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1.
Braz. j. med. biol. res ; 53(7): e9230, 2020. graf
Article in English | LILACS, ColecionaSUS | ID: biblio-1132534

ABSTRACT

As a top leading cause of cancer death in many countries, colorectal cancer (CRC) has drawn increasing attention to the study of the pathological mechanism. According to the "cancer stem cell hypothesis", malignancies originate from a small fraction of cancer cells that show self-renewal properties to initiate and sustain tumor growth and tumor metastasis. Therefore, these cancer stem cells (CSC) probably play important roles in tumor recurrence, metastasis, and drug resistance. Previous research reported that lysine-specific histone demethylase 1 (LSD1) maintains cancer stemness through up-regulating stemness markers SOX2 and OCT4. CD133 is believed to be the most robust surface marker for CRC stem cells, however the regulatory effect of LSD1 on stemness of CD133+ CRC has never been reported. In this study, our objectives included: 1) to isolate pure CD133+ and CD133− cells from SW620 cell line; 2) to investigate the effect of LSD1 on the characteristics of CD133+ stem cancer cells by knocking down the target gene. Results suggested that the SW620 cell line had both CD133+ and CD133− subsets. The CD133+ subset exhibited more CSC-like characteristics compared with the CD133− subset with higher viability, colony formation rate, migration and invasion rate, resistance to anti-cancer drugs, and apoptosis in vitro. The CD133+ also induced faster tumor formation and larger tumors in vivo. In the LSD1-knockdown CD133+ cells, the CSC-like characteristics had been all weakened. We conclude that LSD1 was important for CSCs to maintain their "stemness" features, which could be a potential therapeutic target of CRC.


Subject(s)
Humans , Animals , Rats , Neoplastic Stem Cells/drug effects , Colorectal Neoplasms/pathology , Cell Movement/drug effects , Apoptosis/drug effects , Cell Proliferation/drug effects , Histone Demethylases/pharmacology , Neoplastic Stem Cells/metabolism , Gene Expression Regulation, Neoplastic , Blotting, Western , Colony-Forming Units Assay , Cell Line, Tumor
2.
Biol. Res ; 51: 53, 2018. graf
Article in English | LILACS | ID: biblio-1011397

ABSTRACT

BACKGROUND: Deubiquitination is a posttranslational protein modification prevalent in mammalian cells. Deubiquitinases regulate the functions of the target protein by removing its ubiquitin chain. In this study, the effects of the deubiquitinase USP38's functions on the LSD1 protein and on cell physiology were investigated. MATERIALS AND METHODS: Western blotting, real-time quantitative PCR, immunoprecipitation, denaturing immunoprecipitation and luciferase reporter assays were used to analyze the protein stability, protein interactions and changes in the ubiquitin chain. Cell proliferation assays, colony formation assays, drug treatments and western blotting were used to explore the functions of USP38 in cells. RESULTS: The deubiquitinase USP38 stabilizes protein LSD1 in cells by binding LSD1 and cleaving its ubiquitin chain to prevent the degradation of LSD1 by the intracellular proteasome. USP38 enhances the ability of LSD1 to activate signaling pathways and hence promotes cellular abilities of proliferation and colony formation through interacting with LSD1. Furthermore, USP38 enhances the drug tolerance of human colon cancer cells. CONCLUSIONS: USP38 is an LSD1-specific deubiquitinase that affects cellular physiology through interacting with LSD1.


Subject(s)
Humans , Cells, Cultured/drug effects , Apoptosis/drug effects , Cell Proliferation/drug effects , Histone Demethylases/pharmacology , Ubiquitin-Specific Proteases/pharmacology , Signal Transduction , Blotting, Western , Colony-Forming Units Assay , Immunoprecipitation , Real-Time Polymerase Chain Reaction
3.
Biol. Res ; 51: 13, 2018. graf
Article in English | LILACS | ID: biblio-950899

ABSTRACT

BACKGROUND: Neuroblastoma (NB) represents the most common extracranial solid tumor in children. Accumulating evidence shows that microRNAs (miRs) play an important role in the carcinogenesis of NB. Here, we investigated the biological function of miR-1247 in NB in vitro. METHODS/RESULTS: We found miR-1247 was downregulated in NB tissues and cells using quantitative PCR analysis. Gain- and loss-of-function studies demonstrated that miR-1247 significantly suppressed cell proliferation and induced cell cycle G0/G1 phase arrest and cell apoptosis of NB cells in vitro by using MTT, colony formation assay and Flow cytometry analysis. Luciferase assay suggested ZNF346 was the target of miR-1247 and its expression could be down-regulated by miR-1247 overexpression using Western blotting. Furthermore, downregulation of ZNF346 by siRNA performed similar effects with overexpression of miR-1247 in NB cells. CONCLUSIONS: Our findings suggested miR-1247 directly targeted to repress ZNF346 expression, thus suppressing the progression of NB, which might be a novel therapeutic target against NB.


Subject(s)
Humans , Male , Female , RNA-Binding Proteins/metabolism , MicroRNAs/metabolism , DNA-Binding Proteins/metabolism , Neuroblastoma/metabolism , Phenotype , Time Factors , Tumor Cells, Cultured , Down-Regulation , Gene Expression Regulation, Neoplastic , Child, Preschool , RNA-Binding Proteins/genetics , Colony-Forming Units Assay , MicroRNAs/genetics , Cell Proliferation/genetics , DNA-Binding Proteins/genetics , Real-Time Polymerase Chain Reaction , Flow Cytometry , Neuroblastoma/genetics , Neuroblastoma/pathology
4.
Braz. j. med. biol. res ; 50(12): e6087, 2017. graf
Article in English | LILACS | ID: biblio-888963

ABSTRACT

Using an iron overload mouse model, we explored the protective effect of deferasirox (DFX) and N-acetyl-L-cysteine (NAC) on injured bone marrow hematopoietic stem/progenitor cells (HSPC) induced by iron overload. Mice were intraperitoneally injected with 25 mg iron dextran every 3 days for 4 weeks to establish an iron overload (Fe) model. DFX or NAC were co-administered with iron dextran in two groups of mice (Fe+DFX and Fe+NAC), and the function of HSPCs was then examined. Iron overload markedly decreased the number of murine HSPCs in bone marrow. Subsequent colony-forming cell assays showed that iron overload also decreased the colony forming capacity of HSPCs, the effect of which could be reversed by DFX and NAC. The bone marrow hematopoiesis damage caused by iron overload could be alleviated by DFX and NAC.


