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1.
J. coloproctol. (Rio J., Impr.) ; 41(1): 63-69, Jan.-Mar. 2021. tab, graf
Article in English | LILACS | ID: biblio-1286971

ABSTRACT

Abstract Objective Type-I collagen (Col-I) is one of the main macromolecules of the extracellular matrix, and it is involved in the desmoplastic stromal reaction, an indicator of worse prognosis in cases of colorectal cancer (CRC). The purpose of the present study was to investigate Col-I expression in cases of CRC and adenoma and to correlate with the clinical data and the data regarding the lifestyle of the patients. Methods A retrospective study including 22 patients with adenoma and 15 with CRC treated at a coloproctology service. The clinical and lifestyle data were obtained through medical records, and Col-I expression was investigated by immunohistochemistry. Results Women represented most cases of adenoma (63.64%), whereas CRC was found mainly in men (73.33%) (p=0.0448). Immunoexpression of Col-I showed a basement membrane thickening in areas of lining of epithelium and around the glands in both lesions. The cases of CRC had a quite evident fibrosis process in the stroma. The quantitative analysis demonstrated a higher protein expression in CRCs compared to adenomas (p=0.0109), as well as in female patients (p=0.0214), patients aged ≥ 50 years (p=0.0400), and in those with a positive family history of colorectal disease (p=0.0292). These results suggested a remodeling of the microenvironment of the Worked developed at the Department of Morphology, Centro de Ciências da Saúde, Universidade Federal do Espírito Santo, ES, Brazil. Conclusion The immunohistochemical analysis encourages the performance of more comprehensive studies to ascertain if our results could be a tool for the diagnosis and monitoring of the patients.


Resumo Objetivo O colágeno tipo I (Col-I) é uma das principais macromoléculas da matriz extracelular, e está envolvido na reação desmoplástica estromal, um indicador de pior prognóstico em casos de câncer colorretal (CCR). O objetivo foi investigar a expressão do Col-I emcasos de CCR e adenoma, e correlacioná-la comdados clínicos e de estilo de vida dos pacientes. Metodologia Foi realizado umestudoretrospectivo com22pacientes comadenoma e 15 comCCR tratadosemumserviço de coloproctologia.Os dados dos pacientes foramobtidos dos prontuários médicos, e a expressão do Col-I foi investigada por imunohistoquímica. Resultados As mulheres representaram a maioria dos casos de adenomas (63,64%), enquanto o CCR (73,33%) (p=0,0448) foi mais comum entre os homens. A imunoexpressão de Col-I mostrou espessamento da membrana basal em áreas de revestimento do epitélio e em volta de glândulas em ambas as lesões. O CCR apresentou fibrose no estroma. As análises quantitativas demonstraram maior expressão proteica no CCR (p=0,0109), assim como em mulheres (p=0,0214), pacientes com idade ≥ 50 anos (p=0,0400), e em pacientes com histórico positivo de doença colorretal na família (p=0,0292). Estes resultados sugerem a remodelação do microambiente tumoral na carcinogênese do CCR. As correlações clínico-patológicas positivas mostram uma ligação plausível entre o perfil do paciente e os achados imunohistoquímcos, o que indica uma possível forma de estratificação dos pacientes. Conclusão As análises imunohistoquímicas estimulam a execução de estudos mais abrangentes para confirmar se nossos resultados poderão ser uma ferramenta para o diagnóstico e o monitoramento dos pacientes.


Subject(s)
Humans , Male , Female , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/pathology , Adenocarcinoma/diagnosis , Biomarkers, Tumor/metabolism , Gene Expression Regulation, Neoplastic , Collagen Type I/genetics , Extracellular Matrix/metabolism , Tumor Microenvironment/immunology
2.
J. appl. oral sci ; 29: e20210038, 2021. tab, graf
Article in English | LILACS | ID: biblio-1340106

ABSTRACT

Abstract Potent signaling agents stimulate and guide pulp tissue regeneration, especially in endodontic treatment of teeth with incomplete root formation. Objective This study evaluated the bioactive properties of low concentrations of extracellular matrix proteins on human apical papilla cells (hAPCs). Methodology Different concentrations (1, 5, and 10 µg/mL) of fibronectin (FN), laminin (LM), and type I collagen (COL) were applied to the bottom of non-treated wells of sterilized 96-well plates. Non-treated and pre-treated wells were used as negative (NC) and positive (PC) controls. After seeding the hAPCs (5×103 cells/well) on the different substrates, we assessed the following parameters: adhesion, proliferation, spreading, total collagen/type I collagen synthesis and gene expression (ITGA5, ITGAV, COL1A1, COL3A1) (ANOVA/Tukey; α=0.05). Results We observed greater attachment potential for cells on the FN substrate, with the effect depending on concentration. Concentrations of 5 and 10 µg/mL of FN yielded the highest cell proliferation, spreading and collagen synthesis values with 10 µg/mL concentration increasing the ITGA5, ITGAV, and COL1A1 expression compared with PC. LM (5 and 10 µg/mL) showed higher bioactivity values than NC, but those were lower than PC, and COL showed no bioactivity at all. Conclusion We conclude that FN at 10 µg/mL concentration exerted the most intense bioactive effects on hAPCs.


