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1.
Adv Exp Med Biol ; 1152: 413-427, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31456197

RESUMO

For decades 2D culture has been used to study breast cancer. In recent years, however, the importance of 3D culture to recapitulate the complexity of human disease has received attention. A breakthrough for 3D culture came as a result of a Nature editorial 'Goodbye Flat Biology' (Anonymous, Nature 424:861-861, 2003). Since then scientists have developed and implemented a range of different and more clinically relevant models, which are used to study breast cancer. In this chapter multiple different 3D models will be discussed including spheroids, microfluidic and bio-printed models and in silico models.


Assuntos
Neoplasias da Mama/patologia , Técnicas de Cultura de Células , Feminino , Humanos , Microfluídica , Modelos Anatômicos , Impressão Tridimensional
2.
Braz Oral Res ; 33: e058, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31432925

RESUMO

Cementum is the mineralized tissue covering the tooth root that functions in tooth attachment and post-eruptive adjustment of tooth position. It has been reported to be highly similar to bone in several respects but remains poorly understood in terms of development and regeneration. Here, we investigate whether cementocytes, the residing cells in cellular cementum, have the potential to be protagonist in cementum homeostasis, responding to endocrine signals and directing local cementum metabolism. Cells from healthy erupted human teeth were isolated using sequential collagenase/EDTA digestions, and maintained in standard cell culture conditions. A cementocyte-like cell line was cloned (HCY-23, for human cementocyte clone 23), which presented a cementocyte compatible gene expression signature, including the expression of dentin matrix protein 1 ( DMP1 ), sclerostin ( SOST ), and E11/gp38/podoplanin ( E11 ). In contrast, these cells did not express the odontoblast/dentin marker dentin sialoprotein ( DSPP ). HCY-23 cells produced mineral-like nodules in vitro under differentiation conditions, and were highly responsive to inorganic phosphate (Pi). Within the limits of the present study, it can be concluded that cementocytes are phosphate-responsive cells, and have the potential do play a key role in periodontal homeostasis and regeneration.


Assuntos
Técnicas de Cultura de Células/métodos , Cemento Dentário/citologia , Adolescente , Adulto , Análise de Variância , Proteínas Morfogenéticas Ósseas/análise , Proteínas Morfogenéticas Ósseas/genética , Linhagem Celular , Cemento Dentário/metabolismo , Proteínas da Matriz Extracelular/análise , Proteínas da Matriz Extracelular/genética , Feminino , Imunofluorescência , Expressão Gênica , Marcadores Genéticos/genética , Humanos , Masculino , Glicoproteínas de Membrana/análise , Glicoproteínas de Membrana/genética , Dente Molar/citologia , Fosfatos/farmacologia , Fosfoproteínas/análise , Fosfoproteínas/genética , Sialoglicoproteínas/análise , Sialoglicoproteínas/genética , Fatores de Tempo , Adulto Jovem
3.
Tumour Biol ; 41(8): 1010428319866369, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31402761

RESUMO

Gaining a better understanding of the biological properties of cell-free DNA constitutes an important step in the development of clinically meaningful cell-free DNA-based tests. Since the in vivo characterization of cell-free DNA is complicated by the immense heterogeneity of blood samples, an increasing number of in vitro cell culture experiments, which offer a greater level of control, are being conducted. However, cell culture studies are currently faced with three notable caveats. First, the concentration of cell-free DNA in vitro is relatively low. Second, the median amount and size of cell-free DNA in culture medium varies greatly between cell types. Third, the amount and size of cell-free DNA in the culture medium of a single cell line fluctuates over time. Although these are interesting findings, it can also be a great source of experimental confusion and emphasizes the importance of method optimization and standardization. Therefore, in this study, we compared five commonly used cell-free DNA quantification methods, including quantitative polymerase chain reaction, Qubit Double-Stranded DNA High Sensitivity assay, Quant-iT PicoGreen Assay, Bioanalyzer High Sensitivity DNA assay, and NanoDrop Onec. Analysis of the resulting data, along with an interpretation of theoretical values (i.e. the theoretical detection and quantification limits of the respective methods), enables the calculation of optimal conditions for several important preanalytical steps pertaining to each quantification method and different cell types, including the (1) time-point at which culture medium should be collected for cell-free DNA extraction, (2) amount of cell culture supernatant from which to isolate cell-free DNA, (3) volume of elution buffer, and (4) volume of cell-free DNA sample to use for quantification.