Subject(s)
Animals , Male , Acetylcysteine/pharmacology , Triazoles/pharmacology , Benzoates/pharmacology , Hematopoietic Stem Cells/drug effects , Iron Chelating Agents/pharmacology , Free Radical Scavengers/pharmacology , Iron Overload/prevention & control , Protective Agents/pharmacology , Reference Values , Time Factors , Reproducibility of Results , Treatment Outcome , Reactive Oxygen Species/analysis , Colony-Forming Units Assay , Disease Models, Animal , Flow Cytometry , Hematopoiesis/drug effects , Mice, Inbred C57BL
5.
Article in Portuguese | LILACS | ID: biblio-964823

ABSTRACT

Introdução: As células-tronco mesenquimais (CTM) têm despertado interesse de vários grupos de pesquisa em função do grande potencial de aplicabilidade em terapia celular e medicina regenerativa. Nesse contexto, o tecido adiposo vem recebendo grande destaque como importante fonte para obtenção de CTM. Os protocolos utilizados atualmente para o isolamento das células-tronco derivadas do tecido adiposo (ADSC) empregam, de forma geral, o método de digestão enzimática com colagenase extraída de bactéria (Clostridium histolyticun), que pode conter contaminantes, como endotoxinas e outros peptídeos que, eventualmente, poderão resultar em reações adversas nos procedimentos de terapia celular em pacientes humanos. Objetivo: Pretendeu-se no presente estudo adequar e propor uma nova abordagem empregando a metodologia de dissociação mecânica para isolamento de CTM derivadas de tecido adiposo de ratos. Métodos: As células cultivadas foram analisadas quanto ao potencial de adesão, proliferação e tempo de duplicação celular, por meio de uma curva de crescimento. As células isoladas e cultivadas a partir do tecido adiposo foram também analisadas quanto ao potencial de diferenciação in vitro nas linhagens adipogênica, condrogênica e osteogênica. Resultados: Os resultados mostraram que o tempo de duplicação (velocidade de crescimento) da população celular isolada por dissociação mecânica é mais expressivo quando comparado com a técnica de digestão enzimática. As células isoladas do tecido adiposo apresentaram potencial de diferenciação nas linhagens osteogênica, condrogênica e adipogênica. Conclusão: Os resultados obtidos permitem concluir que a metodologia de dissociação mecânica apresenta-se como uma alternativa viável, de baixo custo e, como tal, extremamente promissora no sentido de permitir que a colagenase de origem bacteriana (Clostridium histolyticun) torne-se um componente prescindível para isolamento e cultivo de células provenientes do tecido adiposo.


Background: Mesenchymal stem cells (MSCs) have attracted interest of several research groups due to the large potential applicability in cell therapy and regenerative medicine. In this context, adipose tissue has received high profile as an important source in order to obtain MSC. The protocols currently suggested for the isolation and culture of adipose- -derived stem cells (ADSC) utilize, in general, the enzymatic digestion method with bacterial collagenase (Clostridium histolyticun) which may contain contaminants such as endotoxin and other peptides that eventually may result in adverse reactions in the cell therapy procedures in human patients. Objective: In this context, it was intended in this study to propose a new methodological approach of mechanical dissociation for isolating and culture of adipose-derived mesenchymal stem cells. Methods: The cultured cells were analyzed for potential adhesion, proliferation and cell doubling time, through a growth curve lineages The cells were also analyzed according to potential for differentiation in adipogenic, chondrogenic and osteogenic lineages. Results: The results showed that the doubling time of the cell population isolated by mechanical dissociation is faster when compared to the enzymatic digestion technique. The isolated cells from adipose tissues howed potential for differentiation in cell lineages osteogenic, adipogenic and chondrogenic. Conclusion: The obtained results allow us to conclude that the methodology of mechanical dissociation, presented in this paper, is a viable, low cost and therefore an extremely promising alternative in order to permit that the bacterial collagenase, from Clostridium histolyticun, become a dispensable component for isolation and cultivation of adipose-derived stem cells.


Subject(s)
Animals , Rats , Stem Cells , Adipose Tissue , Collagenases/isolation & purification , Colony-Forming Units Assay/standards , Rats, Wistar
6.
Acta cir. bras ; 31(1): 59-66, Jan. 2016. graf
Article in English | LILACS | ID: lil-771849

ABSTRACT

PURPOSE: To describe a new technique for isolation of a mesenchymal stem cells (MSCs) population from the olfactory mucosa in rabbits. METHODS: Olfactory stem cells (OSCs) were retrieved from under the cribriform plate of the Ethmoid bone. Several assays were accomplished to characterize the cell population and attest its viability in vitro. The cells were submitted to flow cytometry with the antibodies CD34, CD45, CD73, CD79, CD90 and CD105 and also they were induced to differentiate in three lineages. Functional evaluation involved analysis of in vitro growth behavior, colony forming unit like fibroblasts (CFU-f) and cryopreservation response. Further transduction with Green Fluorescent Protein (GFP) was also performed. RESULTS: The OSCs showed mesenchymal features, as positive response to CD34, CD73 and CD90 antibodies and plasticity. Additionally, these cells have high proliferated rate, and they could be cultured through many passages and kept the ability to proliferate and differentiate after cryopreservation. The positive response to the transduction signalizes the possibility of cellular tracking in vivo. This is a desirable feature in case those cells are used for pre-clinical trials. CONCLUSION: The cells harvested were mesenchymal stem cells and the technique described is therefore efficient for rabbit olfactory stem cells isolation.