Subject(s)
Humans , Extracellular Matrix Proteins , Fibronectins , Cell Adhesion , Cells, Cultured , Laminin , Collagen Type I , Extracellular Matrix
3.
Clinics ; 76: e2145, 2021. tab, graf
Article in English | LILACS | ID: biblio-1153958

ABSTRACT

OBJECTIVES: Arterial embolization of myomas (AEM) is controversial because of the changes that occur in the extracellular matrix (ECM) of the endometrium and its effect on gestational success in infertile patients desiring reproductive capability. Therefore, we performed this study on the expression of genes in the ECM of the endometrium, such as those coding metalloproteinases (MMP), before and 6 months after embolization of the uterine arteries. METHODS: Seven women with leiomyomas were evaluated, and MMP3 and MMP10 levels were measured. The women underwent pelvic nuclear magnetic resonance (NMR), examination, and endometrial biopsy between the 20th and 24th day of the menstrual cycle, and pre- and post-AEM (after 6 months). For data analysis, the Cq comparative method, also known as the 2-ΔΔCT method, was used to calculate the relative quantities of MMP gene expression among the samples collected. RESULTS: There was a significant decrease by 9.52 times in the expression of MMP3 (p=0.007), and a non-significant change in the expression of MMP10 (p=0.22) in post-AEM-treated women than pre-AEM-treated women. CONCLUSIONS: The results suggest that ECM continues to undergo tissue remodeling 6 months after AEM, at least with regard to MMP3 expression, suggesting that AEM affects the ECM for at least 6 months after the procedure.


Subject(s)
Humans , Female , Endometrium , Myoma , Metalloproteases , Extracellular Matrix , Uterine Artery
4.
Rio de Janeiro; s.n; 2021. 135 f p. ilus.
Thesis in Portuguese | LILACS | ID: biblio-1353763

ABSTRACT

O músculo esquelético é responsável pelo movimento e manutenção da postura, e é um órgão produtor de miocinas e altamente metabólico, onde alterações em sua fisiologia podem ter consequências sistêmicas. Esse tecido é alvo para diferentes arboviroses, e mialgia é um sintoma frequentemente relatado. O músculo esquelético é composto majoritariamente por fibras musculares, e uma pequena população de células progenitoras denominadas células satélites (SC), que em caso de lesão podem ser ativadas, proliferam e se diferenciam, sendo capazes de regenerar o tecido muscular. Recentemente nosso grupo demonstrou que SC em proliferação (mioblastos) são infectadas pelo vírus ZIKA (ZIKV), enquanto células diferenciadas e fusionadas (miotubos) não apresentam proteínas virais. O presente trabalho avaliou alterações miogênicas e o perfil transcricional de mioblastos e miotubos humanos após tratamento com ZIKV, com o objetivo de identificar fatores e mecanismos envolvidos na susceptibilidade e resistência destas células à infecção. Confirmamos infecção produtiva do ZIKV nos mioblastos, que apresentaram uma redução no número de células expressando a molécula KI67 em altas concentrações (característico de células em mitose). A análise de sequenciamento mostrou perturbação das vias do ciclo celular em mioblastos infectados, que ainda apresentaram enriquecimento de vias relacionadas à morte celular. Também confirmamos a ausência de infecção produtiva nos miotubos. Interessantemente, verificamos que o ZIKV entra nas células diferenciadas, mas não consegue replicar o RNA viral, e a análise do transcriptoma identificou um enriquecimento de vias e modulação de genes antivirais maior ou exclusivamente nas células diferenciadas em comparação aos mioblastos infectados. Além disso, miotubos expostos ao ZIKV aparentam ter aumento de fusão/hipertrofia. Ao contrário dos mioblastos, miotubos apresentaram enriquecimento de vias relacionadas a organização da matriz extracelular. Dados preliminares do nosso grupo mostraram que o cultivo de mioblastos sobre a isoforma de laminina 511 levou à redução da infecção pelo ZIKV. Contudo, em nossos ensaios, a infecção pelo ZIKV não modulou a expressão de receptores para LM e o bloqueio do receptor de LM, a integrina α6, não reduziu a infecção pelo ZIKV em mioblastos. Nosso trabalho mostrou que a infecção pelo ZIKV induz resposta imune e antiviral, que foram enriquecidos em mioblastos e miotubos, sendo que estes apresentaram uma assinatura única de vias e genes antivirais, que poderiam explicar a resistência frente ao ZIKV.