Assuntos
Ácidos Nucleicos Livres/química , Meios de Cultura/química , Técnicas de Cultura de Células , Corantes Fluorescentes/química , Humanos , Compostos Orgânicos/química
4.
Sheng Wu Gong Cheng Xue Bao ; 35(8): 1374-1381, 2019 Aug 25.
Artigo em Chinês | MEDLINE | ID: mdl-31441608

RESUMO

As one of the top 10 breakthrough and emerging technologies in the world in 2018, cultured meat has attracted extensive attention due to its advantages of traceable origin, food safety and green sustainable development. Europe and the United States have invested a lot of resources to focus on research about cultured meat, which will affect our domestic meat and food market in the future. At present, the challenge of cultured meat production is how to efficiently simulate the growth environment of animal muscle tissue and realize large-scale production in bioreactor. Although cell tissue engineering has been deeply studied and achieved varying successful application, it is still difficult to obtain large-scale cultured meat production due to the high cost and technical requirements. Therefore, the development of efficient and safe cell culture technology is an urgent problem for large-scale cultured meat production, which can effectively reduce costs and achieve industrial application. In this review, we summarize the research progress of animal cell tissue culture technology used for cultured meat, and highlighted the current challenges and possible strategies in further applications.


Assuntos
Técnicas de Cultura de Células , Carne , Animais , Reatores Biológicos , Engenharia Tecidual , Estados Unidos
5.
Adv Exp Med Biol ; 1158: 183-196, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31452141

RESUMO

The term 'mitochondrial dynamics' is commonly used to refer to ongoing fusion and fission of mitochondrial structures within a living cell. A growing number of diseases, from Charcot Marie Tooth Type 2a neuropathies to cancer, is known to be associated with the dysregulation of mitochondrial dynamics, leading to irregularities of mitochondrial network morphology that are associated with aberrant metabolism and cellular dysfunction. Studying these phenomena, and potential pharmacological interventions to correct them, in cultured cells is a powerful approach to developing treatments or cures. Appropriately designed experiments and quantitative approaches for characterizing mitochondrial morphology and function are essential for furthering our understanding. In this chapter, we discuss the importance of cell incubation conditions, choices around imaging modalities, and data analysis tools with respect to experimental outcomes and the interpretation of results from studies of mitochondrial dynamics. We focus primarily on the quantitative analysis of mitochondrial morphology, providing an overview of the available tools and approaches currently being used and discussing some of the strengths and weaknesses associated with each. Finally, we discuss how the ongoing development of imaging and analysis tools continues to improve our ability to study normal and aberrant mitochondrial physiology in vitro and in vivo.


Assuntos
Mitocôndrias , Dinâmica Mitocondrial , Proteínas Mitocondriais , Técnicas de Cultura de Células , Linhagem Celular , Doença de Charcot-Marie-Tooth/fisiopatologia , Humanos , Mitocôndrias/patologia , Mitocôndrias/fisiologia , Dinâmica Mitocondrial/fisiologia , Proteínas Mitocondriais/metabolismo , Neoplasias/fisiopatologia
6.
Zhonghua Gan Zang Bing Za Zhi ; 27(6): 424-429, 2019 Jun 20.
Artigo em Chinês | MEDLINE | ID: mdl-31357757

