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1.
Braz. j. biol ; 83: e248717, 2023. graf
Article in English | LILACS, VETINDEX | ID: biblio-1339344

ABSTRACT

Abstract The human respiratory syncytial virus (hRSV) is the most common cause of severe lower respiratory tract diseases in young children worldwide, leading to a high number of hospitalizations and significant expenditures for health systems. Neutrophils are massively recruited to the lung tissue of patients with acute respiratory diseases. At the infection site, they release neutrophil extracellular traps (NETs) that can capture and/or inactivate different types of microorganisms, including viruses. Evidence has shown that the accumulation of NETs results in direct cytotoxic effects on endothelial and epithelial cells. Neutrophils stimulated by the hRSV-F protein generate NETs that are able to capture hRSV particles, thus reducing their transmission. However, the massive production of NETs obstructs the airways and increases disease severity. Therefore, further knowledge about the effects of NETs during hRSV infections is essential for the development of new specific and effective treatments. This study evaluated the effects of NETs on the previous or posterior contact with hRSV-infected Hep-2 cells. Hep-2 cells were infected with different hRSV multiplicity of infection (MOI 0.5 or 1.0), either before or after incubation with NETs (0.5-16 μg/mL). Infected and untreated cells showed decreased cellular viability and intense staining with trypan blue, which was accompanied by the formation of many large syncytia. Previous contact between NETs and cells did not result in a protective effect. Cells in monolayers showed a reduced number and area of syncytia, but cell death was similar in infected and non-treated cells. The addition of NETs to infected tissues maintained a similar virus-induced cell death rate and an increased syncytial area, indicating cytotoxic and deleterious damages. Our results corroborate previously reported findings that NETs contribute to the immunopathology developed by patients infected with hRSV.


Resumo O vírus sincicial respiratório humano (hRSV) é a causa mais comum de doenças graves do trato respiratório inferior em crianças pequenas em todo o mundo, resultando em grande número de hospitalizações e gastos significativos para os sistemas de saúde. Neutrófilos são recrutados em massa para o tecido pulmonar de pacientes com doenças respiratórias agudas. No local da infecção, eles liberam armadilhas extracelulares de neutrófilos (NETs) que podem capturar e/ou inativar diferentes tipos de microrganismos, incluindo vírus. Evidências demonstraram que o acúmulo de NETs resulta em efeitos citotóxicos diretos nas células endoteliais e epiteliais. Os neutrófilos estimulados pela proteína F do vírus sincicial respiratório (hRSV-F) geram NETs que são capazes de capturar partículas virais, reduzindo assim sua transmissão. No entanto, a produção maciça de NETs obstrui as vias aéreas e aumenta a gravidade da doença. Assim, um maior conhecimento sobre os efeitos das NETs durante as infecções por hRSV é essencial para o desenvolvimento de novos tratamentos específicos e eficazes. Este estudo avaliou os efeitos das NETs no contato prévio ou posterior à infecção de células Hep-2 com hRSV. As células Hep-2 foram infectadas com diferentes quantidades de hRSV (multiplicidade de infecção ou MOI 0,5 ou 1,0), antes ou após a incubação com NETs (0,5-16 μg/mL). Células infectadas e não tratadas mostraram redução da viabilidade celular e intensa coloração com azul de tripano, que foi acompanhada pela formação de sincícios numerosos e grandes. O contato prévio entre as NETs e as células não resultou em efeito protetor. As células em monocamadas mostraram um número e área de sincícios reduzidos, mas a morte celular foi semelhante àquela apresentada por células infectadas e não tratadas. A adição de NETs aos tecidos infectados manteve taxa de morte celular e formação de sincícios semelhantes àqueles induzidos pelo vírus em células não tratadas, indicando danos citotóxicos e deletérios. Nossos resultados corroboram achados relatados anteriormente de que as NETs contribuem para a imunopatologia desenvolvida por pacientes infectados com hRSV.


Subject(s)
Humans , Child, Preschool , Respiratory Syncytial Virus, Human , Respiratory Syncytial Virus Infections , Extracellular Traps , Epithelial Cells , Lung
2.
Braz. j. oral sci ; 21: e226427, jan.-dez. 2022. ilus
Article in English | LILACS, BBO | ID: biblio-1393274

ABSTRACT

Aim: This study aimed to investigate whether non-ionizing radiation emitted by smartphones is likely to cause genotoxic effects on oral epithelial cells. Methods: Thirty adults were distributed into two groups according to the mobile phone brand used, namely Samsung (Samsung, Seoul, South Korea) and Apple (Apple, California, USA). The material was collected with gentle swabbing of the right and left buccal mucosa using a cervical brush, then the micronucleus test was performed. Results: The Mann-Whitney test with a 5% significance level did not reveal statistically significant differences in micronuclei frequency between the exposed and non-exposed sides (p=0.251). The different brands do not seem to cause risks of inducing genetic damage because there were no statistically significant differences between them (p=0.47). Conclusion: Therefore, our results suggest no correlations of micronuclei frequency in the exposed buccal cells of mobile phone users at the exposure standard levels observed


Subject(s)
Humans , Male , Female , Adult , Radiation, Nonionizing/adverse effects , Radio Waves , Micronucleus Tests , Epithelial Cells , Smartphone , Mouth Mucosa , Mutagenicity Tests
3.
Int. j. morphol ; 40(3): 817-823, jun. 2022. ilus
Article in Spanish | LILACS-Express | LILACS | ID: biblio-1385648

ABSTRACT

RESUMEN: Las células epiteliales del amnios (hAECs) son células madre pluripotenciales; tienen capacidad de diferenciarse en células de las tres capas embrionarias. Como tales, se utilizan en algunas terapias regenerativas en medicina. Este estudio tiene por objetivo describir un protocolo de aislación de las células epiteliales del amnios (hAECs) a partir de placentas humanas de partos por cesárea, así como su caracterización y comportamiento in vitro. Se aislaron hAECs de 20 placentas de partos por cesárea con un protocolo optimizado. Se caracterizaron las células mediante citometría de flujo, microscopia óptica y de fluorescencia, y se evaluó la proliferación de las células mediante MTT a los 1, 3, 5 y 7 días con y sin β-mercaptoetanol en el medio de cultivo. El análisis histológico del amnios mostró un desprendimiento prácticamente completo de las células después de la segunda digestión del amnios. El promedio de células obtenidas fue de 10.97 millones de células por gramo de amnios. Las hAECs mostraron una proliferación limitada, la cual no fue favorecida por la adición de β-mercaptoetanol en el cultivo. Se observó un cambio de morfología espontanea de epitelial a mesenquimal después del cuarto pasaje. Las células epiteliales del amnios pueden ser aisladas con un protocolo simple y efectivo, sin embargo, presentan escasa capacidad proliferativa. Bajo las condiciones de este estudio, la adición de β-mercaptoetanol no favorece la capacidad proliferativa de las células.


