente estudo, na comparação das expressões gênicas, o gene TP53 se mostrou elevado na maioria dos casos em tecidos (65%) e em cultura celular primária (100%), enquanto BCL-2, BCL-xL e BAX apresentaram-se variáveis entre os animais analisados. Ademais, nessas situações os resultados sugerem que não ocorreu regulação apoptótica desses genes pelo TP53. O gene P21 mostrou-se, em sua maioria, normal (70%), embora a ausência (6%) e subexpressões (24%) também tenham sido observadas. A análise estatística do gene BCL-xL demonstrou diferenças significativas entre os tecidos dos animais, quando comparadas às culturas celulares, entretanto, para os demais genes, não foi observada diferença estatística entre os grupos. Preliminarmente, os resultados sugerem a presença de alterações nas expressões gênicas dos genes TP53, P21, BAX, BCL-2 e BCL-xL, levando a perda de função desses genes, os quais afetam a tumorigênese do CTVT.
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RESUMEN Los cultivos de células vegetales son sistemas experimentales homogéneos altamente controlables que permiten el estudio de adaptaciones bajo condiciones de estrés hídrico, sin la interferencia de los diferentes tejidos y estados del desarrollo vegetal. Una aproximación para comprender esas adaptaciones, es la aparición de proteínas inducidas, resultado de la alteración en la expresión génica. El presente trabajo analizó la respuesta de cultivos de células de Fouquieria splendens ssp. breviflora, expuestos a ácido abscísico (ABA), mediante la caracterización electroforética en cantidad y calidad de las proteínas inducibles de estrés. Se registraron polipéptidos de bajo peso molecular (< 35kDa), comunes bajo la exposición a 10 mM, seguida la asociación con 20 y 30 mM de ABA, quedando aislada la respuesta de la condición de células en cultivo sin la presencia de éste.
ABSTRACT Plant cell cultures are homogenous experimental systems, highly controllable that allow the study of short and large water stress adaptations without the interference of the different tissues and development ofplants. An approach to understand these adaptations is through the presence of induced proteins; as a result of changes in genetic expression. This work analyze the response of Fouquieria splendens ssp. breviflora cell cultures exposed to abscisic acid (ABA), through the electrophoretic characterization of quantity and quality of stress induced proteins. There were recorded low molecular weight polypeptides (< 35kDa), common in experiments under ABA 10mM, followed by the association with 20 and 30mM ABA conditions, with a particularly response of cell cultures without the stress agent.
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Antecedentes: Los factores de crecimiento utilizados en salud se pueden obtener de una fuente autóloga de primera generación llamada plasma rico en plaquetas (PRP). La diversidad de protocolos para prepararlos genera resultados variables en cuanto al tiempo entre la activación del PRP y sus efectos sobre la proliferación y viabilidad celular. Objetivo: Evaluar proliferación y viabilidad celular de fibroblastos de ligamento periodontal y osteoblastos tratados con PRP en diferentes concentraciones y tiempos de aplicación. Métodos: Se cultivaron líneas celulares de fibroblastos y osteoblastos y se preparó el PRP de sangre venosa de un adulto sano mediante centrifugación, seguido de activación con CaCl2 al 10 %. El efecto sobre la proliferación de las líneas celulares tras la aplicación de PRP y plasma pobre en plaquetas al 1 %, al 3 % y al 5 % se evaluó a las 0, 12, 24, 48 y 72 horas después de su activación mediante MTS. El grupo control consistió en cultivos sin tratamiento. Los datos se analizaron mediante las pruebas de Chi cuadrado, Fischer y McNemar. Resultados: Se observó un aumento de la viabilidad en células tratadas con PRP 24 horas después de su activación en una concentración del 5%. El ensayo de viabilidad celular mostró diferencias estadísticamente significativas entre el grupo experimental y el grupo control (p = 0,05). Conclusión: Los cultivos de fibroblastos y osteoblastos mostraron una tendencia a mayor viabilidad 24 horas después de a la activación con PRP al 5 %.