RESUMO

Objective: To explore the effect of substrate mechanical microenvironment and cell-cell interaction on differentiation of bone marrow mesenchymal stem cells (BMSCs), intrahepatic cellular function and phenotype. Methods: Bone marrow mesenchymal stem cells (BMSCs)-hepatocytes (HCs) and BMSCs-hepatic stellate cells (HSCs) were co-cultured on polyvinyl alcohol (PVA) hydrogel substrates at different stiffness (4.50 ± 0.47 kPa, 19.00 ± 3.51 kPa and 37.00 ± 2.09 kPa) by non-contact co-culture method. Furthermore, the effect of substrate mechanical microenvironment on BMSCs, HCs and HSCs and the activation and proliferation of HCs under different co-cultured condition was studied. A Student's t-test was used to compare the two groups. Results: The expression ofα-smooth muscle actin (α-SMA) and collagenα1- I (Col1A1) in BMSCs and HSCs cultured on its own increased with increase of substrate stiffness. After 72 h, the expression of albumin (ALB) of HCs on three stiff substrates was significantly higher than that of 24 and 48 h. Moreover, the expression of ALB of HCs increased with the increase of substrate stiffness. During the co-culture of BMSCs and HSCs, BMSCs of all three stiffness substrates promoted the expression ofα-SMA, Col1A1 in HSCs, but reduced the expression of PPARγin HSCs cells, thererby promoted the activation of HSCs, with apparent stiffness at 37 kPa. HSCs promoted the expression of ABL in BMSCs at three stiff substrates, but inhibited the expression of alpha-SMA and Col1A1 in BMSCs at 37 kPa, suggesting that co-culture had inhibited the differentiation of BMSCs myofibroblasts, and promoted the differentiation of hepatocyte-like cells, especially at high stiff substrates. In the co-culture of BMSCs and hepatic parenchymal cells, BMSCs had promoted the proliferation of hepatic parenchymal cells at 4.5 kPa. Further, hepatic parenchymal cells had inhibited the expression ofα-SMA in BMSCs, and promoted the expression of Alb, with inhibition of BMSCs differentiation towards myofibroblasts. Conclusion: The differentiation of BMSCs affects the substrate mechanical microenvironment, co-culture of HCs and HSCs. Simultaneously, affecting the function of hepatocytes in relation to the mechanical state of the substrates.


Assuntos
Células da Medula Óssea , Comunicação Celular , Técnicas de Cultura de Células , Diferenciação Celular , Células Estreladas do Fígado , Células-Tronco Mesenquimais , Animais , Células da Medula Óssea/citologia , Comunicação Celular/fisiologia , Técnicas de Cultura de Células/métodos , Células Cultivadas , Microambiente Celular/fisiologia , Células Estreladas do Fígado/citologia , Humanos , Células-Tronco Mesenquimais/citologia , Ratos , Ratos Sprague-Dawley
7.
Biol Res ; 52(1): 39, 2019 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-31358053

RESUMO

In the growth condition(s) of plants, numerous secondary metabolites (SMs) are produced by them to serve variety of cellular functions essential for physiological processes, and recent increasing evidences have implicated stress and defense response signaling in their production. The type and concentration(s) of secondary molecule(s) produced by a plant are determined by the species, genotype, physiology, developmental stage and environmental factors during growth. This suggests the physiological adaptive responses employed by various plant taxonomic groups in coping with the stress and defensive stimuli. The past recent decades had witnessed renewed interest to study abiotic factors that influence secondary metabolism during in vitro and in vivo growth of plants. Application of molecular biology tools and techniques are facilitating understanding the signaling processes and pathways involved in the SMs production at subcellular, cellular, organ and whole plant systems during in vivo and in vitro growth, with application in metabolic engineering of biosynthetic pathways intermediates.


Assuntos
Reguladores de Crescimento de Planta/metabolismo , Fenômenos Fisiológicos Vegetais , Metabolismo Secundário/fisiologia , Estresse Fisiológico/fisiologia , Técnicas de Cultura de Células , Regulação da Expressão Gênica de Plantas/fisiologia , Raízes de Plantas/metabolismo , Brotos de Planta/metabolismo , Plantas/metabolismo , Transdução de Sinais
8.
Adv Exp Med Biol ; 1140: 531-539, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31347069

RESUMO

Stable isotope labeling by amino acids in cell culture (SILAC) is a powerful approach for high-throughput quantitative proteomics. SILAC allows highly accurate protein quantitation through metabolic encoding of whole cell proteomes using stable isotope labeled amino acids. Since its introduction in 2002, SILAC has become increasingly popular. In this chapter we review the methodology and application of SILAC, with an emphasis on three research areas: dynamics of posttranslational modifications, protein-protein interactions, and protein turnover.