Subject(s)
Animals , Rabbits , Cell Separation/methods , Mesenchymal Stem Cells/cytology , Olfactory Mucosa/cytology , /physiology , /physiology , Thy-1 Antigens/physiology , Cells, Cultured , Colony-Forming Units Assay , Cryopreservation , Cell Differentiation/physiology , Cell Plasticity/physiology , Cell Proliferation/physiology , Ethmoid Bone/cytology , Flow Cytometry , Green Fluorescent Proteins/metabolism , Olfactory Mucosa/growth & development
7.
Article in English | WPRIM | ID: wpr-287163

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the effects of panaxadiol saponins component (PDS-C) isolated from total saponins of panax ginseng on proliferation, differentiation and corresponding gene expression profile of megakaryocytes.</p><p><b>METHODS</b>Bone marrow culture of colony forming assay of megakaryocytic progenitor cells (CFU-MK) was observed for the promoting proliferation mediated by PDS-C, and differentiation of megakaryocytic blasts caused by PDS-C was analyzed with flow cytometry in CHRF-288 and Meg-01 cells, as well as proliferation, differentiation-related genes expression profile and protein expression levels were detected by human gene expression microarray and western blot.</p><p><b>RESULTS</b>In response to PDS-C 10, 20 and 50 mg/L, CFU-MK from 10 human bone marrow samples was increased by 28.9%±2.7%, 41.0%±3.2% and 40.5%±2.6% over untreated control, respectively (P <0.01, each). Flow cytometry analysis showed that PDS-C treated CHRF-288 cells and Meg-01 cells significantly increased in CD42b, CD41, TSP and CD36 positive ratio, respectively. PDS-C induced 29 genes up-regulated more than two-fold commonly in both cells detected by human expression microarray representing 4000 known genes. The protein expression levels of ZNF91, c-Fos, BTF3a, GATA-1, RGS2, NDRG2 and RUNX1 were increased with western blot in correspond to microarray results.</p><p><b>CONCLUSION</b>PDS-C as an effective component for hematopoiesis, play the role to enhance proliferation and differentiation of megakaryocytes, also up-regulated expression of proliferation, differentiation-related genes and proteins in vitro.</p>


Subject(s)
Blotting, Western , Bone Marrow Cells , Cell Biology , Cell Differentiation , Cell Proliferation , Cells, Cultured , Colony-Forming Units Assay , Drugs, Chinese Herbal , Pharmacology , Flow Cytometry , Gene Expression Profiling , Ginsenosides , Pharmacology , Humans , Megakaryocytes , Cell Biology , Metabolism , Patents as Topic , Saponins , Pharmacology , Stem Cells , Cell Biology , Transcription Factors , Metabolism , Up-Regulation , Genetics
8.
Article in English | WPRIM | ID: wpr-164159

ABSTRACT

Adipose tissue stem cells (ADSCs) would be an attractive autologous cell source. However, ADSCs require invasive procedures, and has potential complications. Recently, urine stem cells (USCs) have been proposed as an alternative stem cell source. In this study, we compared USCs and ADSCs collected from the same patients on stem cell characteristics and capacity to differentiate into various cell lineages to provide a useful guideline for selecting the appropriate type of cell source for use in clinical application. The urine samples were collected via urethral catheterization, and adipose tissue was obtained from subcutaneous fat tissue during elective laparoscopic kidney surgery from the same patient (n = 10). Both cells were plated for primary culture. Cell proliferation, colony formation, cell surface markers, immune modulation, chromosome stability and multi-lineage differentiation were analyzed for each USCs and ADSCs at cell passage 3, 5, and 7. USCs showed high cell proliferation rate, enhanced colony forming ability, strong positive for stem cell markers expression, high efficiency for inhibition of immune cell activation compared to ADSCs at cell passage 3, 5, and 7. In chromosome stability analysis, both cells showed normal karyotype through all passages. In analysis of multi-lineage capability, USCs showed higher myogenic, neurogenic, and endogenic differentiation rate, and lower osteogenic, adipogenic, and chondrogenic differentiation rate compared to ADSCs. Therefore, we expect that USC can be an alternative autologous stem cell source for muscle, neuron and endothelial tissue reconstruction instead of ADSCs.


Subject(s)
Adult Stem Cells/cytology , Biomarkers/metabolism , Cell Differentiation , Cell Lineage , Cell Proliferation , Cell Separation , Chromosomal Instability , Colony-Forming Units Assay , Humans , Karyotyping , Multipotent Stem Cells/cytology , Subcutaneous Fat, Abdominal/cytology , Transplantation, Autologous , Urine/cytology
9.
Article in Chinese | WPRIM | ID: wpr-357327