Subject(s)
Arbovirus Infections , Muscle Fibers, Skeletal , Myoblasts , Extracellular Matrix , Zika Virus
5.
Article in Chinese | WPRIM | ID: wpr-921829

ABSTRACT

At present, acellular matrix is an effective replacement material for the treatment of skin damage, but there are few systematic evaluation studies on its performance. The experimental group of this study used two decellularization methods to prepare the matrix: one was the acellular matrix which sterilized with peracetic acid first (0.2% PAA/4% ethanol solution) and then treated with hypertonic saline (group A), the other was 0.05% trypsin/EDTA decellularization after γ irradiation (group B); and the control group was soaked in PBS (Group C). Then physical properties and chemical composition of the three groups were detected. Hematoxylin eosin (HE) staining showed that the acellular effect of group B was good. The porosity of group A and B were both above 84.9%. In group A, the compressive modulus of elasticity was (9.94 ± 3.81) MPa, and the compressive modulus of elasticity was (12.59 ± 5.50) MPa in group B. There was no significant difference between group A or B and group C. The total content of collagen in acellular matrix of group A and B was significantly lower than that of group C (1. 662 ± 0.229) mg/g, but there was no significant difference in the ratio of collagen Ⅰ/Ⅲ between group B and group C. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) showed that there was no significant difference in microstructure. Qualitative detection of fibronectin and elastin in each group was basically consistent with that in group C. Therefore, acellular matrix of group B had better performance as scaffold material. The experimental results show that the acellular matrix prepared by γ-ray sterilization and decellularization of 0.05% Trypsin enzyme/EDTA could be used for the construction of tissue-engineered skin. It could also provide reference for the preparation and mounting of heterogeneous dermal acellular matrix. It was also could be used for electrostatic spinning or three-dimensional printed tissue engineered skin scaffold which could provide physical and chemical parameters for it.


Subject(s)
Acellular Dermis , Cells, Cultured , Extracellular Matrix , Porosity , Tissue Engineering , Tissue Scaffolds
6.
Chinese Journal of Biotechnology ; (12): 4024-4035, 2021.
Article in Chinese | WPRIM | ID: wpr-921483

ABSTRACT

Decellularized extracellular matrix (dECM), which contains many proteins and growth factors, can provide three-dimensional scaffolds for cells and regulate cell regeneration. 3D bioprinting can print the combination of dECM and autologous cells layer by layer to construct the tissue structure of carrier cells. In this paper, the preparation methods of tissue and organ dECM bioink from different sources, including decellularization, crosslinking, and the application of dECM bioink in bioprinting are reviewed, with future applications prospected.


Subject(s)
Bioprinting , Extracellular Matrix , Printing, Three-Dimensional , Tissue Engineering , Tissue Scaffolds
7.
Chinese Journal of Biotechnology ; (12): 2668-2677, 2021.
Article in Chinese | WPRIM | ID: wpr-887832

ABSTRACT

Seed cells, biomaterials and growth factors are three important aspects in tissue engineering. Biomaterials mimic extra cellular matrix in vivo, providing a sound environment for cells to grow and attach, so as to maintain cell viability and function. The physicochemical properties and modification molecules of material surface mediate cell behaviors like cell adhesion, proliferation, migration and differentiation, which in turn affect cellular function and tissue regeneration efficacy. Furthermore, the modification molecules of material surface are the direct contact point for cell adhesion and growth. Therefore, the interactions between cells and surface modification molecules are the key to tissue engineering. This review summarizes the effects of surface modification molecules on cell phenotypes and functions.


Subject(s)
Biocompatible Materials , Cell Adhesion , Cell Differentiation , Extracellular Matrix , Tissue Engineering
8.
Acta Physiologica Sinica ; (6): 160-174, 2021.
Article in Chinese | WPRIM | ID: wpr-878245

ABSTRACT

Vascular smooth muscle cell (vSMC) is the predominant cell type in the blood vessel wall and is constantly subjected to a complex extracellular microenvironment. Mechanical forces that are conveyed by changes in stiffness/elasticity, geometry and topology of the extracellular matrix have been indicated by experimental studies to affect the phenotype and function of vSMCs. vSMCs perceive the mechanical stimuli from matrix via specialized mechanosensors, translate these stimuli into biochemical signals controlling gene expression and activation, with the consequent modulation in controlling various aspects of SMC behaviors. Changes in vSMC behaviors may further cause disruption of vascular homeostasis and then lead to vascular remodeling. A better understanding of how SMC senses and transduces mechanical forces and how the extracellular mechano-microenvironments regulate SMC phenotype and function may contribute to the development of new therapeutics for vascular diseases.


Subject(s)
Biophysics , Cells, Cultured , Extracellular Matrix , Humans , Muscle, Smooth, Vascular , Myocytes, Smooth Muscle , Phenotype , Vascular Remodeling
9.
Article in Chinese | WPRIM | ID: wpr-879412

ABSTRACT

Perineuronal nets (PNNs) is a complex network composed of highly condensed extracellular matrix molecules surrounding neurons. It plays an important role in maintaining the performance of neurons and protecting them from harmful substances. However, after spinal cord injury, PNNs forms a physical barrier that surrounds the neuron and limits neuroplasticity, impedes axonal regeneration and myelin formation, and promotes local neuroinflammatory uptake. This paper mainly describes the composition and function of PNNs of neurons and its regulatory effects on axonal regeneration, myelin formation and neuroinflammation after spinal cord injury.