SUMMARY: human amnion epithelial cells (hAECs) are pluripotent stem cells; they have the ability to differentiate into cells of the three embryonic layers, and are used in various regenerative therapies in medicine. This study aims to describe a protocol for the isolation of amnion epithelial cells (hAECs) from human placentas from cesarean delivery, as well as their characterization and culture conditions in vitro. hAECs were isolated from 20 cesarean delivery placentas with an optimized protocol. The cells were characterized by flow cytometry, light and fluorescence microscopy, and the proliferation of the cells was evaluated by MTT at 1, 3, 5 and 7 days with and without β-mercaptoethanol in the culture medium. Histological analysis of the amnion showed a practically complete detachment of the cells of the underlying membrane after the second digestion. The average number of cells obtained was 10.97 million cells per amnion. The hAECs perform a limited proliferation rate, which was not favored by the addition of β-mercaptoethanol in the culture. A spontaneous morphology change from epithelial to mesenchymal morphology is exhibited after the fourth passage. The epithelial cells of the amnion can be isolated with a simple and effective protocol, however, they present little proliferative capacity. Under the conditions of this study, the addition of β-mercaptoethanol does not favor the proliferation of the cells.


Subject(s)
Humans , Cell Separation/methods , Epithelial Cells/cytology , Amnion/cytology , Flow Cytometry , Microscopy
4.
j.tunis.ORL chir. cerv.-fac ; 47(3): 23-28, 2022. tales, figures
Article in French | AIM | ID: biblio-1392584

ABSTRACT

But: Etudier les facteurs influençant le pronostic des carcinomes épidermoïdes du larynx. Méthodes: Etude rétrospective analytique menée sur 100 patients présentant un carcinome épidermoïde primitif du larynx, durant une période de 24 ans (1992­2015). Résultats: La survie globale à 1 an, à 3 ans et à 5 ans a été respectivement de 99 %, de 77 % et de 63 %. La survie sans maladie à 1 an, à 3 ans et à 5 ans a été respectivement de 88 %, de 76 % et de 63 %. L'étude univariée de la survie globale et la survie sans maladie a montré un impact péjoratif de l'atteinte ganglionnaire histologique, de l'engainement péri-nerveux et des limites chirurgicales tumorales (facteurs histo-pronostiques). Dans l'étude multivariée, seuls le stade T, le stade N, l'atteinte sous-glottique, l'atteinte du cartilage thyroïde et le délai de la radiothérapie postopératoire ont présenté un impact significatif sur la survie sans maladie. Aucun facteur n'a présenté d'impact significatif sur la survie globale, en analyse multivariée. L'étude statistique de la récidive n'a montré aucun facteur prédictif. Conclusion: Le stade tumoral et les facteurs histo-pronostiques sont les 2 facteurs pronostiques majeurs. Dans la littérature, Les principaux facteurs prédictifs de récidive sont: le stade tumoral, les limites chirurgicales tumorales et l'extension extra-nodale. Dans notre étude, aucun facteur prédictif n'a été trouvé.


Subject(s)
Humans , Prognosis , Carcinoma , Cell Survival , Epithelial Cells , Squamous Cell Carcinoma of Head and Neck
5.
Article in Chinese | WPRIM | ID: wpr-941015

ABSTRACT

OBJECTIVE@#To establish a culture system for human nasal mucosal organoids with controllable differentiation to reproduce the structure and function of the source tissue through staged expansion-differentiation culture.@*METHODS@#Fresh samples of surgically resected middle turbinate and nasal polyp tissues were collected, from which the nasal mucosa epithelial cells were isolated by enzymatic digestion and filtration for continuous culture at the air-liquid interface for expansion (EO group) or staged culture for expansion and differentiation (DO group). Immunohistochemical staining was used to characterize the structure, cellular composition and ciliary function of nasal mucosal organoids in the two groups. The secretion function of the differentiated nasal mucosal organoids in DO group was evaluated using PAS staining.@*RESULTS@#Both of the two organoid culture systems yielded vacuolar or solid spherical 3D organoids, and their diameters increased progressively with time. On day 16 of culture, more vacuolar organoids occurred in DO group, while more solid spherical organoids were seen in EO group, and the proportion of vacuoles was significantly greater in DO group than in EO group [(54.67±13.26)% vs (21.67±8.57)%, P < 0.05]. Short tandem repeat (STR) test of the nasal mucosal organoids and the source tissue showed a 100% match between them. On day 21 of culture, scanning and transmission electron microscopy of the nasal mucosal organoids identified ultrastructure of cilia in DO group and short villi structure in most of the organoids in EO group. Immunohistochemical staining showed positivity for P63 (basal cells), β-tubulin (ciliated columnar cells), and MUC5AC (goblet cells) in the organoids. Compared with those in EO group, the organoids in DO group showed significantly greater percentages of ciliated cells [(7.95±1.81)% vs (27.04±5.91)%, P < 0.05] and goblet cells [(14.46±0.93)% vs (39.85±5.43)%, P < 0.05) with a similar percentage of basal cells [(56.91±14.12)% vs (53.42±15.77)%, P > 0.05]. The differentiated nasal mucosal organoids in DO group were positively stained for glycogen.@*CONCLUSION@#The staged expansion-differentiation culture method allows more stable and prolonged growth of the cultured cells in vitro to produce organoids with controllable differentiation closely resembling the morphological structure and functions (ciliary function and secretory function) of the source tissue.


