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1.
Electron J Biotechnol ; 49: 5-13, Jan. 2021. ilus, tab, graf
Article in English | LILACS | ID: biblio-1291618

ABSTRACT

BACKGROUND: Opsonization, is the molecular mechanism by which target molecules promote interactions with phagocyte cell surface receptors to remove unwanted cells by induced phagocytosis. We designed an in vitro system to demonstrate that this procedure could be driven to eliminate adipocytes, using peptides mimicking regions of the complement protein C3b to promote opsonization and enhance phagocytosis. Two cell lines were used: (1) THP-1 monocytes differentiated to macrophages, expressing the C3b opsonin receptor CR1 in charge of the removal of unwanted coated complexes; (2) 3T3-L1 fibroblasts differentiated to adipocytes, expressing AQP7, to evaluate the potential of peptides to stimulate opsonization. (3) A co-culture of the two cell lines to demonstrate that phagocytosis could be driven to cell withdrawal with high efficiency and specificity. RESULTS: An array of peptides were designed and chemically synthesized p3691 and p3931 joined bound to the CR1 receptor activating phagocytosis (p < 0.033) while p3727 joined the AQP7 protein (p < 0.001) suggesting that opsonization of adipocytes could occur. In the co-culture system p3980 and p3981 increased lipid uptake to 91.2% and 89.0%, respectively, as an indicator of potential adipocyte phagocytosis. CONCLUSIONS: This in vitro model could help understand the receptor­ligand interaction in the withdrawal of unwanted macromolecules in vivo. The adipocyte-phagocytosis discussed may help to control obesity, since peptides of C3b stimulated the CR1 receptor, promoting opsonisation and phagocytosis of lipidcontaining structures, and recognition of AQP7 in the differentiated adipocytes, favored the phagocytic activity of macrophages, robustly supported by the co-culture strategy.


Subject(s)
Phagocytosis , Complement System Proteins , Adipocytes , In Vitro Techniques , Opsonin Proteins , Coculture Techniques , Foam Cells , Macrophages , Microscopy, Fluorescence
2.
Electron. j. biotechnol ; 44: 33-40, Mar. 2020. graf, tab, ilus
Article in English | LILACS | ID: biblio-1087694

ABSTRACT

BACKGROUND: The preparation of broad bean koji is a key process in the production of Pixian broad bean paste (PBP). Protease is essential for the degradation of proteins during PBP fermentation. To obtain broad bean koji with high protease activity using the cocultivated strains of Aspergillus oryzae QM-6 (A. oryzae QM-6) and Aspergillus niger QH-3 (A. niger QH-3), the optimization of acid and neutral protease activities was carried out using Box­Behnken design with response surface methodology (RSM). RESULTS: The optimum conditions were found to be as follows: inoculation proportion (X1), 3:1 (A. oryzae QM-6: A. niger QH-3, w/w); culture temperature (X2), 33°C; inoculum size (X3), 0.5% (w/w); incubation time (X4), 5 d. The acid and neutral protease activities were 605.2 ± 12.4 U/g and 1582.9 ± 23.7 U/g, respectively, which were in good agreement with the predicted values. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles revealed that the broad bean koji extracellular proteins in the case of cocultivation were richer compared to those in the case of A. oryzae QM-6 or A. niger QH-3 strain only. In addition, the free amino acids (FAAs) in the fermentation product were 55% higher in the cocultivation process than in that involving only A. oryzae QM-6, further confirming the diversity of proteases in the fermentation products. CONCLUSIONS: The optimal conditions of koji-making in PBP were obtained using RSM. The cocultivation of A. oryzae and A. niger increases the overall enzyme activities in the culture medium and the FAAs content, which would thus have potential application in the PBP industry.