ABSTRACT

<p><b>OBJECTIVE</b>Though the rabbit is one of most widely used experimental animals for medical regenerative research, it remains difficult to culture mesenchymal stem cells (MSC) on a in large scale due to the extremely lower number and hematopoietic cell contamination. This study was aimed to establish a novel protocol to generate rabbit MSC by culturing bone marrow plugs instead of bone marrow cells so as to obtain a large amount of MSC with higher proliferation and self-renewal properties.</p><p><b>METHODS</b>The primary MSC were generated from collagenase digested bone marrow plugs and bone marrow cells, respectively. The surface antigen profile of MSC was analyzed with flow cytometry and the cells were induced to differentiate into osteoblasts and adipocytes. The proliferation capacity of MSC were assessed by CCK-8 method. To test their self-renewal property, the colony forming unit-fibroblast assay was performed. Moreover, the cell yields of passage 1, 2, 3 and 4 were calculated.</p><p><b>RESULTS</b>The bone marrow plug-derived MSC shared the typical fibroblast-like morphology same as bone marrow cells derived MSC. Moreover, the ratio of CD45 positive hematopoietic cells in bone marrow plug-derived MSCs was significantly lower than that of bone marrow cell-derived MSC. The results of multi-differentiation experiments showed that bone marrow-plug-derived MSC exhibited similar multi-potent property to their bone marrow counterparts. In addition, the results of CCK-8 and CFU-F assay demonstrated that bone marrow plug-derived MSC grew more robustly and more CFU-F were formed in the culture plates, which indicated that the cells possessed higher proliferation and self-renewal capacities. Promisingly, a larger amount of cells were harvested via using the new protocol.</p><p><b>CONCLUSION</b>The purity and yields of the bone marrow plug-derived MSC are satisfactory compared with previous rabbit MSC isolation methods. The findings may be helpful for the research of regenerative medicine.</p>


Subject(s)
Adipocytes , Animals , Bone Marrow , Bone Marrow Cells , Cell Differentiation , Cell Separation , Colony-Forming Units Assay , Mesenchymal Stem Cells , Osteoblasts , Rabbits
10.
Braz. dent. j ; 25(5): 447-450, Sep-Oct/2014. graf
Article in English | LILACS | ID: lil-731048

ABSTRACT

The radicular cyst is an inflammatory odontogenic cyst of endodontic origin. Radiographically, the lesion appears as a periapical radiolucent image. This report describes a very rare case of a mixed periapical radiographic image diagnosed as a radicular cyst. A 37-year-old female patient presented a mixed, well-circumscribed image located in the periapical region of the left maxillary central incisor, which presented unsatisfactory endodontic treatment. Microscopic examination revealed a cavity lined by non-keratinized squamous epithelium and extensive calcifications in the cystic lumen and lining epithelium. Diagnosis of radicular cyst with extensive calcifications was established. Endodontic retreatment was performed and no radiographic signs of recurrence were observed 18 months after treatment. Although very rare, a radicular cyst should be considered in the differential diagnosis of a mixed periapical image associated to teeth with pulp necrosis.


O cisto radicular é um cisto odontogênico inflamatório de origem endodôntica. Radiograficamente, a lesão se apresenta como uma imagem radiolúcida periapical. Este relato descreve um caso muito raro de uma imagem radiográfica periapical mista diagnosticada como cisto radicular. Uma paciente de 37 anos de idade, do gênero feminino, apresentava uma imagem mista, bem circunscrita, localizada na região periapical do incisivo central superior esquerdo, que apresentava tratamento endodôntico insatisfatório. Avaliação microscópica revelou uma cavidade revestida por epitélio escamoso não-queratinizado e calcificações extensas na cavidade cística e revestimento epitelial. O diagnóstico de cisto radicular com extensas calcificações foi estabelecido. Retratamento endodôntico foi realizado e não foram observados sinais radiográficos de recorrência da lesão após 18 meses de tratamento. Embora muito raro, um cisto radicular deve ser considerado no diagnóstico diferencial de uma imagem periapical mista associada a dentes com necrose pulpar.


Subject(s)
Animals , Mice , Cellular Senescence/physiology , Genes, ras/genetics , MAP Kinase Signaling System/physiology , Nuclear Proteins , /metabolism , Cell Fractionation , Cells, Cultured , Colony-Forming Units Assay , Cell Cycle/physiology , Enzyme Activation , Embryo, Mammalian/physiology , Fibroblasts/cytology , Fibroblasts/metabolism , MAP Kinase Kinase 1 , Mice, Knockout , Microscopy, Fluorescence , Mitogen-Activated Protein Kinase Kinases/metabolism , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Temperature , /metabolism , ras Proteins/metabolism
11.
Article in English | IMSEAR | ID: sea-154624

ABSTRACT

Endogenous erythroid colony (EEC) syndrome comprise of three cardinal features, i.e. ectrodactyly, ectodermal dysplasia and cleft lip. EEC itself has three different forms. Ectrodactyly (absence of one or more digits) can be present with clefting in the proximal portion of hand or foot known as split hand foot malformation (SHFM) or lobster claw deformity. SHFM can be of four types depending upon the different responsible chromosomal loci. SHFM-4 can be present as pure limb malformation (non-syndromic form). In this article, describes a rare case report of lobster claw deformity patient.


Subject(s)
Aged , Colony-Forming Units Assay , Female , Humans , Limb Deformities, Congenital/epidemiology , Nephropidae/etiology
12.
São Paulo; s.n; s.n; 2014. 158 p. ilus, graf, tab.
Thesis in Portuguese | LILACS | ID: biblio-881898