Subject(s)
Axons , Extracellular Matrix , Humans , Nerve Regeneration , Neuronal Plasticity , Neurons , Spinal Cord , Spinal Cord Injuries
10.
Int. j. morphol ; 38(6): 1742-1750, Dec. 2020. tab, graf
Article in English | LILACS | ID: biblio-1134507

ABSTRACT

SUMMARY: Mesenchymal stem cells are present in adult tissues such as the human dental pulp. They are pluripotent and can differentiate into various specialized cell types in vitro through appropriate stimuli. Ameloblasts produce human tooth enamel only during embryonic development before tooth eruption, so endogenous regeneration is not possible. Various efforts have been aimed at generating natural or artificial substitutes for dental enamel with properties similar to the specific components of said tissue. The purpose of this study was to induce human dental pulp stem cells to produce enamel proteins using extracellular matrix derived from the rat tail tendon and pigskin. Primary cultures of human dental pulp stem cells were established and characterized by RT-PCR and immunofluorescence, using mesenchymal cell markers such as CD14, CD40, CD44, CD105, and STRO-1. The cells were then incubated with the extracellular matrix for fourteen days and labeled with specific antibodies to detect the expression of dental enamel proteins such as amelogenin, ameloblastin, enamelisin, tuftelin, and parvalbumin, characteristics of the phenotype of ameloblasts. This work demonstrated a positive effect of the extracellular matrix to induce the expression of enamel proteins in the stem cells of the human dental pulp.


RESUMEN: Las células madre mesenquimales están presentes en los tejidos adultos como la pulpa dental humana. Son pluripotentes y pueden diferenciarse en varios tipos de células especializadas in vitro a través de estímulos adecuados. Los ameloblastos producen esmalte dental humano sólo durante el desarrollo embrionario antes de la erupción dental, por lo que no es posible su regeneración endógena. Varios esfuerzos se han orientado a generar sustitutos naturales o artificiales de esmalte dental con propiedades similares a los componentes específicos de este tejido. El propósito de este estudio fue inducir células madre de pulpa dental humana para producir proteínas del esmalte dental a través del estímulo de matriz extracelular derivada del tendón de la cola de rata y piel de cerdo. Se establecieron cultivos primarios de células madre de pulpa dental humana y se caracterizaron por RT-PCR e inmunofluorescencia utilizando marcadores de células mesenquimales como CD14, CD40, CD44, CD105 y STRO-1. Posteriormente, las células se incubaron con matriz extracelular durante un período de catorce días y se marcaron con anticuerpos específicos para detectar la expresión de proteínas de esmalte dental como amelogenina, ameloblastina, enamelisina, tuftelina y parvalbúmina, las cuales son características del fenotipo de ameloblastos. Este trabajo demostró el efecto positivo que tiene el empleo de la matriz extracelular para inducir la expresión de proteínas de esmalte en las células pluripotenciales de la pulpa dental humana.


Subject(s)
Humans , Stem Cells , Dental Enamel Proteins , Dental Pulp , Extracellular Matrix , Immunophenotyping , Fluorescent Antibody Technique , Cell Culture Techniques , Tissue Engineering
11.
Arq. bras. med. vet. zootec. (Online) ; 72(3): 647-654, May-June, 2020. ilus, tab, graf
Article in English | LILACS, VETINDEX | ID: biblio-1128504

ABSTRACT

The elastic cartilage is composed by chondroblasts and chondrocytes, extracellular matrix and surrounded by perichondrium. It has a low regeneration capacity and is a challenge in surgical repair. One of obstacles in engineering a structurally sound and long-lasting tissue is selecting the most appropriate scaffold material. One of the techniques for obtaining biomaterials from animal tissues is the decellularization that decreases antigenicity. In this work, alkaline solution was used in bovine ear elastic cartilages to evaluate the decellularization and the architecture of the extracellular matrix. The cartilages were treated in alkaline solution (pH13) for 72 hours and lyophilized to be compared with untreated cartilages by histological analysis (hematoxylin-eosin, Masson's trichrome and Verhoeff slides). Areas of interest for cell counting and elastic fiber quantification were delineated, and the distribution of collagen and elastic fibers and the presence of non-fibrous proteins were observed. The results demonstrated that the alkaline solution caused 90% decellularization in the middle and 13% in the peripheral region, and maintenance of the histological characteristics of the collagen and elastic fibers and non-fibrous protein removal. It was concluded that the alkaline solution was efficient in the decellularization and removal of non-fibrous proteins from the elastic cartilages of the bovine ear.(AU)