Subject(s)
Peptide Hydrolases/metabolism , Aspergillus niger , Aspergillus oryzae , Fabaceae/enzymology , Coculture Techniques , Vicia faba , Electrophoresis, Polyacrylamide Gel , Fermentation , Amino Acids
3.
Article in Chinese | WPRIM | ID: wpr-827552

ABSTRACT

OBJECTIVE@#This study aimed to compare the cartilage regeneration of the stromal vascular fraction (SVF) cells and adipose-derived mesenchymal stem cells (ASCs) cocultured with chondrocytes seeded on the scaffolds.@*METHODS@#The cellular morphologies and proliferation capabilities on the scaffolds were evaluated. The scaffolds with the cocul-ture of ASCs/SVF and chondrocytes were implanted into the full thickness cartilage defective rabbit joints for 10 weeks.@*RESULTS@#The cells seeded into the scaffolds showed good adhesion and proliferation. Implantation with SVF and chondrocytes revealed desirable in vitro healing outcomes.@*CONCLUSIONS@#The SVF cells were better than ASCs in terms of the formation of cartilage matrix in a coimplantation model. Without in vitro expansion, the SVF cells are good cell sources for cartilage repair.


Subject(s)
Adipose Tissue , Animals , Cartilage , Chondrocytes , Coculture Techniques , Rabbits , Regeneration
4.
Chinese Journal of Biotechnology ; (12): 652-665, 2020.
Article in Chinese | WPRIM | ID: wpr-827003

ABSTRACT

Co-culture systems consisted of photosynthetic microorganisms and others heterotrophic microbes have attracted great attention in recent years. These systems show many advantages when compared with single culture grown under autotrophic conditions, such as less vulnerable to pollution and more stability, thus have been applied to wastewater treatment, soil remediation, biodegradable harmful substances, and production of high value-added products. In order to explore basic theory and further applications, we summarize here recent progresses in artificial co-culture systems of using photosynthetic microorganisms, to provide a current scientific understanding for the rational design of the co-culture system based on photosynthetic microorganisms using synthetic biology.


Subject(s)
Coculture Techniques , Heterotrophic Processes , Microbiological Techniques , Microbiota , Physiology , Photosynthesis , Physiology , Synthetic Biology
5.
Article in Chinese | WPRIM | ID: wpr-880757

ABSTRACT

OBJECTIVE@#To investigate high-salt exposure-induced polarization of mononuclear macrophages and the changes in proliferation and phenotypic transformation of renal fibroblasts in a co-culture system.@*METHODS@#Cultured mononuclear macrophages were exposed to high salt (161 mmol/L Na +) for 2 h and the surface markers of M0, M1 and M2-type macrophages were detected with RT-qPCR. The culture medium of the macrophages in normal and high-salt groups was collected for detection of the mRNA and protein levels of IL-6 and TGF-β1 using RT-qPCR and ELISA. A co-culture system of high salt-exposed macrophages and renal fibroblasts (NRK-49F) was established using a Transwell chamber, and the changes in proliferation and migration of NRK-49F cells were examined using EdU assay and Transwell assay, respectively. Western blotting was performed to detect the expressions of collagen I, collagen III and collagen α-SMA in NRK-49F cells.@*RESULTS@#The high salt-exposed macrophages showed significantly increased mRNA levels of M2-type macrophage surface markers mannose receptor and arginase (@*CONCLUSIONS@#High-salt exposure induces polarization of mononuclear macrophages into M2-type macrophages and promotes secretion of IL-6 and TGF-β1 by the macrophages to induce the proliferation and phenotypic transformation of NRK-49F cells.


Subject(s)
Cell Proliferation , Coculture Techniques , Fibroblasts , Kidney , Macrophages , Transforming Growth Factor beta1/genetics
6.
Int. j. morphol ; 37(4): 1203-1209, Dec. 2019. graf
Article in English | LILACS | ID: biblio-1040112

ABSTRACT

In vitro modeling of neurodegenerative diseases is now possible by using patient-derived induced pluripotent stem cells (iPS). Through them, it is nowadays conceivable to obtain human neurons and glia, and study diseases cellular and molecular mechanisms, an attribute that was previously unavailable to any human condition. Amyotrophic lateral sclerosis (ALS) is one of the diseases that has gained a rapid advance with iPS technology. By differentiating motor neurons from iPS cells of ALS- patients, we are studying the mechanisms underlying ALS- disease onset and progression. Here, we introduce a cellular platform to help maintain longevity of ALS iPS-motor neurons, a cellular feature relevant for most late-onset human diseases. Long term cultures of patient-derived iPS cells might prove to be critical for the development of personalized-drugs.