Subject(s)
Cell Differentiation , Cells, Cultured , Epithelial Cells , Humans , Nasal Mucosa , Organoids
6.
Article in English | WPRIM | ID: wpr-939855

ABSTRACT

Tumor volume increases continuously in the advanced stage, and aside from the self-renewal of tumor cells, whether the oncogenic transformation of surrounding normal cells is involved in this process is currently unclear. Here, we show that oral squamous cell carcinoma (OSCC)-derived small extracellular vesicles (sEVs) promote the proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of normal epithelial cells but delay their apoptosis. In addition, nuclear-cytoplasmic invaginations and multiple nucleoli are observed in sEV-treated normal cells, both of which are typical characteristics of premalignant lesions of OSCC. Mechanistically, miR-let-7c in OSCC-derived sEVs is transferred to normal epithelial cells, leading to the transcriptional inhibition of p53 and inactivation of the p53/PTEN pathway. In summary, we demonstrate that OSCC-derived sEVs promote the precancerous transformation of normal epithelial cells, in which the miR-let-7c/p53/PTEN pathway plays an important role. Our findings reveal that cancer cells can corrupt normal epithelial cells through sEVs, which provides new insight into the progression of OSCC.


Subject(s)
Carcinoma, Squamous Cell/pathology , Cell Line, Tumor , Cell Movement , Cell Proliferation , Cell Transformation, Neoplastic , Down-Regulation , Epithelial Cells/metabolism , Extracellular Vesicles/pathology , Humans , MicroRNAs/metabolism , Mouth Neoplasms/pathology , PTEN Phosphohydrolase/metabolism , Tumor Suppressor Protein p53/metabolism
7.
Article in English | WPRIM | ID: wpr-939585

ABSTRACT

Objective@#Fine particulate matter (PM 2.5) is an air pollutant that has become of great concern in recent years. Numerous studies have found that PM 2.5 may contribute to lung cancer, but the pathogenesis has not yet been fully elucidated. In this study, we explored the roles of exosomes from bronchial epithelial cells in PM 2.5-promoted lung cancer metastasis.@*Methods@#Exosomes were isolated from cell supernatants. An animal model of lung metastasis (established by tail vein injection of A549-luc) and in vitro studies with lung cancer cell lines were used to investigate the effects of exosomes derived from PM 2.5-treated human bronchial epithelial cells (PHBE-exo).@*Results@#The animal experiments revealed that PHBE-exo-treated mice showed stronger luciferase activity and a larger relative metastatic region in the lungs, thus indicating that PHBE-exo promoted the metastatic potential of lung cancer. Additionally, PHBE-exo promoted the migration, invasion and epithelial-to-mesenchymal transition of lung cancer cells, in a manner mediated by activation of c-Jun N-terminal kinase.@*Conclusion@#These results implied that PM 2.5 may promote the development of lung cancer through exosomes derived from bronchial epithelial cells, thus providing a potential interventional target for lung cancer. These findings broadened our understanding of cancer-promoting mechanisms of environmental pollutants.


Subject(s)
Animals , Epithelial Cells/metabolism , Epithelial-Mesenchymal Transition , Exosomes/metabolism , Humans , Lung Neoplasms/metabolism , Mice , Particulate Matter/toxicity
8.
Acta Physiologica Sinica ; (6): 381-391, 2022.
Article in Chinese | WPRIM | ID: wpr-939573

ABSTRACT

Extracellular matrix (ECM) stiffness is closely related to the physiological and pathological states of breast tissue. The current study was aimed to investigate the effect of silk fibroin/collagen composite hydrogels with adjustable matrix stiffness on the growth and phenotype of normal breast epithelial cells. In this study, the enzymatic reaction of horseradish peroxidase (HRP) with hydrogen peroxide (H2O2) was used to change the degree of cross-linking of the silk fibroin solution. The rotational rheometer was used to characterize the composite hydrogel's biomechanical properties. Human normal mammary epithelial cell line MCF-10A were inoculated into composite hydrogels with various stiffness (19.10-4 932.36 Pa) to construct a three dimensional (3D) culture system of mammary epithelial cells. The CCK-8 assay was applied to detect the cell proliferation rate and active states in each group. Hematoxylin-Eosin (HE) staining and whole-mount magenta staining were used for histological evaluation of cell morphology and distribution. The results showed that with the increase of matrix stiffness, MCF-10A cells exhibited inhibited proliferation rate, decreased formation of acinus structures and increased branching structures. Meanwhile, with the increase of matrix stiffness, the polarity of MCF-10A cells was impeded. And the increase of matrix stiffness up-regulated the expression levels of mmp-2, mmp-3, and mmp-9 in MCF-10A cells. Among the genes related to epithelial-mesenchymal transition (EMT), the expression level of the epithelial marker gene E-cadherin was significantly down-regulated, while the interstitial cell marker gene Vimentin was up-regulated, and the expression levels of Snail, Wnt5b and Integrin β1 in the Wnt pathway were up-regulated. These results suggest that the silk fibroin/collagen composite hydrogels with adjustable matrix stiffness regulates the proliferation and the phenotype of MCF-10A cells. The effects of increased matrix stiffness may be closely related to the changes of the polar structures and function of MCF-10A cells, as well as the occurrence of ECM-remodeling and EMT.