Assuntos
Aminoácidos/química , Técnicas de Cultura de Células , Marcação por Isótopo , Proteômica/métodos , Proteoma
9.
Adv Exp Med Biol ; 1140: 575-583, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31347072

RESUMO

The global measurement of assembly and turnover of protein containing complexes within cells has advanced with the development of pulse stable isotope labelled amino acid approaches. Stable isotope labeling with amino acids in cell culture (SILAC) allows the incorporation of "light" 12-carbon amino acids or "heavy" 13-carbon amino acids into cells or organisms and the quantitation of proteins and peptides containing these amino acid tags using mass spectrometry. The use of these labels in pulse or pulse-chase scenarios has enabled measurements of macromolecular dynamics in cells, on time scales of several hours. Here we review advances with this approach and alternative or parallel strategies. We also examine the statistical considerations impacting datasets detailing mitochondrial assembly, to highlight key parameters in establishing significance and reproducibility.


Assuntos
Aminoácidos/química , Técnicas de Cultura de Células , Marcação por Isótopo , Espectrometria de Massas , Proteínas/análise , Reprodutibilidade dos Testes
10.
Anticancer Res ; 39(7): 3413-3418, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31262863

RESUMO

One aim of cell-based in vitro assays is to identify the best drug candidate to develop using the best tumor cell model. This is challenging in every anticancer drug discovery process. Briefly, we summarize the parameters to be taken into account when performing in vitro cell assays, in order to obtain reliable and reproducible results, which was fundamentally discussed by lecturers at the educational course on preclinical and early-phase clinical pharmacology studies, at the 40th Winter Meeting of the Pharmacology and Molecular Mechanisms Group of the European Organization for Research and Treatment of Cancer. Moreover, specific cellular sensitivity tests are described. In addition to monolayer in vitro cell models for the screening of new potential candidate drugs, three-dimensional tumor/cell tissue models are emerging as new pre-clinical tools that more closely reflect the in vivo microenvironment. Therefore, the use of different in vitro models for drug screening can enhance the predictability and reliability of pre-clinical drug-discovery phases and target validation.


Assuntos
Avaliação Pré-Clínica de Medicamentos , Farmacologia Clínica/métodos , Bioensaio , Técnicas de Cultura de Células , Humanos
11.
Cancer Sci ; 110(9): 2806-2821, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31254429

RESUMO

In human and dogs, bladder cancer (BC) is the most common neoplasm affecting the urinary tract. Dog BC resembles human muscle-invasive BC in histopathological characteristics and gene expression profiles, and could be an important research model for this disease. Cancer patient-derived organoid culture can recapitulate organ structures and maintains the gene expression profiles of original tumor tissues. In a previous study, we generated dog prostate cancer organoids using urine samples, however dog BC organoids had never been produced. Therefore we aimed to generate dog BC organoids using urine samples and check their histopathological characteristics, drug sensitivity, and gene expression profiles. Organoids from individual BC dogs were successfully generated, expressed urothelial cell markers (CK7, CK20, and UPK3A) and exhibited tumorigenesis in vivo. In a cell viability assay, the response to combined treatment with a range of anticancer drugs (cisplatin, vinblastine, gemcitabine or piroxicam) was markedly different in each BC organoid. In RNA-sequencing analysis, expression levels of basal cell markers (CK5 and DSG3) and several novel genes (MMP28, CTSE, CNN3, TFPI2, COL17A1, and AGPAT4) were upregulated in BC organoids compared with normal bladder tissues or two-dimensional (2D) BC cell lines. These established dog BC organoids might be a useful tool, not only to determine suitable chemotherapy for BC diseased dogs but also to identify novel biomarkers in human muscle-invasive BC. In the present study, for the 1st time, dog BC organoids were generated and several specifically upregulated organoid genes were identified. Our data suggest that dog BC organoids might become a new tool to provide fresh insights into both dog BC therapy and diagnostic biomarkers.