Background : Growth factors used in health treatments can be obtained from a first-generation source called platelet-rich plasma. The variety of protocols to prepare PRP produces variable results regarding PRP activation time and its effects on cell proliferation and viability. Purpose: To evaluate proliferation and cell viability of periodontal ligament fibroblasts and osteoblasts stimulated with PRP in several concentrations and times after PRP activation. Methods: An in vitro study was carried out using periodontal ligament fibroblast and osteoblast cell cultures. PRP from venous blood of a healthy adult was prepared through centrifugation and activated with 10% CaCl2. The effect on cell proliferation after application of 1%, 3%, and 5% PRP and platelet-poor plasma was evaluated at 0, 12, 24, 48, and 72 hours after activation through MTS. The control group consisted of culture that did not receive any treatment. Data were analyzed using Chi square, Fisher, and McNemar tests. Results: The cell viability assay showed statistically significant differences between the experimental and the control groups. Cell viability increased in cells treated with 5% PRP 24 hours after activation (p = 0.05). Conclusions: Fibroblast and osteoblast cell lines tended to be more viable 24 hours after activation with 5% PRP.
Subject(s)
Humans , Periodontal Ligament , Cell Proliferation , Osteoblasts , In Vitro Techniques , Platelet-Rich PlasmaABSTRACT
Objective To study the effects of abiotic elicitors methyl jasmonate (MeJA) and salicylic acid (SA) on the alkaloids accumulation and related enzymes metabolism inPinellia ternata suspension cell cultures. Methods Using the leaf petioles-derived suspension cell cultures as the study object, the culture duration, concentrations of MeJA and SA were determined to get the optimal alkaloids accumulation, and the activities of metabolic enzymes IMP dehydrogenase and sAMP synthase were also measured.Results A 9-fold of dried biomass and a 3-fold of alkaloids accumulation were observed inP. ternata suspension cell cultures after culture for 21 d. Both MeJA and SA could significantly promote the accumulation of alkaloids inP. ternata suspension cells. 150 μmol/L MeJA enhanced alkaloids content (4.7 mg/gDW) by 3.6 folds in comparison with control group, whereas 50 μmol/L SA showed a 2.5-fold increase. Meanwhile, 100 μmol/L MeJA and 50 μmol/L SA promoted the increase in IMP dehydrogenase activity by 3.0 and 3.7 fold respectively, and 150 μmol/L MeJA and 100 μmol/L SA showed the increase by 2.6 and 4.4 fold respectively.Conclusion Proper adding exogenous MeJA and SA can promote the accumulation of alkaloids inPinellia ternata suspension cell cultures.
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Introducción: el tejido adiposo humano está compuesto por diferentes tipos celulares, y ha sido objeto de múltiples estudios en los últimos años debido a su acción en diversas funciones. Métodos: se realizó una búsqueda bilbiográfica en PubMed, Scielo, Science Direct and Google Scholars y se reporta la experiencia de los autores. Resultados: los primeros modelos de estudio fueron con tejido de roedores, y permitieron la comprensión del metabolismo de carbohidratos y ácidos grasos y facilitaron el estudio de la biología del adipocito, junto a estudios histológicos y el aislamiento de adipocitos maduros. Posteriormente se realizaron estudios con células precursoras (preadipocitos) que propiciaron el aislamiento de la línea celular 3T3-1L murina. En Latinoamérica, se han realizado diversos estudios con líneas celulares y con células madre mesenquimales precursoras de adipocitos para estudiar el efecto de hormonas y otras sustancias y para genotipificación. En Colombia se han realizado estudios con adipocitos 3T3-L1 para determinar los efectos de medicamentos y sustancias en estas células. En el laboratorio de Fisiología Celular de la Universidad Tecnológica de Pereira el proceso de obtención de muestras ha evidenciado dificultades por tratarse de tejido humano, pero el protocolo de aislamiento y cultivo pudo ser estandarizado a lo largo de seis años de experimentación; se aislaron preadipocitos y adipocitos maduros que permitieron estudiar los efectos de hormonas, realizar caracterización electrofisiológica y estudiar la fisiología del calcio. Conclusiones: este es un campo de investigación muy relevante debido a la implicación de este tipo celular en funciones metabólicas sistémicas y su relación con patologías de alta prevalencia como la obesidad y el síndrome metabólico.
Introduction: The human adipose tissue is composed of different cell types. It has been subject of several studies in the past years due to its role on diverse functions. Methods: A search in the PubMed, Scielo, Science direct and Google Scholars databases was performed and the authors experience was reported. Results: The first model used was rodent fat, which allowed a better comprehension on carbohydrate and fatty acid metabolism and enhanced research in adipocyte cell biology in addition with histological studies and mature adipocyte isolation. Afterwards, learning about precursor cell (pre-adipocytes) promoted the isolation of murine 3T3-L1 cell line. In Latin America research has been conducted using cell lines and adipocyte precursor mesenchymal stem cells to describe effects of hormones and perform DNA sequencing. In Colombia, studies in 3T3-L1 cell line aimed to stablish the effects of different compounds on these cells. In the Cell Physiology Laboratory of the Universidad Tecnológica de Pereira, sample collection process has shown difficulties because the source was human tissue; nevertheless isolation and cell culture protocols were standardized throughout the last six years of experimentation. Pre-adipocytes and mature adipocytes were isolated to study the effects of hormones, perform electrophysiological characterization and study calcium physiology. Conclusions: This is a relevant research field since these cells have important systemic metabolic functions and they have a clear relationship with high-prevalence pathologies such as obesity and the metabolic syndrome.
