Caracterização de células-tronco do tecido adiposo humano

PURPOSE: There is a growing scientific interest in the plasticity and therapeutic potential of adipose-derived stem cells (ASCs), which are multipotent and abundant in adipose tissue and can differentiate in vitro into multiple lineages, including adipocytes, chondrocytes, osteoblasts, neural cells, endothelial cells and cardiomyocytes. The aim of this study was to isolate, cultivate and identify ASCs. METHODS: Human adipose precursor cells were obtained from subcutaneous abdominal tissue. Recently dispersed cells were separated by density centrifugation gradient, cultured and then analyzed. RESULTS: Human ASCs were able to replicate in our culture conditions. The cells maintained their phenotypes throughout the studied period on different passages confirming they suitability for in vitro cultivation. We also induced their adipogenic, osteogenic and chondrogenic differentiation, verifying their mesenchymal stem cells potentiality in vitro. Flow cytometry results showed that these cells expressed CD73, CD90 and CD105, (mesenchymal stem-cells markers), contrasting with the lack of expression of CD16, CD34 and CD45 (hematopoietic cells markers). CONCLUSION: It was possible to isolate human adipose-derived stem cells by in vitro cultivation without adipogenic induction, maintaining their functional integrity and high proliferation levels. The cells demonstrated adipogenic, osteogenic and chondrogenic differentiation potential in vitro.OBJETIVO: Há um interesse científico crescente na plasticidade e potencial terapêutico das células-tronco do tecido adiposo humano, células multipotentes e abundantes no tecido adiposo que podem se diferenciar in vitro em múltiplas linhagens celulares, incluindo adipócitos, condrócitos, osteoblastos, células neurais, endoteliais e cardiomiócitos. O objetivo deste estudo foi isolar, cultivar e identificar células-tronco do tecido adiposo humano. MÉTODOS: Células precursoras humanas do tecido adiposo foram obtidas de tecido abdominal subcutâneo. As células recém-dispersas foram separadas por gradiente de centrifugação por densidade, cultivadas e então analisadas. RESULTADOS: As células-tronco do tecido adiposo humano foram capazes de se replicar nas nossas condições de cultivo e mantiveram seu fenótipo em diferentes passagens durante o estudo, confirmando sua adequabilidade para cultivo in vitro. A diferenciação adipogênica, osteogênica e condrogênica também foi induzida, confirmando seu potencial de células-tronco mesenquimais in vitro. Os resultados de citometria de fluxo evidenciaram a expressão dos marcadores de células-tronco mesenquimais CD73, CD90 e CD105, contrastando com a falta de expressão dos marcadores de células hematopoiéticas CD16, CD34 e CD45. CONCLUSÃO: Foi possível isolar células-tronco do tecido adiposo humano por cultivo in vitro sem indução adipogênica, mantendo sua integridade funcional e altos níveis de proliferação. As células demonstraram potencial de diferenciação adipogênico, osteogênico e condrogênico in vitro.UNIFESP Chairwoman Plastic Surgery Division Head of Department of SurgeryUNIFESP, Chairwoman Plastic Surgery Division Head of Department of SurgerySciEL

Adipose-derived stem cells (ADSC) in the viability of a random pattern dorsal skin flap in rats

PURPOSE:To evaluate the viability of random pattern dorsal skin flaps in rats after injection of adipose-derived stem cells (ADSC).METHODS: Thirty five adult male Wistar EPM rats (weight 250-300 g) were distributed, at random, in two groups. I- Control (flap elevation with injection of saline solution) with fifteen animals and II- Experimental (flap elevation with injection of ADSC ) with fifteen animal. The ADSC were isolated from others five adult male rats. A dorsal skin flap measuring 10x4 cm was raised and a plastic barrier was placed between the flap and its bed in both groups and the injection (cells or saline solution) were perfomed immediately after the surgery. The percentage of flap necrosis was measured on the seventh postoperative day.RESULTS:The ADSC were able to replicate in our culture conditions. We also induced their adipogenic, osteogenic and chondrogenic differentiation to verify their mesenchymal stem cells potentiality in vitro. The results were statistically significant showing that the ADSC decreased the area of necrosis (p<0.05).CONCLUSIONS:The cells demonstrated adipogenic, osteogenic and chondrogenic differentiation potential in vitro. The administration of adipose-derived stem cells was effective to increase the viability of the random random pattern dorsal skin flaps in rats.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Federal University of São Paulo Department of SurgeryUniversidade Estadual de Santa Cruz Department of SurgeryUNIFESPUNIFESP, Department of SurgeryUNIFESPCNPq: 312356/2009-9SciEL