ABSTRACT

Células tumorais têm sua proliferação e mobilidade modificada por diversos fatores de crescimento, citocinas e mediadores inflamatórios, dentre os quais a amilóide sérica A (SAA). Estudos prévios do nosso grupo mostraram o efeito direto da SAA em processos de proliferação, migração e invasão de células de glioblastoma multiforme (GBM), A172 e T98G. Neste estudo nós complementamos resultados prévios de migração e invasão; avaliamos SAA como possível indutora de moléculas importantes para a invasividade do tumor, como as MMP-2 e -9 e ROS; realizamos ensaio clonogênico com a intenção de investigar uma possível contribuição da rSAA no estágio inicial de desenvolvimento do tumor; avaliamos o impacto da hipóxia na expressão dos diferente genes da SAA; estimulamos as células com indutores hepáticos clássicos da SAA e analisamos a possibilidade destes induzirem os diferentes genes da SAA em células tumorais; avaliamos possíveis receptores e vias de sinalizações envolvidas nos processos de proliferação, migração e invasão. Construímos knockdowns (KDs) dos genes da SAA de fase aguda (SAA1 e 2) e constitutiva (SAA4) e avaliamos a função de cada um deles para a morfologia e para os processos de proliferação, migração e invasão de GBM. Por fim investigamos SAA como possível biomarcadora de gliomas em amostras clínicas. Nossos resultados sugerem que rSAA afetou a atividade das MMP-2 e -9 e a produção de ROS em ambos GBM, mas não se mostrou clonogênica. As citocinas IL-6, TNF-α e IL-1ß, mas não a hipóxia, foram capazes de induzir os diferentes genes da SAA. A adição de rSAA às culturas celulares estimulou a transrição dos diferentes genes da SAA, sugerindo a ativação de mecanismos intracelulares retroalimentados. Efeitos pró-tumorais da rSAA parecem ser viabilizados via RAGE, enquanto efeitos anti-tumorais parecem ser induzidos via TLR-4. Pela primeira vez mostramos que SAA induz aumento de RAGE. KDs da SAA inibiram proliferação, migração e invasão, sugerindo que SAA seja um produto tumoral importante para a manutenção do fenótipo invasivo de GBM. A adição de SAA exógena reverteu grande parte dos efeitos nas células T98G KD, enquanto células A172 KD responderam parcialmente à rSAA. KDs da SAA sugerem a mesma como mantenedora da morfologia das células de GBM. De maneira inédita mostramos que o gene SAA4 até então descrito como um gene constitutivo de função desconhecida é importante para a proliferação, migração e invasão de GBM. Nós especulamos que os efeitos diferenciados induzidos por rSAA nos GBM estejam associados à natureza multiligante da SAA e às diferenças genéticas dos GBM. Pacientes com GBM apresentaram aumento significativo na transcrição e expressão de SAA1 no tecido tumoral, bem como aumento sérico de SAA. A correlação na expressão de SAA1 com moléculas importantes para progressão tumoral, como CXCR4, CXCR7, CD163 e HIF-1α também a identificam como uma proteína associada à malignidade


Tumor cells have their proliferation and migration modified by several growth factors, cytokines and inflammatory mediators, such as serum amyloid A (SAA). Previous studies from our group showed the direct effect of SAA on proliferation, migration and invasion of glioblastoma multiforme (GBM) cells, A172 and T98G. In this study we complemented previous migration and invasion data; evaluated SAA as possible inducer of MMP-2, -9 and ROS; performed clonogenic assay to investigate a possible contribution of rSAA in the early stage of tumor development; evaluated the impact of hypoxia on the expression of different genes of SAA; stimulated the cells with classics inducers of hepatic SAA and analyzed the possibility of these different genes to induce SAA in tumor cells; evaluated possible receptors and signaling pathways involved in proliferation, migration and invasion processes. SAA knockdowns (KDs) were made for acute phase (SAA1 and 2) and constitutive protein (SAA4) and evaluated their role in cell proliferation, migration, morfology and invasion. Finally it was investigated SAA as a possible biomarker of glioma grade in clinical samples. Our results suggest that rSAA affects MMP-2 and -9 activity and ROS production in both GBM, but did not affect clonogenicity. IL-6, TNF-α and IL-1ß, but not hypoxia, were able to induce SAA expression. rSAA addition to cell cultures stimulated transcription of the three different SAA genes, suggesting the activation of intracellular feedback mechanisms. Pro-tumor effects of rSAA seem to occur via RAGE and anti-tumor effects appear to be induced via TLR-4. This was de first time that induction of RAGE triggered by rSAA was shown. Proliferation, migration and invasion were inhibited in SAA KDs, suggesting that SAA is an important tumoral product for the maintenance of the invasive phenotype of GBM. The addition of exogenous SAA largely reversed the effects on SAA KDs T98G cells, whereas SAA KDs A172 cells partially responded to the rSAA. The findings with SAA KDs suggest that SAA affect cell morphology. Another new contribution from our study was that SAA4, a constitutive gene with unknown function, was important for the proliferation, migration and invasion of GBM and it can be induced by rSAA, IL-6, TNF-α and IL-1ß. We speculate that the different effects induced by rSAA in GBM are associated with the affinity of SAA to different receptors and the different genetic backgrounds of GBM. Patients with GBM showed a significant increase in the transcription and expression of SAA1 in tumor tissue as well as increased serum SAA. The correlation between the expression of SAA1 with important molecules for tumor progression, such as CXCR4, CXCR7, CD163 and HIF-1α also identified SAA as a protein associated with malignancy


Subject(s)
Serum Amyloid A Protein/analysis , Glioblastoma/metabolism , Tumor Stem Cell Assay/methods , Cell Hypoxia , Cell Movement , Colony-Forming Units Assay , Cell Proliferation , Gene Knockdown Techniques/methods
13.
Article in Chinese | WPRIM | ID: wpr-349687

ABSTRACT

The aim of this study was to investigate the effect of GW003 on the ability of granulocyte colony forming in vitro of bone marrow cells. The bone marrow samples was collected from normal rhesus, the patients with leukemia in stages of remission and chemotherapy respectively, and the nucleated cells were separated and cultured for 12 days after addition of different concentrations of GW003 or rhG-CSF, or G-CSF mutant. Then the amount of colony-forming unit-granulocyte-macrophage was counted. The results indicated that GW003 could enhance the ability of bone marrow nucleated cells of rhesus to forming CFU-GM in vitro, and its effect was much better than that of rhG-CSF or G-CSF mutant at the same concentration(®). The GW003 showed dose-response relationship to CFU-GM level (r = R(2) = 0.965, P = 0.003, in a certain concentration), the GW003 also could enhance CFU-GM formation of marrow nucleated cells in leukemic patients, especially for patients receiving chemotherapy. The GW003 could relieve the marrow suppression caused by chemotherapy significantly. It is concluded that the GW003 can significantly improve the ability of bone marrow cells to form granulocyte colony in vitro as well as effectively alleviate bone marrow suppression.