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Choibá (Dipteryx oleifera Benth) es un árbol de la familia Fabaceae (Papilionoideae), con una distribución geográfica reportada desde Nicaragua hasta Colombia a una altura de hasta 1000 msnm. Crece en bosque húmedo, muy húmedo o premontano húmedo. Esta especie es considerada vulnerable debido a la sobreexplotación de su madera, ya que es un árbol altamente apetecido por esta y por sus frutos. Su almendra almacena una buena cantidad de aceites con potencial para la industria alimentaria, lo que podría resultar en una nueva fuente alimenticia, por lo cual el cultivo in vitro de vegetales con el propósito de producir compuestos de interés, marca un punto de partida para reducir el uso del suelo y lograr componentes bioactivos bajo condiciones controladas. En este trabajo, como una primera etapa experimental, se evaluó el crecimiento celular en suspensiones, a partir de callo inducido en explantes de cotiledón; se ensayaron 6 tratamientos diferentes, la mitad de estos con MS como medio basal y la otra mitad con B5, cada uno de los dos grupos con un control y la combinación hormonal de 2.5 mg/L de 2,4-D y 1 mg/L de BAP o kinetina, suplementado con adenina, biotina, glutamina y ácido pantoténico y 30 g/L de sacarosa, bajo completa oscuridad. Se encontró que dos tratamientos con MS en combinación con 2.5 mg/L de 2,4-D y 1 mg/L de kinetina o BAP fueron los mejores.
Choibá (Dipteryx olifera) is a tree of the Fabaceae family, with a geographical distribution reported from Nicaragua to Colombia, nearly 1.000 msnm in a tropical rain forest. This species is a highly desired tree for its timber and fruits, the kernel store a lot of important oils for the food industry, resulting in a new possible food source, so we are making in vitro cultivation of vegetables with the purpose of producing compounds of interest and that mark a starting point on the reduction of land use and it achieves bioactive components under controlled conditions. In this work, as a first experimental step was evaluated the tissue cell growth in suspension, using fragments of cotyledon and testing 6 different treatments, the half of those with MS as basal medium and the other half with B5, each of the two groups with one control and hormone combination of 2.5 mg/L 2,4-D and 1 mg/L BAP or kinetin, supplemented with adenine, biotin, pantothenic acid and glutamine and 30 g l sucrose under complete darkness. It was found that two treatments with MS in combination with 2.5 mg/L 2,4-D and 1 mg/L kinetin or BAP were the best.
Subject(s)
Biomass , Dipteryx , Diffusion Chambers, Culture , FabaceaeABSTRACT
Introducción: la presencia de microorganismos residuales post-preparación químico-quirúrgico de los conductos radiculares, sugiere el empleo de medicación intracanal eficaz y biológicamente compatible con los tejidos periapicales. Objetivo: comparar la citotoxicidad de medicaciones de uso intracanal en fibroblastos gingivales humanos. Métodos: estudio de tipo experimental. Con cuatro diferentes grupos: control (G1), Clorhidrato de Clindamicina 2g asociado a fosfato de dexametasona 0,32g (Clindex) vehiculado en solución alcohólica (G2), Clindex Vehiculado en polietilenoglicol 400 (G3), y NDP: paramonoclorofenol asociado a fosfato de dexametasona 0,32g vehiculado en polietilenoglicol 400 y solución salina (G4). Las células fueron estimuladas con las medicaciones por 24, 48 y 72 horas y la viabilidad celular fue evaluada usando la técnica de análisis de MTT y la lectura fue realizada en el espectrofotómetro de ELISA. Resultados: fueron analizados por medio de la actividad mitocondrial y mostraron que el G2 obtuvo los mejores resultados en los tres tiempos estudiados, seguido por el G3 y el G4. Se realizó análisis estadístico (ANOVA y complementado con la prueba de Tukey) mostraron diferencias significante a nivel de 1% G2 y G3 cuando se compararon con el G4. Conclusión: la combinación Clindamicina dexametasona independiente del vehículo presentó mayor viabilidad celular que el paramonoclorofenol asociado a fosfato de dexametasona.