Actualmente es posible modelar in vitro enfermedades neurodegenerativas humanas mediante el uso de células madre pluripotentes inducidas (iPS) derivadas del paciente. A través de ellas, es hoy concebible obtener neuronas y glía humanas, y estudiar mecanismos celulares y moleculares de enfermedades, un atributo que anteriormente no era posible para ninguna condición humana. La esclerosis lateral amiotrófica (ELA) es una de las enfermedades que se ha beneficiado con la tecnología de iPS. Al diferenciar neuronas motoras de células iPS obtenidas de pacientes con ELA, hemos iniciado estudios sobre los mecanismos que subyacen a la aparición y progresión de la enfermedad. Aquí, presentamos el desarrollo de una plataforma celular que permite extender la longevidad de las neuronas motoras derivadas de iPS, una característica relevante para la mayoría de las enfermedades humanas de inicio tardío. Los cultivos a largo plazo de células iPS provenientes de pacientes pueden ser determinantes en el desarrollo de terapias asociadas a la medicina de precisión.


Subject(s)
Humans , Animals , Mice , Induced Pluripotent Stem Cells/cytology , Amyotrophic Lateral Sclerosis/metabolism , Immunohistochemistry , Cell Line , Coculture Techniques , Amyotrophic Lateral Sclerosis/pathology , Amyotrophic Lateral Sclerosis/therapy
7.
Asia Pacific Allergy ; (4): e4-2019.
Article in English | WPRIM | ID: wpr-750170

ABSTRACT

Food allergy is a growing global public health concern. As treatment strategies are currently limited to allergen avoidance and emergency interventions, there is an increasing demand for appropriate models of food allergy for the development of new therapeutics. Many models of food allergy rely heavily on the use of animals, and while useful, many are unable to accurately reflect the human system. In order to bridge the gap between in vivo animal models and clinical trials with human patients, human models of food allergy are of great importance. This review will summarize the commonly used human ex vivo and in vitro models of food allergy and highlight their advantages and limitations regarding how accurately they represent the human in vivo system. We will cover biopsy-based systems, precision cut organ slices, and coculture systems as well as organoids and organ-on-a-chip. The availability of appropriate experimental models will allow us to move forward in the field of food allergy research, to search for effective treatment options and to further explore the cause and progression of this disorder.


Subject(s)
Allergens , Anaphylaxis , Animals , Biological Phenomena , Coculture Techniques , Emergencies , Food Hypersensitivity , Humans , In Vitro Techniques , Models, Animal , Models, Theoretical , Organoids , Public Health
8.
Article in English | WPRIM | ID: wpr-761749

ABSTRACT

Acanthamoeba castellanii has ubiquitous distribution and causes primary acanthamoebic keratitis (AK). AK is a common disease in contact lens wearers and results in permanent visual impairment or blindness. In this study, we observed the cytopathic effect, in vitro cytotoxicity, and secretion pattern of cytokines in human corneal epithelial cells (HCECs) induced by A. castellanii trophozoites and/or cysts. Morphological observation revealed that panked dendritic HCECs co-cultured with amoeba cysts had changed into round shape and gradually died. Such changes were more severe in co-culture with cyst than those of co-cultivation with trophozoites. In vitro cytotoxicity assay revealed the highest cytotoxicity to HCECs in the co-culture system with amoeba cysts. A. castellanii induced the expression of IL-1α, IL-6, IL-8, and CXCL1 in HCECs. Secreted levels of IL-1α, IL-6, and IL-8 in HCECs co-cultured with both trophozoites and cysts were increased at an early incubation time (3 and 6 hr). These results suggested that cytopathic changes and pro-inflammatory cytokines release of HCECs in response to A. castellanii, especially amoebic cysts, are an important mechanism for AK development.


Subject(s)
Acanthamoeba castellanii , Acanthamoeba , Amoeba , Blindness , Coculture Techniques , Cytokines , Epithelial Cells , Humans , In Vitro Techniques , Interleukin-6 , Interleukin-8 , Keratitis , Trophozoites , Vision Disorders
9.
Article in English | WPRIM | ID: wpr-719450