Assuntos
Técnicas de Cultura de Células/métodos , Doenças do Cão/patologia , Organoides/patologia , Neoplasias da Bexiga Urinária/veterinária , Bexiga Urinária/patologia , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Doenças do Cão/tratamento farmacológico , Doenças do Cão/genética , Doenças do Cão/urina , Cães , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Masculino , Organoides/efeitos dos fármacos , Organoides/metabolismo , Análise de Sequência de RNA , Regulação para Cima , Bexiga Urinária/citologia , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/urina , Urina/citologia , Urotélio/citologia
12.
Mol Biol (Mosk) ; 53(3): 467-475, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31184612

RESUMO

Placenta is a highly specialized organ that is necessary for successful gestation. Several models of the placental barrier are used to study how it functions, including the transplacental transport of xenobiotics. One of these models, human choriocarcinoma cell line BeWo is widely used in vitro. Notably, cancerous BeWo cells form multilayer structures that normally are not found in the human placenta. Here, we aim to develop techniques suitable for monitoring BeWo b30 cells in culture. To assess the state of BeWo b30 cells growing on a membrane, we use impedance spectroscopy, which allows us to estimate the number of cell layers by the change in the electrical parameters of the biological system. In mature BeWo b30 cell cultures, we also note a significant increase in the expression of genes encoding metallothioneins (particularly, MT1B, MT1F, and MT2A) and syncytins (ERVW-1 and ERVFRD-1), which can be used as biomarkers reflecting the development of mature phenotypic characteristics, namely, trophoblastic invasion and formation of the syncytium.


Assuntos
Coriocarcinoma/genética , Coriocarcinoma/patologia , Espectroscopia Dielétrica , Perfilação da Expressão Gênica , Modelos Biológicos , Placenta/citologia , Placenta/metabolismo , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Feminino , Humanos , Gravidez
13.
Life Sci ; 232: 116598, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31247209

RESUMO

Hematopoietic stem cells (HSCs) are a rare cell population in adult bone marrow, mobilized peripheral blood, and umbilical cord blood possessing self-renewal and differentiation capability into a full spectrum of blood cells. Bone marrow HSC transplantation has been considered as an ideal option for certain disorders treatment including hematologic diseases, leukemia, immunodeficiency, bone marrow failure syndrome, genetic defects such as thalassemia, sickle cell anemia, autoimmune disease, and certain solid cancers. Ex vivo proliferation of these cells prior to transplantation has been proposed as a potential solution against limited number of stem cells. In such culture process, MSCs have also been shown to exhibit high capacity for secretion of soluble mediators contributing to the principle biological and therapeutic activities of HSCs. In addition, endothelial cells have been introduced to bridge the blood and sub tissues in the bone marrow, as well as, HSCs regeneration induction and survival. Cell culture in the laboratory environment requires cell growth strict control to protect against contamination, symmetrical cell division and optimal conditions for maximum yield. In this regard, microfluidic systems provide culture and analysis capabilities in micro volume scales. Moreover, two-dimensional cultures cannot fully demonstrate extracellular matrix found in different tissues and organs as an abstract representation of three dimensional cell structure. Microfluidic systems can also strongly describe the effects of physical factors such as temperature and pressure on cell behavior.


Assuntos
Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/citologia , Animais , Células da Medula Óssea/citologia , Técnicas de Cultura de Células/métodos , Técnicas de Cocultura , Células Endoteliais/citologia , Sangue Fetal/citologia , Humanos , Células-Tronco Mesenquimais/citologia
14.
Cancer Immunol Immunother ; 68(7): 1195-1209, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31177329

RESUMO

The anti-tumor efficacy of TCR-engineered T cells in vivo depends largely on less-differentiated subsets such as T cells with naïve-like T cell (TN) phenotypes with greater expansion and long-term persistence. To increase these subsets, we compared the generation of New York esophageal squamous cell carcinoma-1 (NY-ESO-1)-specific T cells under supplementation with either IL-2 or IL-7/IL-15. PBMCs were transduced with MS3II-NY-ESO-1-siTCR retroviral vector. T cell generation was adapted from a CD19-specific CART cell production protocol. Comparable results in viability, expansion and transduction efficiency of T cells under stimulation with either IL-2 or IL-7/IL-15 were observed. IL-7/IL-15 led to an increase of CD4+ T cells and a decrease of CD8+ T cells, enriched the amount of TN among CD4+ T cells but not among CD8+ T cells. In a 51Cr release assay, similar specific lysis of NY-ESO-1-positive SW982 sarcoma cells was achieved. However, intracellular cytokine staining revealed a significantly increased production of IFN-γ and TNF-α in T cells generated by IL-2 stimulation. To validate these unexpected findings, NY-ESO-1-specific T cell production was evaluated in another protocol originally established for TCR-engineered T cells. IL-7/IL-15 increased the proportion of TN. However, the absolute number of TN did not increase due to a significantly slower expansion of T cells with IL-7/IL-15. In conclusion, IL-7/IL-15 does not seem to be superior to IL-2 for the generation of NY-ESO-1-specific T cells. This is in sharp contrast to the observations in CD19-specific CART cells. Changes of cytokine cocktails should be carefully evaluated for individual vector systems.