Subject(s)
Adult , Animals , Bone Marrow Cells , Cell Line, Tumor , Colony-Forming Units Assay , Female , Granulocyte Colony-Stimulating Factor , Pharmacology , Granulocyte-Macrophage Progenitor Cells , Cell Biology , Granulocytes , Humans , Macaca mulatta
14.
Braz. oral res ; 27(4): 369-375, Jul-Aug/2013. tab, graf
Article in English | LILACS | ID: lil-679221

ABSTRACT

The purpose of this study was to evaluate the influence of the substratum position and the saliva acquired pellicle (AP) on Candida albicans biofilm development. Poly(methylmethacrylate) (PMMA) disks were fabricated and randomly allocated to experimental groups: HNP (disks placed in a horizontal position and uncoated by pellicle), VNP (disks placed in a vertical position and uncoated by pellicle), HCP (disks placed in a horizontal position and coated by pellicle), and VCP (disks placed in a vertical position and coated by pellicle). Disks were placed in a 24-well plate and a suspension of 107 cells/mL of Candida albicans was added to each well for biofilm development. The plates were aerobically incubated at 35°C. The biofilms were evaluated at 1.5 (adhesion time point), 24, 48, 72, and 96 hours. The number of viable cells was quantified in terms of the colony-forming units per milliliter (CFU/mL). Metabolic activity was measured by the XTT assay. The biofilm structure was analyzed by scanning electron microscopy. The data were analyzed by three-way ANOVA followed by Tukey's test, with significance set at 5%. The vertical groups showed less biofilm formation and lower metabolic activity than the horizontal groups (p< 0.05). Significant differences in cell viability and metabolic activity were observed between the adhesion and other time points (p< 0.05), but these variables were not affected by the presence of the pellicle (p > 0.05). It can be concluded that the substratum position influenced biofilm development.


Subject(s)
Biofilms/growth & development , Candida albicans/growth & development , Dental Pellicle/microbiology , Saliva/microbiology , Analysis of Variance , Colony-Forming Units Assay , Microscopy, Electron, Scanning , Polymethyl Methacrylate , Random Allocation , Surface Properties , Time Factors
15.
Braz. j. med. biol. res ; 46(1): 39-51, 11/jan. 2013. tab, graf
Article in English | LILACS | ID: lil-665801

ABSTRACT

Imatinib mesylate (IM) is used to treat chronic myeloid leukemia (CML) because it selectively inhibits tyrosine kinase, which is a hallmark of CML oncogenesis. Recent studies have shown that IM inhibits the growth of several non-malignant hematopoietic and fibroblast cells from bone marrow (BM). The aim of the present study was to evaluate the effects of IM on stromal and hematopoietic progenitor cells, specifically in the colony-forming units of granulocyte/macrophage (CFU-GM), using BM cultures from 108 1.5- to 2-month-old healthy Swiss mice. The results showed that low concentrations of IM (1.25 µM) reduced the growth of CFU-GM in clonogenic assays. In culture assays with stromal cells, fibroblast proliferation and α-SMA expression by immunocytochemistry analysis were also reduced in a concentration-dependent manner, with a survival rate of approximately 50% with a dose of 2.5 µM. Cell viability and morphology were analyzed using MTT and staining with acrydine orange/ethidium bromide. Most cells were found to be viable after treatment with 5 µM IM, although there was gradual growth inhibition of fibroblastic cells while the number of round cells (macrophage-like cells) increased. At higher concentrations (15 µM), the majority of cells were apoptotic and cell growth ceased completely. Oil red staining revealed the presence of adipocytes only in untreated cells (control). Cell cycle analysis of stromal cells by flow cytometry showed a blockade at the G0/G1 phases in groups treated with 5-15 µM. These results suggest that IM differentially inhibits the survival of different types of BM cells since toxic effects were achieved.


Subject(s)
Animals , Male , Mice , Antineoplastic Agents/pharmacology , Benzamides/pharmacology , Hematopoietic Stem Cells/drug effects , Mesenchymal Stem Cells/drug effects , Piperazines/pharmacology , Pyrimidines/pharmacology , Cell Proliferation , Cell Survival , Cells, Cultured , Colony-Forming Units Assay , Fibroblasts , Hematopoietic Stem Cells/cytology , Immunohistochemistry , Mesenchymal Stem Cells/cytology
16.
Article in English | WPRIM | ID: wpr-152457

ABSTRACT

Non healing chronic wounds are difficult to treat in patients with diabetes and can result in severe medical problems for these patients and for society. Negative-pressure wound therapy (NPWT) has been adopted to treat intractable chronic wounds and has been reported to be effective. However, the mechanisms underlying the effects of this treatment have not been elucidated. To assess the vasculogenic effect of NPWT, we evaluated the systemic mobilization of endothelial progenitor cells (EPCs) during NPWT. Twenty-two of 29 consecutive patients who presented at the clinic of Seoul National Universty Hospital between December 2009 and November 2010 who underwent NPWT for diabetic foot infections or skin ulcers were included in this study. Peripheral blood samples were taken before NPWT (pre-NPWT) and 7-14 days after the initiation of NPWT (during-NPWT). Fluorescence-activated cell sorting (FACS) analysis showed that the number of cells in EPC-enriched fractions increased after NPWT, and the numbers of EPC colony forming units (CFUs) significantly increased during NPWT. We believe that NPWT is useful for treating patients with diabetic foot infections and skin ulcers, especially when these conditions are accompanied by peripheral arterial insufficiency. The systemic mobilization of EPCs during NPWT may be a mechanism for healing intractable wounds in diabetic patients with foot infections or skin defects via the formation of increased granulation tissue with numerous small blood vessels.