Introduction: the presence of residual microorganisms after chemical-surgical preparation of root canals suggests the use of effective intracanal medication, biologically compatible with periapical tissues. Objective: compare the cytotoxicity of intracanal medications in gingival human fibroblasts. Methods: an experimental study was conducted with four different groups: control (G1), Clindamycin Hydrochloride 2g associated to dexamethasone phosphate 0.32g (Clindex) vehicled in alcoholic solution (G2), Clindex vehicled in polyethylene glycol 400 (G3), and NDP: paramonochlorophenol associated to dexamethasone phosphate 0.32g vehicled in polyethylene glycol 400 and saline solution (G4). The cells were stimulated with the medications for 24, 48 and 72 hours. Cell viability was evaluated with the technique of MTT analysis. Readings were performed in an ELISA spectrophotometer. Results: the groups were analyzed in terms of mitochondrial activity. G2 had the best results in the three times studied, followed by G3 and G4. A statistical analysis was performed (ANOVA and complemented by Tukey's test). Significant differences of 1% were found when comparing G2 and G3 with G4. Conclusion: the Clindamycin dexamethasone combination, irrespective of the vehicle, showed greater cell viability than paramonochlorophenol associated to dexamethasone phosphate.
ABSTRACT
In order to assess the anticancer action of extracts obtained by latex from Calotropis procera and Pedilanthus tithymaloides, samples were collected from adult plants. Soluble proteins were extracted with 16 uL of 50 mM sodium acetate pH 5/ug integral latex and centrifugation at 16,000 x g for 15 min, the supernatant was named "latex crude extract" (LCE). The "latex methanolic extract" (LME) was obtained on dried latex. Both extracts were tested in vitro by cytotoxic and cytostatic activity in Jurkat cell cultures. Cellular viability, proliferation, necrosis and apoptosis were evaluated. LCE and LME of C. procera were found with cytotoxic and cytostatic activity after 24 incubation hours (p < 0,05) with doses from 1ug/mL. The LCE and LME of P. tithymaloides presented cytotoxic effect (p < 0,05) from 50 ug/mL and from 1ug/mL, respectively.
Con el objetivo de evaluar el potencial anticanceroso de extractos de látex de Calotropis procera y Pedilanthus tithymaloides se colectaron muestras de plantas adultas. Las proteínas solubles fueron extraídas con 16 uL de acetato de sodio 50 mM pH 5/ug de látex integral y centrifugación a 16.000 x g durante 15 min, denominándose al sobrenadante extracto crudo de látex (ECL). El extracto metanólico de látex (EML) se obtuvo sobre látex deshidratado. Ambos extractos fueron probados en su actividad citotóxica y citostática in vitro sobre cultivos de células Jurkat. Se realizaron estudios de viabilidad, proliferación, necrosis y apoptosis celular. El ECL y el EML de C. procera presentaron actividad citotóxica y citostática después de 24 y 48 horas de incubación (p < 0,05) con dosis desde 1 ug/mL. Los ECL y EML de P. tithymaloides presentaron efectos citotóxicos (p < 0,05) a partir de 50 ug/mL y desde 1 ug/mL respectivamente.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Calotropis/chemistry , Euphorbia/chemistry , Plant Extracts/pharmacology , Apoptosis , Cell Culture Techniques , Cell Survival , Jurkat Cells , Latex , Methanol , Cell ProliferationABSTRACT
Objetivo: Construir e implementar un control interno para la detección molecular de micoplasmas en cultivos celulares. Materiales y métodos: Se alinearon secuencias del RNA ribosomal 16S de las principales especies de micoplasmas reportadas como contaminantes de cultivos, y diseñaron oligonucleótidos para la detección de las mismas. Para la construcción del control interno se amplificó una secuencia espadadora, además de las secuencias de hibridación de los oligonucleótidos usados para detección. La amplificación desde dicho control generó un fragmento con tamaño diferente al obtenido desde una muestra positiva. Posteriormente, para la evaluación del efecto del control interno sobre la amplificación de una muestra positiva se realizó la prueba sobre diluciones seriadas de la muestra, en presencia y ausencia del control interno. Finalmente, se ensayó el protocolo utilizando sobrenadantes de cultivo provenientes de diferentes laboratorios. Resultados: Se observó alta homogeneidad al comparar los géneros Mycoplasma y Acholeplasma; no obstante se realizó el diseño de oligonucleótidos degenerados para aumentar teóricamente la eficiencia en la detección de todas las especies. Se demostró que la aplicación del control interno no afecta la sensibilidad de detección de la prueba. Los resultados obtenidos con muestras problema resaltan la importancia del control interno al realizar la prueba desde sobrenadantes de cultivo. Conclusiones: El uso del control interno evita la obtención de resultados falsos negativos, generados por presencia de inhibidores en la muestra. La implementación de la prueba beneficiará los laboratorios en cuyas investigaciones utilicen cultivos celulares y permitirá ejercer un control de calidad, lo cual hará más confiables los resultados/productos obtenidos.