Assuntos
Antígenos de Neoplasias/metabolismo , Engenharia Celular/métodos , Imunoterapia Adotiva/métodos , Proteínas de Membrana/metabolismo , Neoplasias/terapia , Receptores de Antígenos Quiméricos/imunologia , Antígenos CD19/metabolismo , Antígenos de Neoplasias/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/transplante , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/transplante , Técnicas de Cultura de Células/métodos , Linhagem Celular Tumoral , Meios de Cultura , Humanos , Interleucina-15/imunologia , Interleucina-2/imunologia , Interleucina-7/imunologia , Proteínas de Membrana/imunologia , Neoplasias/imunologia , Receptores de Antígenos Quiméricos/genética
15.
Altern Lab Anim ; 47(1): 19-29, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31237165

RESUMO

Ex vivo organ cultures represent unique research models, as they combine the advantages of cell cultures with those of animal models. Being able to mimic in vivo situations through the use of organ cultures provides an excellent opportunity to investigate cellular processes, molecular pathways and cell-cell interactions, as well as structural and synaptic organisation. Human and animal organ cultures are now well established and comprise sensitive, easy-to-manipulate experimental systems that raise minimal ethical concerns. The eye, in particular, is a very complex organ that is not easy to reproduce in vitro. However, a lot of research has been dedicated to the development of suitable ocular organ cultures. This review covers the various ex vivo retinal organ culture systems available for use in ophthalmology research and compares them with commonly used animal models. In particular, bovine and porcine retinal organ culture systems are described, because the size, anatomy, physiology and vessel morphology of bovine and porcine eyes are similar to the human eye in an undisputed way, thus making them good models. In addition, these animals are widely used by the food industry and the eyes are considered surplus material. A short overview of murine, rat, rabbit, cat, canine and simian retinal organ cultures is also provided.


Assuntos
Técnicas de Cultura de Órgãos , Retina , Animais , Técnicas de Cultura de Células , Humanos , Modelos Animais , Modelos Biológicos , Retina/citologia , Suínos
16.
Life Sci ; 231: 116549, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31200002

RESUMO

BACKGROUND: Long non-coding RNA (lncRNA) is emerging as an important regulator in various physiological and pathological processes. Recently, it was found that lncRNA long non-coding RNA tumor suppressor candidate 7 (TUSC7) could play tumor suppressive roles in several cancers. However, the function and underlying regulatory mechanism of lncRNA TUSC7 in endometrial carcinoma (EC) remains largely unclear. METHODS: The expression levels of TUSC7 and microRNAs-616 (miR-616) were analyzed by real-time PCR and in situ hybridization. Cell cycle and cell metastasis associated protein expressions were determined by western blotting. Cell proliferation, cycle and metastasis were determined by CCK-8 cell viability, colony formation, flow cytometer, wound scratch and transwell assays respectively in vitro. RNA pull-down, luciferase and western blotting assays were used to examine the target relationship between TUSC7 and miR-616 or that between miR-616 and suppressors of cytokine signaling 4 (5) (SOCS4 (SOCS5)). The functional effects of TUSC7 through sponging miR-616 were further examined using a xenograft tumor mouse model in vivo. RESULTS: TUSC7 was downexpressed in EC tissues and cell lines, and TUSC7 upregulation could remarkably inhibit cell proliferation, cycle progression and metastasis in EC cells. Mechanistic investigations demonstrated that TUSC7 can interact with miR-616 and decrease its expression, thereby upregulating the expression of miR-616's targets SOCS4 (SOCS5). Additionally, in vivo experiments using a xenograft tumor mouse model revealed that TUSC7 can serve as a tumor suppressor through sponging miR-616, and upregulating SOCS4 (SOCS5) in EC. CONCLUSIONS: In this study, a newly identified regulatory mechanism of lncRNA TUSC7/miR-616/ SOCS4 (SOCS5) axis was systematically studied, which may hold promise as a promising target for EC treatment.