Subject(s)
Adolescent , Adult , Aged , Aged, 80 and over , Case-Control Studies , Child , Colony-Forming Units Assay , Cytokines/genetics , Diabetic Foot/surgery , Endothelial Cells/metabolism , Endothelium, Vascular/cytology , Female , Humans , Male , Middle Aged , Negative-Pressure Wound Therapy , Stem Cells/metabolism
17.
Article in Chinese | WPRIM | ID: wpr-332761

ABSTRACT

This study aimed to investigate the expression of Flk-1 on distinct hematopoietic precursor cells in E10.5 mouse AGM region. By flow cytometry, we found that < 10% of Flk-1(+) cells of E10.5 AGM region co-expressed CD41 and CD45/Ter119. Then, E10.5 AGM cells were fractionated into two subsets, the CD31(+)CD45(-)Ter119(-)Flk-1(+)CD41(+) cells (R1, putative immature hematopoietic cells) and the CD31(+)CD45(-)Ter119(-)Flk-1(+)CD41(-) cells (R2, putative endothelial cells), followed by methylcellulose-based CFU-C assay and OP9-based stromal co-culture to examine their myeloid or/and lymphoid potential in vitro. The results showed that only R1 cells could give rise to typical hematopoietic colonies in CFU-C assay. In contrast, after co-cultured with OP9 for 7-9 days, both subsets could generate abundant hematopoietic progenitor cells (CD45(+)c-Kit(+)), myeloid cells (Gr-1(+)/Mac-1(+)), erythroid cells (Ter119(+)), and B lymphocytes (CD19(+)). It is concluded that both maturing CD41(+)CD45(-) hematopoietic percursor cells and homogenic endothelial cells express Flk-1 in E10.5 AGM region. It requires further functional assay in vivo to clarify whether the hematopoietic stem cells (HSCs) and their precursors retain Flk-1 expression at this developmental stage.


Subject(s)
Animals , Cells, Cultured , Coculture Techniques , Colony-Forming Units Assay , Embryo, Mammalian , Cell Biology , Endothelial Cells , Cell Biology , Female , Hematopoietic Stem Cells , Cell Biology , Male , Mesonephros , Mice , Mice, Inbred C57BL , Vascular Endothelial Growth Factor Receptor-2 , Metabolism
18.
Article in English | WPRIM | ID: wpr-347113

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the effects of sodium copper chlorophyllin (SCC) on the proliferation, differentiation and immunomodulatory function of mesenchymal stem cells (MSCs) from mice with aplastic anemia.</p><p><b>METHODS</b>A mouse model of aplastic anemia was established by exposure of BALB/c mice to sublethal doses of 5.0 Gy Co60 γ radiation, followed by transplantation of 2×10(6) lymph node cells from DBA/2 donor mice within 4 h after radiation. Aplastic anemic BALB/c mice were randomly divided into six groups: the treated groups, which received 25, 50, or 100 mg/kg/day SCC, respectively; a positive control group treated with cyclosporine A (CsA); and an untreated model control group (model group); while, the non-irradiated mice as the normal control group. SCC or CsA were administered by gastrogavage for 20 days, starting on day 4 after irradiation. Peripheral blood cells were counted and colony-forming fibroblasts (CFU-F) in the bone marrow were assayed. The ability of MSCs to form calcium nodes after culture in osteoinductive medium was also observed. The immunosuppressive effect of MSCs on T lymphocytes was analyzed by enzyme-linked immunosorbent assay and flow cytometry, to evaluate the efficacy of SCC in mice with aplastic anemia.</p><p><b>RESULTS</b>Peripheral blood white cell and platelet counts were increased by medium and high SCC doses, compared with the untreated control. CFU-Fs were also increased compared with the untreated control, and the numbers of calcium nodes in MSCs in osteoinductive medium were elevated in response to SCC treatment. The percentage of Forkhead box protein 3 (FOXP3(+)) T cells was increased in T cell-MSC cocultures, and the cytokine transforming growth factor β1 was up-regulated in SCC-treated groups.</p><p><b>CONCLUSION</b>The results of this study suggest that SCC not only promotes the proliferation and differentiation of MSCs, but also improves their immunoregulatory capacity in mice with aplastic anemia.</p>


Subject(s)
Anemia, Aplastic , Blood , Pathology , Therapeutics , Animals , Anthraquinones , Metabolism , Biomarkers , Metabolism , Bone Marrow Cells , Pathology , Calcium , Metabolism , Cell Differentiation , Cell Proliferation , Chlorophyllides , Pharmacology , Colony-Forming Units Assay , Female , Immunosuppression Therapy , Leukocyte Count , Male , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells , Cell Biology , Metabolism , Mice , Mice, Inbred BALB C , Mice, Inbred DBA , Osteoblasts , Pathology , Platelet Count , T-Lymphocytes
19.
AJMB-Avicenna Journal of Medical Biotechnology. 2013; 5 (2): 104-117
in English | IMEMR | ID: emr-142798