Objective: to construct and implement an internal control for molecular detection of mycoplasma in cell cultures. Materials and Methods: Sequences of 16S ribosomal RNA of the major species of mycoplasma reported as contaminants in cell cultures were aligned, and oligonucleotides for detection were designed. For the construction of the internal control, a spacer sequence as well as sequences for hybridization of the oligonucleotides used for detection, were amplified. Amplification from the internal control and positive samples generated fragments with different sizes. Subsequently, to evaluate the effect of the internal control on the amplification of a positive sample, the assay was performed on serial dilutions of the sample in the presence and absence of the internal control. Finally, the protocol was tested using culture supernatants from different laboratories. Results: High homogeneity was observed when Mycoplasma and Acholeplasma genera were compared; however, degenerated oligonucleotides were designed to increase the efficiency of detection in all the analyzed species. It was shown that implementation of the internal control does not affect the sensitivity of detection. The results obtained with cell cultures samples highlighted the importance of the internal control. Conclusions: The use of an internal control facilitates the detection of false negative results generated by the presence of inhibitors in the sample. Implementation of the test as a quality control will benefit laboratories using cell cultures, making the results and / or products obtained more reliable.
ABSTRACT
Background: To assess the increased cellular immunity of Peripheral Blood Mononuclear Cells (PBMC) derived LAK cells from endometriosis patients towards endometriosis cell cultures after stimulation with IL-2. Methods: This study is a quasi-experimental study of pre and post treatment using controls. Phenotype evaluation of CD3+CD4+, CD3+CD8+ and CD56+ effector cells of PBMC from endometriosis patients and controls was performed. Cytotoxicity test of PBMC from endometriosis patients and control towards Daudi, K562 cell line and endometriosis cell cultures using 51Chromium release assay was also carried out. Results: Phenotype evaluation of PBMC from endometriosis patients (n = 10) and controls (n = 6) were done prior to and after IL-2 stimulation. Before IL-2 stimulation, CD3+CD4+, CD56+ from endometriosis group (n = 10) tend to be lower than control (n=6) whereas CD3+CD8+ were higher in endometriosis group than controls. After IL-2 stimulation, CD3+ CD8+, CD56+ of PBMC from endometriosis group were significantly increased (p < 0.05). Cytotoxicity test revealed a significant increase (p < 0.05) in both PBMC’s effector cells from endometriosis and control group towards target cells, Daudi, and K562 cell lines after IL-2 stimulation. PBMC’s effector cells cytotoxicity from both endometriosis and control towards target endometriosis cell cultures were also elevated after IL-2 stimulation. Conclusion: LAK cells derived IL-2 stimulated PBMC from endometriosis patients increased cellular immunity towards endometriosis cell cultures.
Subject(s)
Endometriosis , Killer Cells, Lymphokine-ActivatedABSTRACT
La producción de metabolitos secundarios en cultivos celulares de plantas puede ser de interés para obtener compuestos difíciles de sintetizar o aislar de otras fuentes, lo cual generalmente se relaciona con un alto valor económico, aunque también puede ser útil para ayudar a dilucidar las vías metabólicas involucradas en la síntesis de estos compuestos. En este trabajo se presenta una descripción general de las antocianinas, un grupo de pigmentos de gran importancia para la industria, complementada con la referencia de los trabajos científicos recientes que se han publicado sobre la producción in vitro de las mismas. Con relación a esto último, se hace una descripción del efecto de cambios en las condiciones de cultivo, de la adición de precursores, del uso de reguladores de crecimiento, así como de la utilización de inductores y factores de estrés sobre la producción de estos compuestos. Finalmente, se hace mención al uso de raíces en cabellera, en inglés hairy roots, obtenidas mediante el uso de Agrobacterium rhizogenes, para la producción de estos compuestos.