A cartilagem elástica é composta por condroblastos e condrócitos, matriz extracelular e envolta por pericôndrio. Possui uma baixa capacidade de regeneração e é um desafio em reparos cirúrgicos. Um dos obstáculos na engenharia de tecido estruturalmente sólido e de longa duração é a seleção do material de arcabouço mais adequado. Uma das técnicas para obtenção de biomateriais oriundos de tecidos animais é a descelularização, que diminui a antigenicidade. Neste trabalho, foi utilizada solução alcalina em cartilagem elástica auricular bovina para avaliar a descelularização e a arquitetura da matriz extracelular. As cartilagens foram tratadas em solução alcalina (pH13) durante 72 horas e liofilizadas, e comparadas com cartilagens não tratadas por análise histológica (hematoxilina-eosina, tricrômio de Masson e Verhoeff). Foram determinadas as áreas de interesse para contagem celular e quantificação de fibras elásticas, observada a distribuição de colágeno e fibras elásticas e a presença de proteínas não fibrosas. Os resultados demonstraram que a solução alcalina causou 90% de descelularização na região central e 13% na região periférica, manutenção das características histológicas do colágeno e fibras elásticas e remoção das proteínas não fibrosas. Concluiu-se que a solução alcalina foi eficiente na descelularização e retirada de proteínas não fibrosas de cartilagens elásticas da orelha de bovinos.(AU)


Subject(s)
Biocompatible Materials , Chondrocytes , Tissue Engineering/veterinary , Elastic Cartilage , Extracellular Matrix , Cattle , Cartilage , Eosine Yellowish-(YS) , Alkalies
12.
Rev. bras. oftalmol ; 79(1): 71-80, Jan.-Feb. 2020. tab, graf
Article in Portuguese | LILACS | ID: biblio-1092653

ABSTRACT

Resumo Atualmente a membra amniótica (MA) tem obtido importância devido à comprovada capacidade de reduzir inflamação, auxiliar a cicatrização e epitelização, possuindo propriedades antimicrobianas e antivirais, além de baixa imunogenicidade. As indicações de seu uso na oftalmologia têm aumentado muito nas duas últimas décadas. Objetivo: Descrever a estrutura básica e as propriedades biológicas da MA em relação aos componentes da sua matriz extracelular e fatores de crescimento, as consequências de diferentes técnicas empregadas na sua preservação e esterilização, métodos para remoção do epitélio e a comparação dos custos dos diferentes meios de conservação atualmente empregados. Métodos: Pesquisa nas bases de dados do Portal da Biblioteca Virtual em Saúde (BVS), Pubmed, Cochrane, Scielo e Lilacs com as palavras-chave: membrana amniótica, transplante, reconstrução da córnea, doenças da conjuntiva. Resultados: A literatura é vasta na descrição dos efeitos de diversos agentes e técnicas na preparação da MA, dentre elas sua preservação, esterilização e desepitelização. A membrana desnuda tem sido a escolha para a reconstrução da superfície ocular, pois facilita a cicatrização. Em relação aos agentes conservantes, o glicerol é o meio mais utilizado mundialmente pelo baixo custo e facilidade de manuseio. Conclusão: A comparação das diversas técnicas nos guia na elaboração de protocolos de preparo da MA para uso oftalmológico. A membrana desnuda facilita a cicatrização em relação a com células epiteliais. O glicerol é o meio de conservação mais utilizado pelo baixo custo e facilidade de manuseio.


Abstract Currently, the amniotic membrane (AM) has obtained importance due to its ability to reduce inflammation, helping in the healing and epithelialization processes, having antimicrobial and antiviral properties and low immunogenicity. Its indications in ophthalmology have increased considerably in the past two decades. Objective: To describe the basic structure and biological properties of the AM, the components of the extracellular matrix and growth factors, the consequences of different techniques used in its preservation, and sterilization methods for the epithelium removal. To compare the costs of the different preservation solutions currently employed. Study design: literature review. Methods: Research in BVS databases, PubMed, Cochrane, Scielo and Lilacs with keywords: amniotic membrane transplantation, corneal reconstruction, conjunctival diseases. Results: The literature is vast in describing the effects of different agents and techniques used in the preparation of MA, including its preservation, sterilization and desepithelization. The naked membrane is the choice to reconstruct the ocular surface, as it facilitates the healing course. Regarding the preservatives, glycerol is the most used worldwide due its low cost and easy handling. Conclusion: Comparing different techniques guides us in developing a MA preparation protocol for ophthalmic use. The naked membrane facilitates the healing process compared with the presence of epithelial cells. The glycerol is the most used preservation method because of its low cost and easy handling.