Subject(s)
Collagen/metabolism , Epithelial Cells/metabolism , Fibroins/pharmacology , Humans , Hydrogels/metabolism , Hydrogen Peroxide , Phenotype
9.
Article in Chinese | WPRIM | ID: wpr-935804

ABSTRACT

Objective: To construct a recombinant lentiviral vector for mouse miR-204 overexpression, and to verify the targeted regulation of miR-204 and DVL3 in silica (SiO(2)) -induced mouse lung epithelial cells (MLE-12 cells) . Methods: In October 2019, the pre-miR-204 gene was amplified from the mouse genome by the polymerase chain reaction (PCR) method. After sequencing, the amplified product was cloned into the pLenti-CMV-EGFP lentiviral vector. The positive clones were identified by PCR screening and sequencing. The miR-204 overexpressed lentiviral vector was transfected into 293T cells, and lentiviral packaging and titer determination were performed. The experiment was divided into SiO(2) control group, virus control group, and miR-204 virus group, and the expressions of miR-204 and DVL3 gene were detected by real-time PCR. Results: The miR-204 lentiviral expression vector Lv-miR-204-5p was constructed and identified correctly by PCR and sequencing, and a virus dilution with a titer of 9.57×10(8) IU/ml was obtained. The results of real-time PCR showed that the expression of miR-204 in MLE-12 cells of the miR-204 virus group was higher than that of SiO(2) control group and virus control group, and the expression of DVL3 gene was lower than that of SiO(2) control group and virus control group, the differences were statistically significant (P<0.05) . Conclusion: Overexpression of miR-204 by lentiviral vector may inhibit the expression of DVL3 gene in silica-induced mouse lung epithelial cells.


Subject(s)
Animals , Epithelial Cells , Genetic Vectors , Lentivirus/metabolism , Lung , Mice , MicroRNAs/metabolism , Silicon Dioxide/toxicity , Transfection
10.
Article in Chinese | WPRIM | ID: wpr-928380

ABSTRACT

OBJECTIVE@#To investigate the effect and possible mechanism of BDNF-AS on renal tubular epithelial cell injury induced by high glucose.@*METHODS@#Human renal tubular epithelial cells HK-2 were cultured in vitro and transfected with BDNF-AS small interfering RNA or miR-145-5p mimic, or co-transfected with BDNF-AS small interfering RNA and miR-145-5p inhibitor, respectively. The cells were then intervened with 30 mmol/L glucose for 24 hours. The expression of BDNF-AS and miR-145-5p were detected by RT-qPCR. Cell proliferation was detected by CCK-8, and apoptosis was detected by flow cytometry. The expression of Bcl-2 and Bax proteins were detected by Western blotting, and the levels of IL-1β and IL-6 in cell culture supernatant were detected by enzyme-linked immunosorbent assay. Dual luciferase reporter gene experiment was used to verify the regulatory relationship of BDNF-AS with miR-145-5p.@*RESULTS@#High glucose promoted the expression of BDNF-AS in HK-2 cells (P<0.05), but inhibited that of miR-145-5p (P<0.05). Interfering with BDNF-AS or overexpression of miR-145-5p decreased the inhibition rate, apoptosis rate and expression of Bax protein, IL-1β and IL-6 of HK-2 cells induced by high glucose (P<0.05), but promoted the expression of Bcl-2 protein (P<0.05). Interfering with miR-145-5p reversed the effect of interfering with BDNF-AS on the proliferation, apoptosis rate and the expression of IL-1β and IL-6 of HK-2 cells induced by high glucose. BDNF-AS could target and down-regulate miR-145-5p.@*CONCLUSION@#Interfering with BDNF-AS may promote the proliferation of renal tubular epithelial cells induced by high glucose and inhibit cell apoptosis and the expression of inflammatory factor by down-regulating miR-145-5p.


Subject(s)
Apoptosis , Brain-Derived Neurotrophic Factor/genetics , Cell Proliferation , Epithelial Cells , Glucose , Humans , MicroRNAs/genetics
11.
Acta Physiologica Sinica ; (6): 80-92, 2022.
Article in Chinese | WPRIM | ID: wpr-927584

ABSTRACT

Human amniotic epithelial cells (hAECs) are epithelial cells located on the placental amnion near the fetus. Different from other placental-derived stem cells, hAECs are derived from embryonic epiblast, and have been considered as seed cells for regenerative medicine. hAECs possess embryonic stem cell-like multi-differentiation capabilities and adult stem cell-like immunomodulatory properties. Compared with other types of stem cells, special properties of hAECs make them unique, including easy isolation, abundant cell numbers, non-tumorigenicity after transplantation, and the obviation of ethical debates. During the past two decades, the therapeutic potential of hAECs has been extensively investigated in various diseases. Accumulating evidence has demonstrated that hAECs contribute to repairing and remodeling the function of damaged tissues and organs through different molecular mechanisms. This article provides an in-depth review of the biological characteristics of hAECs, summarizes the research status of hAECs, and discusses the clinical application prospects of hAEC-based cell therapy.


Subject(s)
Amnion , Cell Differentiation , Epithelial Cells , Female , Humans , Placenta , Pregnancy , Stem Cells
12.
Acta Physiologica Sinica ; (6): 28-38, 2022.
Article in Chinese | WPRIM | ID: wpr-927578

ABSTRACT

Acute kidney injury (AKI) is a common critical clinical disease characterized by a sharp decline of renal function. Ischemia-reperfusion (IR) is one of the main causes of AKI. The mortality of AKI remains high due to the lack of early diagnosis and cause specific treatment. IR rapidly initiates innate immune responses, activates complement and innate immune cells, releasing a large number of injury-related molecules such as high mobility group box-1 (HMGB1), inflammatory mediators such as caspase-3, and then recruits immune inflammatory cells including M1 macrophages (Mϕ) to the microenvironment of injury, causing apoptosis and necrosis of renal tubular epithelial cells (TECs). Dead cells and associated inflammation further activate the adaptive immune system, which not only aggravates tissue damage, but also initiates M2 Mϕ participated inflammatory clearance, tissue repair and regeneration. Mϕ, professional phagocytes, and TECs, semi-professional phagocytes, can phagocytose around damaged cells including apoptotic Mϕ and TECs, which are key innate immune cells to regulate the outcome of injury, repair or fibrosis. In recent years, it has been found that erythropoietin (EPO) not only binds to the homodimeric receptor (EPOR)2 to induce erythropoiesis, but also binds to the heterodimeric receptor EPOR/βcR, also known as innate repair receptor, which plays renoprotective roles. Properdin is the only positive regulator in the complement activation of alternative pathway. It also can effectively identify and bind to early apoptotic T cells and facilitate phagocytic clearing by Mϕ through a non-complement activation-dependent mechanism. Our previous studies have shown that Mϕ and TECs associated with EPO and its receptors and properdin are involved in IR injury and repair, but the underlying mechanism needs to be further explored. As an important carrier of cell-to-cell signal transmission, exosomes participate in the occurrence and development of a variety of renal diseases. The role of exosomes involved in the interaction between Mϕ and TECs in IR-induced AKI is not fully defined. Based on the available results in the role of Mϕ and TECs in renal IR-induced AKI, this review discussed the role of Mϕ polarization and interaction with TECs in renal IR injury, as well as the participation of EPO and its receptors, properdin and exosomes.