Assuntos
Neoplasias do Endométrio/genética , Neoplasias do Endométrio/metabolismo , MicroRNAs/genética , RNA Longo não Codificante/metabolismo , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Animais , Apoptose , Técnicas de Cultura de Células , Ciclo Celular , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Neoplasias do Endométrio/patologia , Feminino , Xenoenxertos , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Invasividade Neoplásica , RNA Longo não Codificante/biossíntese , RNA Longo não Codificante/genética , Proteínas Supressoras da Sinalização de Citocina/genética
17.
Plant Physiol Biochem ; 141: 133-141, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31163340

RESUMO

Ruscus aculeatus is a threatened medicinal plant whose main bioactive components, the ruscogenins, have long been used in the treatment of hemorrhoids and varicose veins, but recently demonstrated activity against some types of cancer. Plant cell biofactories could constitute an alternative to the whole plant as a source of ruscogenins. In this pipeline, despite the in vitro recalcitrance of R. aculeatus, after many attempts we developed friable calli and derived plant cell suspensions, and their ruscogenin production was compared with that of organized in vitro plantlet and root-rhizome cultures. Root-rhizomes showed a higher capacity for biomass and ruscogenin production than the cell suspensions and the yields were greatly improved by elicitation with coronatine. Although ruscogenins accumulate in plants mainly in the root-rhizome, it was demonstrated that the aerial part could play an important role in their biosynthesis, as production was higher in the whole plant than in the root-rhizome cultures.


Assuntos
Biotecnologia/métodos , Ruscus/metabolismo , Espirostanos/metabolismo , Aminoácidos/farmacologia , Biomassa , Técnicas de Cultura de Células , Indenos/farmacologia , Irã (Geográfico) , Luz , Células Vegetais , Extratos Vegetais , Raízes de Plantas , Plantas Medicinais , Rizoma , Saponinas , Sementes/metabolismo , Técnicas de Cultura de Tecidos
18.
Nat Commun ; 10(1): 2612, 2019 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-31197141

RESUMO

Primary microcephaly is caused by mutations in genes encoding centrosomal proteins including WDR62 and KIF2A. However, mechanisms underlying human microcephaly remain elusive. By creating mutant mice and human cerebral organoids, here we found that WDR62 deletion resulted in a reduction in the size of mouse brains and organoids due to the disruption of neural progenitor cells (NPCs), including outer radial glia (oRG). WDR62 ablation led to retarded cilium disassembly, long cilium, and delayed cell cycle progression leading to decreased proliferation and premature differentiation of NPCs. Mechanistically, WDR62 interacts with and promotes CEP170's localization to the basal body of primary cilium, where CEP170 recruits microtubule-depolymerizing factor KIF2A to disassemble cilium. WDR62 depletion reduced KIF2A's basal body localization, and enhanced KIF2A expression partially rescued deficits in cilium length and NPC proliferation. Thus, modeling microcephaly with cerebral organoids and mice reveals a WDR62-CEP170-KIF2A pathway promoting cilium disassembly, disruption of which contributes to microcephaly.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Cinesina/metabolismo , Microcefalia/patologia , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fosfoproteínas/metabolismo , Proteínas Repressoras/metabolismo , Animais , Técnicas de Cultura de Células , Proteínas de Ciclo Celular/genética , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Cílios/metabolismo , Modelos Animais de Doenças , Feminino , Técnicas de Inativação de Genes , Humanos , Masculino , Camundongos , Camundongos Knockout , Microcefalia/genética , Proteínas Associadas aos Microtúbulos/genética , Proteínas do Tecido Nervoso/genética , Células-Tronco Neurais/citologia , Células-Tronco Neurais/patologia , Neuroglia/citologia , Neuroglia/patologia , Organoides/patologia , Fosfoproteínas/genética , RNA Interferente Pequeno/metabolismo
19.
Cornea ; 38(9): 1175-1181, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31169610