Subject(s)
Humans , Tissue Preservation/methods , Conjunctival Diseases/surgery , Corneal Diseases/surgery , Tissue and Organ Harvesting/methods , Eye Diseases/surgery , Amnion/transplantation , Tissue Banks/standards , Tissue Donors/supply & distribution , Wound Healing , Biological Dressings/standards , Biological Products/standards , Tissue and Organ Procurement/standards , Cryopreservation/methods , Sterilization/methods , Collagen/metabolism , Intercellular Signaling Peptides and Proteins/metabolism , Extracellular Matrix/metabolism , Amnion/cytology , Amnion/microbiology , Amnion/ultrastructure
13.
Electron. j. biotechnol ; 43: 55-61, Jan. 2020. tab, ilus, graf
Article in English | LILACS | ID: biblio-1087522

ABSTRACT

Background: Matrix metalloproteinase 12 (MMP12), a member of MMPs, can take lots of roles including extracellular matrix component degradation, viral infection, inflammation, tissue remodeling and tumorigenesis. To explore the transcriptional regulation of MMP12 gene, a sensitive luciferase reporter HEK293 cell line for endogenous MMP12 promoter was generated by CRISPR/Cas9 technology. Results: The HEK293-MMP12-T2A-luciferase-KI cell line was successfully established by CRISPR/Cas9 technology. The sequencing results indicated that one allele of the genome was proven to have a site-directed insertion of luciferase gene and another allele of the genome was confirmed to have additional 48 bp insertion in this cell line. The cell line was further demonstrated to be a sensitive reporter of the endogenous MMP12 promoter by applying transcription factors STAT3, AP-1 and SP-1 to the cell line. The reporter cell line was then screened with bioactive small molecule library, and a small molecule Tanshinone I was found to significantly inhibit the transcriptional activity of MMP12 gene in HEK293-MMP12-T2A-luciferase-KI cell line by luciferase activity assay, which was further confirmed to inhibit the expression of MMP12 mRNA in wild-type HEK293 cells. Conclusions: This novel luciferase knock-in reporter system will be helpful for investigating the transcriptional regulation of MMP12 gene and screening the drugs targeting MMP12 gene.


Subject(s)
Humans , Matrix Metalloproteinase 12/genetics , CRISPR-Cas Systems , Luciferases/genetics , Transcription, Genetic , Cell Communication , Cell Line , Promoter Regions, Genetic/genetics , Cell Culture Techniques , Extracellular Matrix , Gene Knock-In Techniques , Clustered Regularly Interspaced Short Palindromic Repeats
14.
São Paulo; s.n; 2020. 50 p. ilust.
Thesis in Portuguese | LILACS, Inca | ID: biblio-1178971

ABSTRACT

Introdução: A matriz extracelular (MEC) desempenha um papel importante na função celular, sendo que seu componente mais importante são as fibras de colágeno (COL). Nos últimos anos, a Microscopia de Geração Segundo Harmônico (em inglês, second harmonic generation microscopy; SHG) tem sido utilizada para a análise de COL em vários tipos de cânceres ginecológicos, como o da mama e ovário. Desta forma, tem-se enfatizado o valor da quantificação do colágeno por este método como meio de aprofundar o conhecimento da fisiopatologia e do prognóstico na avaliação destas neoplasias. Objetivo: No presente estudo caracterizamos as fibras de COL utilizando-se a microscopia de SHG objetivando avaliar diferenças entre carcinoma de células escamosas e lesões pré-neoplásicas da vulva, bem como verificar se há relação entre características dessas fibras com parâmetros clinicopatológicos associados a prognóstico. Método: Trata-se de um estudo de coorte horizontal e retrospectivo. Foram incluídos 52 pacientes, entre os anos de 2000 a 2010, sendo 40 com Carcinoma de Células Escamosas (CCE) Vulvar; 12 pacientes com Neoplasia Intraepitelial Vulvar (NIV); e, para o controle de comparação interna, foram considerados tecidos distantes ao tumor sem presença de lesão tumoral e/ou NIV. Para análises laboratoriais, foi selecionado um espécime representativo do processo patológico para revisão diagnóstica; e, posteriormente, submetido ao estudo do COL usando a microscopia de SHG. Os tecidos adjacentes normais e intratumorais foram selecionados em secções, para a avaliação do COL, e corados em Hematoxilina/Eosina (H&E) e analisados por microscopia confocal. Parâmetros relacionados ao colágeno, como quantidade, organização e uniformidade, em áreas de lesões neoplásicas e pré-neoplásicas da vulva, foram comparados com áreas distantes de aspecto normal das mesmas pacientes. Resultado/conclusão: Foi observado um evidente decréscimo nos valores dos parâmetros do COL no estroma associado às lesões pré-neoplásicas e neoplásicas da vulva. Além disto, aumentos na quantidade e uniformidade das fibras do colágeno tumorais se associaram à presença de metástases linfonodais, um reconhecido parâmetro de pior prognóstico no carcinoma de vulva