The production of secondary metabolites in plant cell cultures may be of interest for obtaining compounds that are difficult to synthesize or isolate from other sources, which is usually associated with high economic value of the substances, but may also be useful to help elucidating the metabolic pathways involved in the synthesis of such compounds. This paper presents a general description of anthocyanins, a group of pigments of great importance to the industry, complemented by referring the scientific papers that have been recently published on their in vitro production. Regarding the latter, a description of the effect of changes in growing conditions, of the addition of precursors, of the use of growth regulators, and of the utilization of elicitors and stressors on the production of these compounds, is done. Finally, this review mentions the use of hairy roots obtained by the use of Agrobacterium rhizogenes for the production of these compounds.
ABSTRACT
BACKGROUND: Dengue virus (DENV) infection is increasing in prevalence and severity globally. The severity of dengue is influenced by several factors including the immune response, viral and host genetic factors. METHOD: The DENV serotypes were determined in 770 serum samples from dengue immunoglobulin (Ig) M antibody positive (n = 469), dengue IgM negative (n = 185) and dengue antibody negative (n = 116) patients with suspected dengue who presented during (n = 150) or after (n = 620) the acute phase of illness during 2003-2007. Dengue antibodies were detected by enzyme-linked immunosorbent assays and DENV RNA by reverse transcriptase-polymerase chain reaction (RT-PCR) performed on serum and cell culture supernatants of C6/36 mosquito cells inoculated with acute phase serum (n = 150). RESULTS: Based on serological profiles, 41% of acute phase sera and 66% of post acute sera were from patients with current primary or secondary dengue, while 41% and 35% of acute and post-acute phase sera, respectively, were from patients with secondary dengue or past exposure only. Dengue virus RNA was found in 20/770 samples (2.6%). Only 1.5% (9/620) of sera collected after the acute phase of illness tested positive for DENV RNA compared with 2.6% (4/150) of sera collected during the acute phase and 7.3% of cell culture supernatants inoculated with acute phase serum (11/150, p = 0.001). All four serotypes including DENV-1 (3/20, 15%), DENV-2 (7/20, 35%), DENV-3 (3/20, 15%) and DENV-4 (7/20, 35%) were identified over the five-year period. These results also showed that DENV- 1, 2 and 4 were present during 2007 and that DENV-2 and DENV- 4 were the likely causative viruses of the 2007-2008 dengue outbreak in Jamaica. The three strains of DENV-3 were isolated from infants less than three years of age with primary infection during 2006. CONCLUSION: This study highlights the increasing threat of dengue and severe dengue disease to the Jamaican population. Preventative measures including laboratory surveillance and vector control should be strictly maintained at the highest level.
ANTECEDENTES: La infección por virus del dengue (DENV) está creciendo en prevalencia y severidad a nivel global. La severidad del dengue está influida por varios factores, incluyendo la respuesta inmunológica así como factores virales y genéticos del huésped. MÉTODO: Los serotipos del DENV fueron determinados en 770 muestras de suero de pacientes positivos al anticuerpo inmunoglobulina M (Ig) contra el dengue (n = 469), negativos a la IgM contra el dengue (n =185), y negativos al anticuerpo contra el dengue (n =116). Estos pacientes, con sospecha de dengue, se presentaron durante (n = 150) o después (n = 620) de la fase aguda de la enfermedad en el período de 2003-2007. Los anticuerpos contra el dengue fueron detectados mediante ensayos inmunoabsorbentes ligados a enzimas (ELISA) y DENV ARN mediante reacción en cadena de la polimerasa con transcriptasa inversa (RT-PCR) realizados sobre suero y sobrenadantes del cultivo de células C6/36 de mosquitos, inoculadas con suero de la fase aguda (n =150). RESULTADOS: De acuerdo con los perfiles serológicos, 41% de los sueros de la fase aguda y 66% de los sueros de la fase post-aguda, provenían de pacientes con dengue primario o secundario corriente, mientras que el 41% y el 23% de los sueros de las fases agudas y post-agudas, provenían de pacientes con dengue secundario o exposición pasada solamente. Se halló ARN del virus del dengue en 20/770 muestras (2.6%). Sólo el 1.5% (9/620) de los sueros recogidos tas la fase aguda de la enfermedad resultaron positivos al ARN del DENV, en comparación con 2.6% (4/150) de los sueros recogidos durante la fase aguda y 7.3% de los sobrenadantes del cultivo celular inoculados con suero de la fase aguda (11/150, p = 0.001). Los cuatro serotipos incluyendo DENV-1 (3/20, 15%), DENV-2 (7/20, 35%), DENV-3 (3/20, 15%) y DENV-4 (7/20, 35%) fueron identificados en un período de cinco años. Estos resultados también mostraron que DENV-1, 2 y 4 estuvieron presentes durante 2007 y que DENV- 2 y DENV-4 fueron probablemente los virus causantes del brote de dengue en 2007-2008 en Jamaica. Las tres cepas de DENV-3 fueron aisladas de infantes menores de tres años de edad con infección primaria, durante 2006. CONCLUSIÓN: Este estudio señala la creciente amenaza del dengue y el carácter severo de esta enfermedad para la población jamaicana. Medidas preventivas, incluyendo la vigilancia de laboratorios y el control de vectores, deben mantenerse al más alto nivel.