RESUMO

PURPOSE: To investigate if the peripheral corneal endothelium that is discarded after the preparation of preloaded Descemet membrane endothelial keratoplasty (DMEK) grafts for transplantation could be successfully used for corneal endothelial cell culture. METHODS: Complete Descemet membrane-endothelial complex (11.00 mm) was peeled from research-grade tissues (n = 15). The periphery (2.75 mm) of clinical-grade tissues (n = 15) deemed for preloaded DMEK transplants was gently peeled and preserved for 48 hours in tissue culture media, followed by centrifugation at 1000 rpm for 5 minutes. After enzymatic digestion, the cells from each group were plated in 2 different wells of an 8-well chamber slide. Media were refreshed and the confluence rate was monitored every alternate day. Live/dead staining and the expression of ZO-1, Tag1A3, Tag2A12, and Ki-67 markers were used to assess the viability, morphology, tight-junctions, cell area, and number of proliferative cells. The Wilcoxon and Student's t test were applied, where P < 0.05 was deemed statistically significant. RESULTS: Average endothelial cell density at confluence was 2,352 cells/mm2 from complete endothelium and 2,510 cells/mm from peripheral endothelium (P = 0.0351). The confluence rate (%), hexagonality (%), polymorphism (%), cell area (µm), and Ki-67 positivity (%) did not differ between both groups (P > 0.05). All the antibodies were expressed in both groups at confluence. CONCLUSIONS: The discarded peripheral endothelial cells obtained after preparing a preloaded DMEK graft for clinical application has a huge reservoir of healthy endothelial cells having proliferative potential. Using these discarded tissue pieces from donor tissues will significantly increase the primary source of healthy donor endothelial cells for regenerative treatments, which are otherwise difficult to obtain.


Assuntos
Técnicas de Cultura de Células/métodos , Ceratoplastia Endotelial com Remoção da Lâmina Limitante Posterior , Células Endoteliais/citologia , Epitélio Posterior/citologia , Doadores de Tecidos , Contagem de Células , Lâmina Limitante Posterior/cirurgia , Humanos
20.
Cell Prolif ; 52(4): e12587, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31206838

RESUMO

OBJECTIVES: Cellular aggregates are readily applicable in cell-based therapy. The effects of agitation and inoculation density on the aggregation of cells in spinner flask and the molecular mechanism of aggregation were investigated. MATERIALS AND METHODS: The aggregation kinetics of cells in spinner flask was evaluated with bovine articular chondrocytes (bACs), rabbit bone marrow-derived mesenchymal stem cells (rMSCs) and their mixture. The morphology of cellular aggregates was studied with scanning electron microscopy and gene expression of cell adhesion-related molecules was analysed. RESULTS: It was shown that suspension culture in spinner flask induced the aggregation of bACs and rMSCs. Both cells exhibited increased aggregation rate and aggregate size with decreasing agitation rate and increasing cell inoculation density. Additionally, aggregate size increased with extended culture time. By analysing gene expression of integrin ß1 and cadherin, it was indicated that these molecules were potentially involved in the aggregation process of bACs and rMSCs, respectively. Aggregates composed of both bACs and rMSCs were also prepared, showing rMSCs in the core and bACs in the periphery. CONCLUSIONS: Cellular aggregates were prepared in dynamic suspension culture using spinner flask, the key parameters to the aggregation process were identified, and the molecular mechanism of aggregation was revealed. This would lay a solid foundation for the large-scale production of cellular aggregates for cell-based therapy, such as cartilage regeneration.


Assuntos
Condrócitos/citologia , Células-Tronco Mesenquimais/citologia , Animais , Medula Óssea/metabolismo , Medula Óssea/fisiologia , Bovinos , Adesão Celular/fisiologia , Moléculas de Adesão Celular/metabolismo , Contagem de Células/métodos , Técnicas de Cultura de Células/métodos , Terapia Baseada em Transplante de Células e Tecidos/métodos , Células Cultivadas , Condrócitos/metabolismo , Expressão Gênica/fisiologia , Células-Tronco Mesenquimais/metabolismo , Coelhos
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