Background: The extracellular matrix (ECM) plays an important role in cellular function, being collagen fibers its most important component. Over the last few years, second harmonic generation (SHG) microscopy has been used for the analysis of collagen fibers in several types of cancers, including breast and ovarian cancer. The value of collagen parameters obtained using this technique has been advocated to gain insights on the physiopathology and on the prognostic evaluation of cancer. Objective: Herein, we have characterized collagen fibers using the SHG microscopy, to evaluate differences between vulvar cancer and preneoplastic lesions, and to find possible associations between characteristics of collagen fibers with clinicopathological parameters related to prognosis. Methods: This is a retrospective and horizontal cohort study. Were included 52 patients, between the years 2000 to 2010, 40 of whom with Vulva Squamous Cell Carcinoma (VSCC); 12 patients with Vulvar Intraepithelial Neoplasia (VIN); and, for the control of internal comparison, tissues distant to the tumor without tumor lesion and / or VIN were considered. For laboratory analysis, a specimen representative of the pathological process was selected for diagnostic review and subsequently subjected to the study of collagen using SHG microscopy. The normal and intratumoral adjacent tissues were selected in sections, for collagen evaluation, and stained in Hematoxylin/Eosin (H&E) and analyzed by confocal microscopy. Collagen parameters, quantity, organization, and uniformity, of stroma adjacent to neoplastic or preneoplastic lesions were compared with values obtained in normal appearing tissue distant from those lesions from the same patients. Results/Conclusion: There was an evident decrease in the values of collagen fiber parameters in the stroma associated to tumors. Increased quantity and uniformity of tumor associated collagen fibers were associated with the presence of lymph node metastases, which suggest a prognostic value of such parameters in the evaluation of vulvar cancer


Subject(s)
Humans , Female , Adult , Middle Aged , Aged , Precancerous Conditions , Vulvar Neoplasms , Collagen , Extracellular Matrix , Second Harmonic Generation Microscopy , Neoplasm Metastasis
15.
Mem. Inst. Oswaldo Cruz ; 115: e200007, 2020. graf
Article in English | SES-SP, LILACS, SES-SP | ID: biblio-1135242

ABSTRACT

BACKGROUND Behavioral and neurochemical alterations associated with toxoplasmosis may be influenced by the persistence of tissue cysts and activation of an immune response in the brain of Toxoplasma gondii-infected hosts. The cerebral extracellular matrix is organised as perineuronal nets (PNNs) that are both released and ensheath by some neurons and glial cells. There is evidences to suggest that PNNs impairment is a pathophysiological mechanism associated with neuropsychiatric conditions. However, there is a lack of information regarding the impact of parasitic infections on the PNNs integrity and how this could affect the host's behavior. OBJECTIVES In this context, we aimed to analyse the impact of T. gondii infection on cyst burden, PNNs integrity, and possible effects in the locomotor activity of chronically infected mice. METHODS We infected mice with T. gondii ME-49 strain. After thirty days, we assessed locomotor performance of animals using the open field test, followed by evaluation of cysts burden and PNNs integrity in four brain regions (primary and secondary motor cortices, prefrontal and somesthetic cortex) to assess the PNNs integrity using Wisteria floribunda agglutinin (WFA) labeling by immunohistochemical analyses. FINDINGS AND MAIN CONCLUSIONS Our findings revealed a random distribution of cysts in the brain, the disruption of PNNs surrounding neurons in four areas of the cerebral cortex and hyperlocomotor behavior in T. gondii-infected mice. These results can contribute to elucidate the link toxoplasmosis with the establishment of neuroinflammatory response in neuropsychiatric disorders and to raise a discussion about the mechanisms related to changes in brain connectivity, with possible behavioral repercussions during chronic T. gondii infection.


Subject(s)
Animals , Mice , Cerebellum/metabolism , Toxoplasmosis/pathology , Toxoplasmosis, Animal , Extracellular Matrix/metabolism , Motor Neurons/cytology , Neurons/pathology , Toxoplasma , Cerebellum/cytology , Toxoplasmosis/metabolism , Disease Models, Animal , Motor Neurons/metabolism , Neurons/metabolism
16.
Article in Korean | WPRIM | ID: wpr-811256

ABSTRACT

PURPOSE: Diabetic nephropathy is one of the most important diabetic complications prompted by chronic hyperglycemia, characterized by glomerulosclerosis, tubular fibrosis, and it eventually causes kidney failure. Nobiletin is a polymethoxyflavone present in tangerine and other citrus peels, and has anti-cancer and anti-inflammatory effects. This study investigated the effects of nobiletin on glomerular fibrosis through inhibition of the transforming growth factor (TGF)-β1-Src-caveolin-1 pathway.METHODS: Human renal mesangial cells (HRMC) were incubated in media containing 33 mM glucose with or without 1–20 uM nobiletin for 3 day. The cellular expression levels of fibrogenic collagen IV, fibronectin, connective tissue growth factor (CTGF), TGF-β1, Src and caveolin-1 were all examined. In addition, TGF-β1, Src and caveolin-1 proteins were screened to reveal the relationship among TGF-β1-Src-caveolin-1 signaling in glomerular fibrosis.RESULTS: High glucose promoted the production of collagen IV, fibronectin and CTGF in HRMC, which was inhibited in a dose dependent manner by 1–20 uM nobiletin. The Western blot data showed that high glucose elevated the expression of TGF-β1, Src, caveolin-1 and Rho GTPase. When nobiletin was treated to the HRMC exposed to high glucose, the expression of TGF-β1-Src-caveolin-1 was dampened. Finally, TGF-β1-Src-caveolin-1 signaling pathway was activated in high glucose-exposed HRMC, and such activation was encumbered by nobiletin.CONCLUSION: These result demonstrated that nobiletin blunted high glucose-induced extracellular matrix accumulation via inhibition of the TGF-β1-Src-caveolin-1 related intracellular signaling pathway. Nobiletin may be a potent renoprotective agent to counteract diabetes-associated glomerular fibrosis that leads to kidney failure.