Subject(s)
Acute Kidney Injury/metabolism , Animals , Epithelial Cells/metabolism , Humans , Ischemia/metabolism , Kidney , Macrophages/physiology , Mice , Mice, Inbred C57BL , Reperfusion , Reperfusion Injury
13.
Chinese Medical Journal ; (24): 606-618, 2022.
Article in English | WPRIM | ID: wpr-927543

ABSTRACT

BACKGROUND@#Gene promoter methylation is a major epigenetic change in cancers, which plays critical roles in carcinogenesis. As a crucial regulator in the early stages of B-cell differentiation and embryonic neurodevelopment, the paired box 5 (PAX5) gene is downregulated by methylation in several kinds of tumors and the role of this downregulation in esophageal squamous cell carcinoma (ESCC) pathogenesis remains unclear.@*METHODS@#To elucidate the role of PAX5 in ESCC, eight ESCC cell lines, 51 primary ESCC tissue samples, and eight normal esophageal mucosa samples were studied and The Cancer Genome Atlas (TCGA) was queried. PAX5 expression was examined by reverse transcription-polymerase chain reaction and western blotting. Cell apoptosis, proliferation, and chemosensitivity were detected by flow cytometry, colony formation assays, and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assays in ESCC cell lines with PAX5 overexpression or silencing. Tumor xenograft models were established for in vivo verification.@*RESULTS@#PAX5 methylation was found in 37.3% (19/51) of primary ESCC samples, which was significantly associated with age (P = 0.007) and tumor-node-metastasis stage (P = 0.014). TCGA data analysis indicated that PAX5 expression was inversely correlated with promoter region methylation (r = -0.189, P = 0.011 for cg00464519 and r = -0.228, P = 0.002 for cg02538199). Restoration of PAX5 expression suppressed cell proliferation, promoted apoptosis, and inhibited tumor growth of ESCC cell lines, which was verified in xenografted mice. Ectopic PAX5 expression significantly increased p53 reporter luciferase activity and increased p53 messenger RNA and protein levels. A direct interaction of PAX5 with the p53 promoter region was confirmed by chromatin immunoprecipitation assays. Re-expression of PAX5 sensitized ESCC cell lines KYSE150 and KYSE30 to fluorouracil and docetaxel. Silencing of PAX5 induced resistance of KYSE450 cells to these drugs.@*CONCLUSIONS@#As a tumor suppressor gene regulated by promoter region methylation in human ESCC, PAX5 inhibits proliferation, promotes apoptosis, and induces activation of p53 signaling. PAX5 may serve as a chemosensitive marker of ESCC.


Subject(s)
Animals , Carcinoma, Squamous Cell/genetics , Cell Line, Tumor , Cell Proliferation/genetics , Epithelial Cells/metabolism , Esophageal Neoplasms/genetics , Esophageal Squamous Cell Carcinoma/genetics , Gene Expression Regulation, Neoplastic , Humans , Mice , PAX5 Transcription Factor/genetics , Tumor Suppressor Protein p53/genetics , Xenograft Model Antitumor Assays
14.
Chinese Medical Journal ; (24): 324-332, 2022.
Article in English | WPRIM | ID: wpr-927534

ABSTRACT

BACKGROUND@#Sweat secreted by eccrine sweat glands is transported to the skin surface through the lumen. The eccrine sweat gland develops from the initial solid bud to the final gland structure with a lumen, but how the lumen is formed and the mechanism of lumen formation have not yet been fully elucidated. This study aimed to investigate the mechanism of lumen formation of eccrine gland organoids (EGOs).@*METHODS@#Human eccrine sweat glands were isolated from the skin for tissue culture, and the primary cultured cells were collected and cultured in Matrigel for 14 days in vitro. EGOs at different development days were collected for hematoxylin and eosin (H&E) staining to observe morphological changes and for immunofluorescence staining of proliferation marker Ki67, cellular motility marker filamentous actin (F-actin), and autophagy marker LC3B. Western blotting was used to detect the expression of Ki67, F-actin, and LC3B. Moreover, apoptosis was detected using a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) apoptosis assay kit, and the expression of poly (ADP-ribose) polymerase and Caspase-3 was detected by Western blot. In addition, 3-methyladenine (3MA) was used as an autophagy inhibitor to detect whether the formation of sweat glands can be effectively inhibited.@*RESULTS@#The results showed that a single gland cell proliferated rapidly and formed EGOs on day 4. The earliest lumen formation was observed on day 6. From day 8 to day 14, the rate of lumen formation in EGOs increased significantly. The immunofluorescence and Western blot analyses showed that the expression of Ki67 gradually decreased with the increase in days, while the F-actin expression level did not change. Notably, the expression of autophagy marker LC3B was detected in the interior cells of EGOs as the apoptosis signal of EGOs was negative. Compared with the control group, the autophagy inhibitor 3MA can effectively limit the formation rate of the lumen and reduce the inner diameter of EGOs.@*CONCLUSION@#Using our model of eccrine gland 3D-reconstruction in Matrigel, we determined that autophagy rather than apoptosis plays a role in the lumen formation of EGOs.