ABSTRACT

Despite major progress in our general knowledge related to the application of adult stem cells, finding alternative sources for bone marrow Mesenchymal Stem Cells [MSCs] has remained to be challenged. In this study successful isolation, multilineage differentiation, and proliferation potentials of sheep MSCs derived from bone marrow, adipose tissue, and liver were widely investigated. The primary cell cultures were prepared form tissue samples obtained from sheep 30-35 day fetus. Passage-3 cells were plated either at varying cell densities or different serum concentrations for a week. The Population Doubling Time [PDT], growth curves, and Colony Forming Unit [CFU] of MSCs was determined. The stemness and trilineage differentiation potential of MSCs were analyzed by using molecullar and cytochemical staining approaches. The data was analyzed through one way ANOVA using SigmaStat [ver. 2]. The highest PDT and lowest CFU were observed in adipose tissue group compared with other groups [p<0.001]. Comparing different serum concentrations [5, 10, 15, and 20%], irrespective of cell sources, the highest proliferation rate was achieved in the presence of 20% serum [p<0.001]. Additionally, there was an inverse relation between cell seeding density at culture initiation and proliferation rate, except for L-MSC at 300 cell seeding density. All three sources of fetal sheep MSCs had the identical trilineage differentiation potential. The proliferative capacity of liver and bone marrow derived MSCs were similar at different cell seeding densities except for the higher fold increase in B-MSCs at 2700 cells/cm2 density. Moreover, the adipose tissue derived MSCs had the lowest proliferative indices


Subject(s)
Animals , Bone Marrow Cells/cytology , Cell Differentiation/physiology , Cells, Cultured , Colony-Forming Units Assay , Liver/cytology , Cells , Adipose Tissue/cytology , Microscopy, Electron, Scanning Transmission , Cell Culture Techniques
20.
West Indian med. j ; 61(9): 861-864, Dec. 2012. ilus, tab
Article in English | LILACS | ID: lil-694356

ABSTRACT

OBJECTIVE: Diabetes mellitus is a chronic noncommunicable disease with high prevalence in the North American and Caribbean region. Diabetic Foot Syndrome which is an associated complication can lead to the development of wounds and ulcers which can become infected. Justicia secunda, a plant known locally in Barbados as Bloodroot used in folklore for wound healing, was selected to test its ability to aid diabetic wound healing by antimicrobial activity. It was therefore tested against the bacteria Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27853, and Enterococcus feacalis (clincal strain) which are commonly found in diabetic wounds. METHODS: The plant was collected by local users. Methanol and acetone extracts of the plant were prepared with use of soxhlet extraction. The antimicrobial activity was assessed with the use of a modified KirbyBaurer method. Concentrations of 200 mg/ml, 100 mg/ml, 10 mg/ml, and 1 mg/ml of the extract were used, with a standard ciprofloxacin 5 µg positive control, and a 5% dimethyl sulfoxide (DMSO) solution negative control. RESULTS: The J secunda methanol and acetone extracts with an extraction yield of 15.3% and 0.75%, respectively yielded no activity within the concentration range against the three strains of bacteria tested. In comparison with the positive control, relative inhibition zone diameter (RIZD) values of 0% resulted for both the negative control and the extracts, with the positive control having a value of 100%. CONCLUSION:The in vitro screen of the extracts prepared from J secunda, yielded no antimicrobial activity against the three strains of bacteria tested and therefore does not support the folklore claims by this mechanism of action.


OBJETIVO: La diabetes mellitus es una enfermedad crónica no transmisible, de alta prevalencia en la región de Norte América y el Caribea. El síndrome de pie diabético es una complicación asociada, que puede llevar al desarrollo de heridas y úlceras, con la consiguiente posibilidad de infección. Justicia segunda es una planta conocida localmente en Barbados como "bloodroot" (sanguinaria canadensis) y usada en la medicina folklórica para la curación de heridas. Esta planta fue seleccionada para analizar su capacidad de ayudar a curar las heridas de diabéticos por su actividad antimicrobiana. Por lo tanto, se la sometió a prueba frente a bacterias Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27853, y Enterococcus feacalis (cepa clínica) que normalmente se encuentran en las heridas del diabético. MÉTODOS: La planta fue proporcionada por usuarios locales. Usando un extractor Soxhlet, se prepararon extractos de metanol y acetona a partir de la planta. La actividad antimicrobiana se evaluó mediante el método de KirbyBauer modificado. Se usaron concentraciones de 200 mg/ml, 100 mg/ml, 10 mg/ml, y 1 mg/ml del extracto, con un control positivo de 5 µg de ciprofloxacina estándar, y un control negativo de una solución de dimetil sulfóxido (DMSO) al 5%. RESULTADOS: Los extractos de metanol y acetona de J secunda con un rendimiento de extracción de 15.3% y 0.75% respectivamente, no arrojaron actividad alguna dentro del rango de la concentración contra las tres cepas de bacterias sujetas a prueba. Comparado con el control positivo, el diámetro de la zona de inhibición relativa (RIZD) arrojó valores de 0%, tanto para el control negativo como para los extractos, con un valor de 100% para el control positivo. CONCLUSIÓN: El tamizaje in vitro de los extractos preparados de J secunda, no arrojó actividad antimicrobiana alguna contra las tres cepas de bacterias analizadas, y por consiguiente no sustenta la afirmación de la medicina folklórica en relación con este mecanismo de acción.


Subject(s)
Humans , Anti-Bacterial Agents/therapeutic use , Bacterial Infections/drug therapy , Diabetic Foot/drug therapy , Medicine, Traditional , Microbial Sensitivity Tests , Phytotherapy , Plant Extracts/therapeutic use , Sanguinaria , Wound Healing/drug effects , Wound Infection/drug therapy , Barbados , Colony-Forming Units Assay , Ciprofloxacin/pharmacology , Dose-Response Relationship, Drug , Enterococcus faecalis/drug effects , Plants, Medicinal , Pseudomonas aeruginosa/drug effects , Staphylococcus aureus/drug effects
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