Subject(s)
Adolescent , Adult , Child, Preschool , Female , Humans , Infant , Male , Middle Aged , Young Adult , Dengue Virus/classification , Dengue/virology , Serotyping , Antibodies, Viral/blood , Dengue Virus/genetics , Immunoglobulin G/blood , Immunoglobulin M/blood , Jamaica , RNA, Viral/blood , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Our objective was to determine the presence of chromosomal abnormalities in primary cultures of ovarian surface epithelial cells in women of different ages with no history of cancer. Throughout conventional cytogenetic techniques, we analyzed chromosome spreads of cultured ovarian epithelial cells from 10 donors who were 50 or more years old (B) and 16 controls between 20 and 49 years old (A), belonging to the mestizo population in Bogota DC, Colombia. Of the 26 cultures that were analyzed in passage 1, 61.5% had an abnormal chromosome complement (62.5% in A, and 60% in B). Abnormalities included polyploidies, endoduplications and monosomies. Deletions in chromosomes 3 and 11 were found in just one metaphase. None of the samples showed weaknesses or breakpoints. After transforming and applying the exact students t-test for variance heterogeneity, we found significant differences in the frequency of metaphases, that were higher in A than in B (p=0.05), and in the frequency of polyploidies, which were higher in B than in A (p=0.044). Through the application of the Mann-Whitney test, we determined that the frequency of endoduplications was higher in A than in B (p=0.126), without reaching significant differences. There were no significant differences in the frequency of monosomies. The level of significance was set at p £ 0.05. Taking into account that polyploidization is a marker of chromosomal instability and that the risk of cancer arising from the ovarian surface epithelium augments substantially after menopause, the increase in the frequency of age-associated polyploidies could be used as a predictor of ovarian cancer in women from an ethnically homogeneous population as the mestizo one in Bogota DC.
El objetivo del presente trabajo fue determinar la presencia de anormalidades cromosómicas en cultivos primarios de células del epitelio superficial ovárico en mujeres de diferentes edades, sin antecedentes de cáncer. Mediante técnicas de citogenética convencional fueron analizados extendidos de células epiteliales ováricas histológicamente normales, provenientes de cultivos primarios de 10 donantes de 50 o más años (B) y de 16 donantes entre 20 y 49 años que se utilizaron como grupo control (A), pertenecientes a la población mestiza de Bogotá DC, Colombia. De 26 cultivos examinados en pase 1, 61,5% presentó complemento cromosómico anormal, 62,5% en A y 60% en B. Las anomalías numéricas halladas, todas en mosaico, incluyeron poliploidías, endoduplicaciones y monosomías. En una única célula en metafase de un cultivo, se presentaron deleciones en los cromosomas 3 y 11. Ninguna muestra presentó fragilidades o roturas. Previa aplicación de transformaciones, con la prueba exacta t-student para varianzas heterogéneas, se encontraron diferencias significativas en la frecuencia de células con metafase normal, mayor en A que en B (p=0,05) y en la de poliploidías, mayor en B que en A (p=0,044). Con la prueba exacta de Mann-Whitney se determinó que la frecuencia de endoduplicaciones en A fue mayor que en B (p=0,126), sin alcanzar diferencias significativas y que no hubo diferencias significativas en la frecuencia de monosomías. El nivel de significación fue p £ 0,05. Si se tiene en cuenta que la poliploidización es un marcador de inestabilidad cromosómica y, que además, el riesgo de aparición de cáncer derivado del epitelio superficial del ovario aumenta sustancialmente después de la menopausia, el incremento en la frecuencia de poliploidías asociado con la edad podría ser utilizado como predictor de cáncer ovárico en mujeres de una población étnicamente homogénea como la población mestiza de Bogotá DC.