Subject(s)
Apoptosis , Autophagy , Eccrine Glands , Epithelial Cells , Humans , Organoids
15.
Article in Chinese | WPRIM | ID: wpr-936322

ABSTRACT

OBJECTIVE@#To explore the role of heat shock protein 90α (HSP90α) and endoplasmic reticulum (ER) stress pathway in allergic airway inflammation induced by house dust mite (HDM) in bronchial epithelial cells.@*METHODS@#A HDM- induced asthmatic cell model was established in human bronchial epithelial (HBE) cells by exposure to a concentration gradient (200, 400 and 800 U/mL) of HDM for 24 h. To test the effect of siHSP90α and HSP90 inhibitor 17-AAG on HDM-induced asthmatic inflammation, HBE cells were transfected with siHSP90α (50 nmol, 12 h) or pretreated with 17-AAG (900 nmol, 6 h) prior to HDM exposure (800 U/mL) for 24 h, and the changes in the expression of HSP90α and ER stress markers were assessed. We also tested the effect of nasal drip of 17-AAG, HDM, or their combination on airway inflammation and ER stress in C57BL/6 mice.@*RESULTS@#In HBE cells, HDM exposure significantly up-regulated the expression of HSP90α protein (P=0.011) and ER stress markers XBP-1 (P=0.044), ATF-6α (P=0.030) and GRP-78 (P=0.027). Knocking down HSP90α and treatment with 17-AAG both significantly inhibited HDM-induced upregulation of XBP-1 (P=0.008). In C57BL/6 mice, treatment with 17-AAG obviously improved HDM-induced airway inflammation and significantly reduced the number of inflammatory cells in the airway (P=0.014) and lowered the levels of IL-4 (P=0.030) and IL-5 (P=0.035) in alveolar lavage fluid. Immunohistochemical staining showed that the expressions of XBP-1 and GRP-78 in airway epithelial cells decreased significantly after the treatment of 17-AAG.@*CONCLUSIONS@#HSP90α promotes HDM-induced airway allergic inflammation possibly by upregulating ER stress pathway in bronchial epithelial cells.


Subject(s)
Animals , Asthma/metabolism , Endoplasmic Reticulum Stress , Epithelial Cells , Inflammation/metabolism , Mice , Mice, Inbred C57BL , Pyroglyphidae
16.
Article in Chinese | WPRIM | ID: wpr-936288

ABSTRACT

OBJECTIVE@#To explore the expression of CCN5 in endometriotic tissues and its impact on proliferation, migration and invasion of human endometrial stromal cells (HESCs).@*METHODS@#We collected ovarian endometriosis samples from 20 women receiving laparoscopic surgery and eutopic endometrium samples from 15 women undergoing IVF-ET for comparison of CCN5 expression. Cultured HESCs were transfected with a recombinant adenovirus Ad-CCN5 for CCN5 overexpression or with a CCN5-specific siRNA for knocking down CCN5 expression, and the changes of cell proliferation, migration and invasion were evaluated using CCK-8 assay, wound healing assay and Transwell chamber assay. RT-qPCR and Western blotting were used to examine the expression levels of epithelial-mesenchymal transition (EMT) markers including E-cadherin, N-cadherin, Snail-1 and vimentin in HESCs with CCN5 overexpression or knockdown.@*RESULTS@#CCN5 expression was significantly decreased in ovarian endometriosis tissues as compared with eutopic endometrium samples (P < 0.01). CCN5 overexpression obviously inhibited the proliferation, migration and invasion of HESCs, significantly increased the expression of E-cadherin and decreased the expressions of N-cadherin, Snail-1 and vimentin (P < 0.01). CCN5 knockdown significantly enhanced the proliferation, migration and invasion of HESCs and produced opposite effects on the expressions of E-cadherin, N-cadherin, Snail-1 and vimentin (P < 0.01).@*CONCLUSION@#CCN5 can regulate the proliferation, migration and invasion of HESCs and thus plays an important role in EMT of HESCs, suggesting the potential of CCN5 as a therapeutic target for endometriosis.


Subject(s)
Cell Movement , Cell Proliferation , Endometriosis/metabolism , Endometrium/metabolism , Epithelial Cells , Epithelial-Mesenchymal Transition , Female , Humans , Stromal Cells
17.
Article in Chinese | WPRIM | ID: wpr-936230

ABSTRACT

Objective: Transcriptome sequencing and bioinformatics analysis were performed on the gene expression of nasal epithelial cells in patients with seasonal allergic rhinitis (AR) and perennial AR, so as to obtain the differences in the gene expression of nasal epithelial cells between seasonal AR and perennial AR. Methods: The human nasal epithelial cell line(HNEpC) was cultured in vitro, treated with 100 μg/ml mugwort or house dust mite (HDM) extracts for 24 hours. Total cell RNA was extracted, and quantitative real-time polymerase chain reaction (qPCR) was used to detect the expression of cytokines, including IL-6, IL-8, IL-33 and thymic stromal lymphopoietin (TSLP). From November 2019 to November 2020, 3 seasonal AR patients, 3 perennial AR patients, and 3 healthy controls who attended the Department of Otolaryngology Head and Neck Surgery, China-Japan Union Hospital of Jilin University were analyzed. The patients' primary nasal epithelial cells were cultured in vitro, treated with corresponding allergens for 24 hours. Total RNA was extracted for transcriptome sequencing, and the sequencing results were analyzed by bioinformatics. Results: The qPCR results showed that the cytokines IL-6, IL-8, IL-33 and TSLP of HNEpC treated with mugworts extracts and HDM extracts had the same trend of change. After the nasal epithelial cells from patients with seasonal AR and perennial AR were treated with corresponding allergens, there were differences in biological processes and signal pathways between those and control. Gene ontology (GO) enrichment analysis showed that the differentially expressed genes (DEG) in AR patients allergic to mugwort were mainly enriched in the oxidation-reduction process, the negative regulation of apoptosis process, and the cell adhesion; the DEG in AR patients allergic to HDM were mainly enriched in cell adhesion, the negative regulation of cell proliferation and the response to drug. Enrichment analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway showed that the DEG of AR patients allergic to mugwort were significantly enriched in arachidonic acid metabolism, p53 signaling pathway and transforming growth factor β (TGF-β) signaling pathway, while the DEG of AR patients allergic to HDM were mainly enriched in cells cycle, Fanconi anemia pathway and DNA replication. Gene Set Enrichment Analysis (GSEA) showed that the inflammatory response, TNF-α/NF-κB signaling pathway and IL-2/STAT5 signaling pathway were significantly up-regulated in AR patients allergic to mugwort, indicating the promotion of inflammatory response; and AR patients allergic to HDM had significant down-regulation of G2M, E2F, and MYC, indicating the inhibition of cell proliferation. The protein-protein interaction network showed that TNF and CDK1 were the most interacting proteins in mugwort and HDM allergic AR patients, respectively. Conclusion: Seasonal AR and perennial AR may affect the different biological processes and signal pathways of nasal epithelial cells, leading to differences in the occurrence and development of AR.