Subject(s)
Blotting, Western , Caveolin 1 , Citrus , Collagen , Connective Tissue Growth Factor , Diabetes Complications , Diabetic Nephropathies , Extracellular Matrix , Fibronectins , Fibrosis , Glucose , GTP Phosphohydrolases , Humans , Hyperglycemia , Mesangial Cells , Renal Insufficiency , Transforming Growth Factors
17.
Article in English | WPRIM | ID: wpr-811201

ABSTRACT

Matrix metallopeptidase 3 or MMP3, is a zinc-dependent proteolytic enzyme that is involved in various physiological processes via modification of the extracellular matrix. In particular, its over-expression has been associated with cancer metastasis and tumor growth in various cancers including breast cancer. MMP3 gene expression is regulated by several factors such as DNA polymorphisms which also serve as risk factors for breast cancer. As such, DNA polymorphisms of MMP3 have the potential to be utilized as genetic biomarkers for prediction and prognosis of metastatic breast cancer. Presently, genome-wide association studies of MMP3 gene polymorphisms which are associated with breast cancer risk and patient survival in a variety of populations are reviewed. In order to understand the potential role of MMP3 polymorphisms as genetic markers for breast cancer metastasis, the domain structure of MMP3, the regulation of its expression and its role in breast cancer metastasis are also briefly discussed in this review. The emergence of MMP3 gene polymorphisms as prognostic biomarker candidates for breast cancer metastasis may contribute towards improving targeted therapies and categorization of breast cancer cases in order to provide a better and more accurate prognosis.


Subject(s)
Biomarkers , Breast Neoplasms , Breast , DNA , Extracellular Matrix , Gene Expression , Genetic Markers , Genome-Wide Association Study , Humans , Matrix Metalloproteinase 3 , Neoplasm Metastasis , Physiological Phenomena , Prognosis , Risk Factors
18.
Article in English | WPRIM | ID: wpr-811137

ABSTRACT

Renal fibrosis is considered to be the final common outcome of chronic kidney disease. Dipeptidyl peptidase-4 (DPP-4) inhibitors have demonstrated protective effects against diabetic kidney disease. However, the anti-fibrotic effect of evogliptin, a DPP-4 inhibitor, has not been studied. Here, we report the beneficial effects of evogliptin on unilateral ureteral obstruction (UUO)-induced renal fibrosis in mice. Evogliptin attenuated UUO-induced renal atrophy and tubulointerstitial fibrosis. Immunohistochemistry and Western blotting demonstrated that evogliptin treatment inhibits pro-fibrotic gene expressions and extracellular matrix production. In vitro findings showed that the beneficial effects of evogliptin on renal fibrosis are mediated by inhibition of the transforming growth factor-β/Smad3 signaling pathway. The present study demonstrates that evogliptin is protective against UUO-induced renal fibrosis, suggesting that its clinical applications could extend to the treatment of kidney disease of non-diabetic origin.


Subject(s)
Animals , Atrophy , Blotting, Western , Diabetic Nephropathies , Dipeptidyl-Peptidase IV Inhibitors , Extracellular Matrix , Fibrosis , Gene Expression , Immunohistochemistry , In Vitro Techniques , Kidney Diseases , Kidney Failure, Chronic , Mice , Renal Insufficiency, Chronic , Transforming Growth Factor beta , Ureter , Ureteral Obstruction
19.
Article in Chinese | WPRIM | ID: wpr-826347

ABSTRACT

Idiopathic pulmonary fibrosis(IPF)is a progressive lung disease characterized by pulmonary interstitial fibrosis and pulmonary dysfunction.Cell microenvironment is mainly composed of cell components,extracellular matrix,extracellular regulators,and liquid substances.Changes in microenvironment components are closely related to IPF.This article elaborates the roles of cell microenvironments including cytokines,mesenchymal cells,extracellular matrix,and unfolded proteins in the pathogenesis of IPF.


Subject(s)
Cellular Microenvironment , Extracellular Matrix , Humans , Idiopathic Pulmonary Fibrosis , Lung
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