Development of experimental in vitro burn model

PURPOSE:To propose an experimental burn model in NIH-3T3 cell line.METHODS: Induction of thermal injury in cultures of mouse fibroblast - NIH-3T3- cell line and determination of cell viability by MTT and imunofluorescence.RESULTS: The heating of the Petri dish increased proportionally to the temperature of the base and the time of exposure to microwave. In this in vitro burn model, using the cell line NIH-3T3 was observed drastic cellular injury with significant changes in cell viability and activity. It showed drastically modified cell morphology with altered membrane, cytoskeleton and nucleus, and low cellularity compared to the control group.CONCLUSION: The burn model in vitro using the cell line NIH-3T3 was reproductive and efficient. This burn model was possible to determine significant changes in cell activity and decreased viability, with drastic change in morphology, cell lysis and death.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Federal de São Paulo (UNIFESP)Universidade Estadual de Santa Cruz Department of Biological SciencesUNIFESPUNIFESPSciEL

Evaluation of antimicrobial and antitumoral activity of Garcinia mangostana L. (mangosteen) grown in Southeast Brazil

PURPOSE:To characterize the anatomy of the fruit and leaf and the presence of phytocompounds. To evaluate the antitumor and antimicrobial activity of ethanolic extract of Garcinia mangostana L. (mangosteen) cultivated in southeastern Brazil.METHODS:Anatomical characterization and histochemical reactions were performed for structural identification and the presence of phytocompounds. Preparation of ethanolic extract of the fruit, leaf and resin of mangosteen. Culture B16-F10 melanoma cells for treatment with mangosteen ethanolic extract to determine cell viability by MTT and genotoxic effect by comet assay. Evaluation by antimicrobial activity against Staphylococcus aureus and Escherichia coli by agar diffusion test and by determination of Minimum Inhibitory Concentration (MIC).RESULTS:Our results showed many secretory canals in resin fruit and leaf; identifying lipids, starch, lignin and phenolic compounds. The leaf extract induced genotoxicity and apoptosis in B16-F10 cells, since the fragmentation of DNA in the comet assay. The ethanolic extract of mangosteen obtained in the resin, leaf and fruit showed antimicrobial activity against Staphylococcus aureus and Escherichia coli with a MIC at 0.1 mg/mL.CONCLUSION: In conclusion, we have demonstrated both antimicrobial and antitumor activity of ethanol extract of mangosteen emphasizing its therapeutic potential in infectious diseases and in cancer, such as melanoma.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Bahia Research Foundation (FAPESB)Universidade Estadual de Santa Cruz Department of Biological SciencesUniversidade Estadual de Santa Cruz Departament of Agricultural and Environmental SciencesUNIFESPUNIFESPSciEL

Antioxidant and cytotoxic activity of diplotropis incexis rizzini & a. Mattos seed oil in b16-f10 murine melanoma / Actividade antioxidantes e citóxicos de diplotropis incexis rizzini & a. Mattos semente de petróleo em b16-f10 murina melanoma

Introduction: Diplotropis incexis is popularly known as sucupira. To extract its oil, which is used for the treatment of eczema, inflammation, skin patches, rheumatism, and to treat hemorrhages, it is necessary to breakdown and decoct the seeds. The aim was to evaluate the antioxidant activity and cytotoxic effect of Diplotropis incexis oil (DIO) in B16-F10 murine melanoma cells. Materials and methods: DIO was obtained from the breakdown of seeds, Soxhlet extraction, and rotary evaporation. Antioxidant activity was measured by radical scavenging. Quantification of phenolic groups was performed using a standard curve of gallic acid and quercetin. DIO toxicity was determined between 0.01 – 100 µg/mL by photometry (MTT) and cytometry. Finally, cell morphology was examined by confocal microscopy. Results: The DIO showed antioxidant potential, being about 335x less potent than the gallic acid control. 109 mg of total phenols and 381 mg of flavonoids were found for every 100g of crude oil. The DIO showed high cytotoxicity against the B16-F10 line, presenting IC50 = 10 µg/mL for cells treated for 48 hours. The cells underwent drastic morphological changes, presenting changes in membrane structure and drastic changes in the cytoskeleton, due to F-actin filament disarrangement. Conclusions: The first reports of DIO activity showed that it has moderate antioxidant activity with the presence of phenolic groups in its composition. In addition to having cytotoxic activity in the B16-F10 cell lines, it is able to promote decreased cell viability and morphological changes depending on concentration and time spent in contact with oil