Subject(s)
Allergens , Animals , Computational Biology , Cytokines/metabolism , Epithelial Cells/metabolism , Gene Expression , Humans , Interleukin-33/metabolism , Interleukin-6/metabolism , Interleukin-8 , Nasal Mucosa/metabolism , Plant Extracts/metabolism , Pyroglyphidae , RNA/metabolism , Rhinitis, Allergic/metabolism , Rhinitis, Allergic, Perennial , Rhinitis, Allergic, Seasonal , Seasons
18.
Article in English | WPRIM | ID: wpr-928985

ABSTRACT

OBJECTIVES@#Bladder cancer is one of the most common urothelial tumors with high incidence and mortality rates. Although it has been reported that microRNA (miR)-133b can regulate tumorigenesis of bladder cancer, the mechanism remains unclear. Sex-determining region Y-box transcription factor 4 (SOX4) exhibits an important role in tumorigenesis, but it is unclear whether SOX4 and miR-133b are associated with regulation of pathogenesis of bladder cancer. This study aims to determine the expressions of SOX4 and miR-133b in bladder cancer tissues and cells, investigate their effects on the proliferation, colony formation, and invasion of bladder cancer cells, and to explore the association between miR-133b and SOX4 in regulating biological featurss of bladder cancer cells.@*METHODS@#The bladder cancer and adjacent tissue samples of 10 patients who underwent surgical resection in the Second Xiangya Hospital of Central South Universty from Januray to June 2015 were obtained. The levels of miR-133b were tested by real-time PCR, and the protein levels of SOX4 were evaluated using Western blotting in bladder cancer tissues, matched adjacent tissues, and cell lines. The correlation between miR-133b expression and SOX4 expression in bladder cancer tissues was analyzed. Using the online database TargetScan, the relationship between SOX4 and miR-133b was predicted. MiR-133b mimics, miR-133b inhibitor, and short hairpin RNA (shRNA)-SOX4 were transfected into T24 cells by Lipofectamine 2000. The relationship between miR-133b and SOX4 was also verified by a dual-luciferase reporter assay. The proliferation of T24 cells cultured for 0, 12, 48, 72, and 96 h was evaluated by cell counting kit-8 (CCK-8) assay. The colony formation capacity of bladder cancer cells was tested after 14-day culture, and cell invasion capacity was evaluated with Transwell invasion assay.@*RESULTS@#Bladder cancer tissue and bladder cancer cells had low level of miR-133b but high level of SOX4, compared with matched adjacent tissues and normal bladder epithelial cells. A negative correlation between miR-133b mRNA and SOX4 protein levels in bladder cancer tissues was also found (r=-0.84). The results of online database TargetScan showed that miR-133b targets at SOX4, and overexpression of miR-133b significantly attenuated the expression of SOX4 in T24 cells. Both overexpression of miR-133b and knockdown of SOX4 significantly inhibited the proliferation, colony formation, and invasion capacity of bladder cancer cells in vitro. SOX4 down-regulation restored the effects of miR-133b inhibitor on the proliferation, colony formation, and invasion capacity of T24 cells.@*CONCLUSIONS@#The up-regulation of SOX4 contributes to the progression of bladder cancer, and miR-133b can regulate the proliferation, colony formation, and invasion of bladder cancer cells via inhibiting SOX4.


Subject(s)
Carcinogenesis/genetics , Cell Line, Tumor , Cell Movement/genetics , Cell Proliferation/genetics , Epithelial Cells/metabolism , Gene Expression Regulation, Neoplastic , Humans , MicroRNAs/genetics , SOXC Transcription Factors/genetics , Urinary Bladder , Urinary Bladder Neoplasms/genetics
19.
São Paulo; s.n; s.n; 2022. 172 p. tab, graf.
Thesis in English | LILACS | ID: biblio-1378625

ABSTRACT

The solar ultraviolet (UV) radiation that reaches the Earth is composed of 95% of UVA (320 to 400 nm) and 5% of UVB (280 to 320 nm) radiation. UVB is carcinogenic, generating potentially mutagenic DNA lesions. The solar UVA radiation also causes DNA damage, but this fact does not fully account for its biological impact. UVA is absorbed by non-DNA cellular chromophores, generating reactive oxygen species such as singlet oxygen. Knowing the proteome mediates stress responses in cells, here we investigated the cellular effects of a non-cytotoxic dose of UVA radiation, equivalent to about 20 minutes of midday sun exposure, on the proteome of human keratinocytes. Using a combination of mass spectrometry-based proteomics, bioinformatics, and conventional biochemical assays, we analyzed two aspects of UVA-induced stress: spatial remodeling of the proteome in subcellular compartments 30 minutes after stress and long-term changes in protein levels and secretion (24 hours and 7 days postirradiation). In the first part of this thesis, we quantified and assigned subcellular localization for over 3000 proteins, of which about 600 potentially redistribute upon UVA exposure. Protein redistributions were accompanied by redox modulations, mitochondrial fragmentation and DNA damage. In the second part of the work, our results showed that primary human keratinocytes enter senescence upon exposure to a single dose of UVA, mounting antioxidant and inflammatory responses. Cells under UVA-induced senescence further elicit paracrine responses in neighboring premalignant HaCaT epithelial cells via inflammatory mediators. Altogether, these results reiterate the role of UVA radiation as a potent metabolic stressor in the skin