Subject(s)
Aged , Female , Humans , Middle Aged , Chromosome Aberrations , Epithelial Cells/ultrastructure , Ovary/cytology , Age Factors , Aneuploidy , Cell Transformation, Neoplastic/genetics , Cells, Cultured/ultrastructure , Disease Susceptibility , Karyotyping , Metaphase , Mitotic Index , Ovarian Neoplasms/genetics , PostmenopauseABSTRACT
Antecedentes. En general, los pacientes alérgicos, después de un año de inmunoterapia (IT), muestran mejoría en la frecuencia e intensidad de los síntomas. Objetivo. Demostrar que la IT tiene efectos moduladores sobre parámetros clínicos, celulares y moleculares de alergia de acuerdo con las edades. Metodología. Se incluyeron 81 pacientes alérgicos asmáticos: 28 infantes, 36 niños, 17 adultos y 27 controles. En los niños y jóvenes se evaluó el pico flujo espiratorio (PEF), reactividad cutánea, se registró eosinofilia sanguínea y de exudado de mucosa nasal, liberación de enzima ß-hexosaminidase (ß-H) de basófilos, proliferación de PBMC estimuladas con alérgeno específico o PHA; se cuantificaron las concentraciones de IgE, IL-13 y sCD23 en sueros y de IFN-γ en sobrenadantes de cultivos de PBMC. Resultados. Niños y adultos sin IT empeoraron sus reducidos valores de PEF. Niños y adultos después de IT mostraron incremento en los volúmenes de PEF (p<0,0001). Todos los grupos con IT redujeron la reactividad cutánea (p<0,0001, p<0,0001 y p<0,02) y la liberación de B-H de basófilos desafiados con el alérgeno específico (p<0,0001, p<0,0001 y p<0,003). Niños y adultos con IT observaron disminución en los niveles de IgE, ambos p<0,05, y redujeron los niveles de células eosinófilos nasales (p<0,0001 y p<0,01). Solamente los niños tratados con IT redujeron los niveles de células eosinófilas sanguíneas (p<0,0001). Las PBMC de infantes y niños con IT disminuyeron los índices de proliferación desafiadas con el alérgeno específico, ambos p<0,05. Los pacientes alérgicos observaron menores niveles de IFN-γ (p<0,02) y más altos niveles de IL-13 (p<0,05) y sCD23 (p<0,001) que los hallados en controles. Conclusiones. La IT demostró efectividad terapéutica en pacientes asmáticos y cambios en parámetros clínicos celulares y moleculares. Algunos de esos parámetros fueron indicadores de la evolución de la enfermedad y ellos fueron diferentes de acuerdo con las edades(AU)
Background: Most allergic patients, after a year of specific immunotherapy (SIT), show improvement in the frequency and intensity of the symptoms. Objective. The objective was to demonstrate that specific immunotherapy has modulating effects on clinical, cellular and molecular parameters, of allergy. Methods. The study included 81 asthmatic allergic patients and 27 healthy controls from 2 to 50 years of age. Peak expiratory flow, skin reactivity, blood and nasal smear eosinophilia was conducted. ß- hexosaminidase enzyme (B-H) release assays and Peripheral Blood Mononuclear Cells cultures stimulated with specific allergens, were realized. Total IgE, sCD23 and IL-13 were measured in serum and IFN- γ, in culture supernatants. Results. In all the cases a reduction in the frequency and intensity of the asthma attacks and a decrease in medical consultations and antiallergic drugs consumption were observed. The patients without SIT diminished low PEF values. Children and adult people with SIT increased the volumes of peak expiratory flow, (p<0.0001). Skin reactivity was reduced in all the SIT groups'. Skin reactivity were p < 0.0001, p<0.0001 y p<0.02) respectively, and B-H released were p<0.0001, p<0.0001 y p<0.003) to each group. The infants without SIT increased IgE levels. Nasal smear eosinophilia fell from children and adults, p < 0.0001. In Infants and children with SIT, the cellular expansion by the allergen was reduced (p<0.05). In allergic patients IFN-γ values were lower than controls, (p < 0.02). IFN -γ synthesis did not vary after a year of allergen -specific immunotherapy. Allergic patients observed higher serum levels of IL-13 (p<0.05) and sCD23 (p<0.001) than controls. Conclusions. SIT after a year demonstrated therapeutic effectiveness in asthmatic patients with changes in multiple parameters: clinical, cellular and molecular. (AU)