A radiação ultravioleta (UV) solar que atinge a superfície terrestre é composta por 95% de radiação UVA (320 a 400 nm) e 5% de radiação UVB (280 a 320 nm). A radiação UVB é carcinogênica e gera lesões potencialmente mutagênicas no DNA. A radiação UVA solar também gera danos no DNA, mas a genotoxicidade dessa radiação não explica inteiramente o seu impacto biológico. Atualmente, sabe-se que a radiação UVA é absorvida por cromóforos celulares, gerando espécies reativas de oxigênio, como o oxigênio singlete. Sabendo que o proteoma é um mediador de respostas ao estresse celular, nós investigamos os efeitos celulares de uma dose não-citotóxica de radiação UVA, equivalente a cerca de 20 minutos de exposição ao sol, no proteoma de queratinócitos humanos. Utilizando espectrometria de massas, bioinformática e ensaios bioquímicos convencionais, nós analisamos dois aspectos do estresse induzido por radiação UVA: o remodelamento espacial do proteoma 30 minutos depois do estresse e alterações nos níveis e na secreção de proteínas no longo prazo (24 horas e 7 dias depois da irradiação). Na primeira parte desta tese, nós quantificamos e atribuímos classificações de localização subcelular a mais de 3000 proteínas. Dentre essas proteínas, 600 tem potencialmente a sua distribuição subcelular alterada em resposta à radiação. As redistribuições subcelulares são acompanhadas de modulações redox, fragmentação mitocondrial e danos no DNA. Na segunda parte da tese, os nossos resultados mostraram que queratinócitos humanos primários entram em senescência sob exposição a uma única dose de radiação UVA, montando respostas antioxidantes e pró-inflamatórias. Células sob senescência induzida por UVA, por sua vez, desencadeiam respostas parácrinas em queratinócitos pré-tumorais (células HaCaT) por meio de mediadores inflamatórios. Em conjunto, esses resultados reiteram o papel da radiação UVA como um potente estressor metabólico em células da pele


Subject(s)
Skin , Ultraviolet Rays/adverse effects , Keratinocytes/chemistry , Proteomics/classification , Radiation Dosage , Mass Spectrometry/methods , DNA , Epithelial Cells/classification , Genotoxicity/adverse effects , HaCaT Cells/classification , Antioxidants/adverse effects
20.
Arq. gastroenterol ; 58(3): 353-358, July-Sept. 2021. tab
Article in English | LILACS | ID: biblio-1345299

ABSTRACT

ABSTRACT BACKGROUND: The Prex2 protein is a member of the Rac family proteins that belongs to small G proteins with a critical role in cell migration, cell proliferation, and apoptosis through its effects on PI3K cell signaling pathway and phosphatase activity of PTEN protein. The effect of PREX2 gene expression has been shown in some cancer cells. A survey of PREX2 gene expression in gastric antral epithelial cells of gastric cancer patients with Helicobacter pylori various genotypes infection can conduct to better understanding H. pylori infection's carcinogenesis. METHODS: In a case-control study, PREX2 gene expression was evaluated in gastric antral biopsy samples on four groups of patients referred to Sanandaj hospitals, including gastritis with (n=23) and without (n=27) H. pylori infection and gastric cancer with (n=21) and without (n=32) H. pylori infection. Each gastric biopsy sample's total RNA was extracted and cDNA synthesized by using Kits (Takara Company). The PREX2 gene expression was measured using the relative quantitative real-time RT-PCR method and ΔΔCt formula. RESULTS: The PREX2 gene expression increased in gastric antral biopsy samples of gastritis and gastric cancer patients with H. pylori infection (case groups) than patients without H. pylori infection (control groups) 2.38 and 2.27 times, respectively. The patients with H. pylori vacA s1m1 and sabB genotypes infection showed a significant increase of PREX2 gene expression in gastric cancer antral epithelial cells. CONCLUSION: H. pylori vacA s1m1 and sabB genotypes have the positive correlations with PREX2 gene expression in gastric antral epithelial cells of gastritis and gastric cancer patients.


RESUMO CONTEXTO: A proteína Prex2 é membro das proteínas da família Rac que pertencem a pequenas proteínas G com um papel crítico na migração celular, na proliferação celular e na apoptose através de seus efeitos na via de sinalização celular PI3K e atividade fosfatase da proteína PTEN. O efeito da expressão genética PREX2 tem sido mostrada em algumas células cancerosas. Um levantamento da expressão genética PREX2 em células epiteliais antrais gástricas de pacientes infectados com vários genótipos de Helicobacter pylori pode conduzir a um melhor entendimento da carcinogênese da infecção por H. pylori. MÉTODOS: Em estudo de caso-controle, a expressão genética PREX2 foi avaliada em amostras de biópsia antral gástrica em quatro grupos de pacientes encaminhados aos hospitais de Sanandaj, incluindo gastrite com (n=23) e sem (n=27) infecção por H. pylori e de câncer gástrico com (n=21) e sem (n=32) infecção por H. pylori. O RNA total de cada amostra de biópsia gástrica foi extraído e cDNA sintetizado por meio de kits (Takara Company). A expressão genética PREX2 foi medida utilizando-se o método RT-PCR em tempo real quantitativo relativo e a fórmula ΔΔCt. RESULTADOS: A expressão genética PREX2 aumentou em amostras de biópsia antral gástrica de pacientes com gastrite e câncer gástrico com infecção por H. pylori (grupos de casos) em relação aos sem infecção por H. pylori (grupos de controle) 2,38 e 2,27 vezes, respectivamente. Os pacientes com infecção por genótipos H. pylori vacA s1m1 e sabB apresentaram um aumento significativo da expressão genética PREX2 em células epiteliais antrais de câncer gástrico. CONCLUSÃO: Os genótipos H. pylori vacA s1m1 e sabB têm correlações positivas com a expressão genética PREX2 em células epiteliais antrais gástricas de pacientes com câncer gástrico e gastrites.


Subject(s)
Humans , Helicobacter Infections , Guanine Nucleotide Exchange Factors/genetics , Gastritis/genetics , Gastritis/microbiology , Case-Control Studies , Helicobacter pylori , Epithelial Cells/metabolism , Epithelial Cells/microbiology , Gastric Mucosa
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