ABSTRACT

BACKGROUND/AIMS: This study aimed to determine the regulatory role of N-acetyl-l-cysteine (NAC), an antioxidant, in interleukin 17 (IL-17)-induced osteoclast differentiation in rheumatoid arthritis (RA). METHODS: After RA synovial fibroblasts were stimulated by IL-17, the expression and production of receptor activator of nuclear factor κ-B ligand (RANKL) was determined by real-time polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA). Osteoclastogenesis was also determined after co-cultures of IL-17-stimulated RA synovial fibroblasts, Th17 cells and various concentrations of NAC with monocytes. After human peripheral CD4⁺ T cells were cultured with NAC under Th17 condition, IL-17, interferon γ, IL-4, Foxp3, RANKL, and IL-2 expression and production was determined by flow cytometry or ELISA. RESULTS: When RA synovial fibroblasts were stimulated by IL-17, IL-17 stimulated the production of RANKL, and NAC reduced the IL-17-induced RANKL production in a dose-dependent manner. NAC decreased IL-17-activated phosphorylation of mammalian target of rapamycin, c-Jun N-terminal kinase, and inhibitor of κB. When human peripheral blood CD14⁺ monocytes were cultured with macrophage colony-stimulating factor and IL-17 or RANKL, osteoclasts were differentiated, and NAC reduced the osteoclastogenesis. After human peripheral CD4⁺ T cells were co-cultured with IL-17-pretreated RA synovial fibroblasts or Th17 cells, NAC reduced their osteoclastogenesis. Under Th17 polarizing condition, NAC decreased Th17 cell differentiation and IL-17 and RANKL production. CONCLUSIONS: NAC inhibits the IL-17-induced RANKL production in RA synovial fibroblasts and IL-17-induced osteoclast differentiation. NAC also reduced Th17 polarization. NAC could be a supplementary therapeutic option for inflammatory and bony destructive processes in RA.


Subject(s)
Acetylcysteine , Arthritis, Rheumatoid , Coculture Techniques , Enzyme-Linked Immunosorbent Assay , Fibroblasts , Flow Cytometry , Humans , Interferons , Interleukin-17 , Interleukin-2 , Interleukin-4 , JNK Mitogen-Activated Protein Kinases , Macrophage Colony-Stimulating Factor , Monocytes , Osteoclasts , Osteogenesis , Phosphorylation , RANK Ligand , Real-Time Polymerase Chain Reaction , Sirolimus , T-Lymphocytes , Th17 Cells
10.
Article in English | WPRIM | ID: wpr-742308

ABSTRACT

Trichomoniasis is a common sexually transmitted infection caused by Trichomonas vaginalis, which actually does not exist a vaccine for control or prevention. Thus, the identification of new and potent immunogens in T. vaginalis, which can contribute to the development of a vaccine against this parasite, is necessary. Therefore, the aim of this work was to evaluate the potential of a recombinant Transient Receptor Potential-like channel of T. vaginalis (TvTRPV), as a promising immunogen in BALB/c mice. First, TvTRPV was cloned and expressed as a recombinant protein in Escherichia coli BL21 cells and purified by nickel affinity. Next, BALB/c mice were immunized and the antibody levels in mice serum and cytokines from the supernatant of macrophages and from co-culture systems were evaluated. Recombinant TvTRPV triggered high levels of specific total IgG in sera from the immunized mice. Also, a statistically significant increase of cytokines: IL-1β, IL-6, and TNF-α after stimulation with the corresponding antigens in vitro, was identified. Moreover, co-cultures using CD4⁺ T cells from immunized mice were able to identify higher levels of IL-10 and IFN-γ. These results were useful to validate the immunogenicity of TvTRPV in BALB/c mice, where IL-10-IFN-γ-secreting cells could play a role in infection control, supporting the potential of TvTRPV as a promising target for vaccine against T. vaginalis.


Subject(s)
Animals , Calcium , Clone Cells , Coculture Techniques , Cytokines , Escherichia coli , Immunoglobulin G , In Vitro Techniques , Infection Control , Interleukin-10 , Interleukin-6 , Macrophages , Mice , Nickel , Parasites , Sexually Transmitted Diseases , T-Lymphocytes , Trichomonas vaginalis , Trichomonas
11.
Acta Physiologica Sinica ; (6): 575-580, 2019.
Article in Chinese | WPRIM | ID: wpr-777154

ABSTRACT

The aim of the present study was to investigate the effect of salidroside (Sal) on inflammatory activation induced by lipopolysaccharide (LPS) in the co-culture of rat alveolar macrophages (AM) NR 8383 and type II alveolar epithelial cells (AEC II) RLE-6TN. CCK-8 colorimetric method was used to detect cell proliferation percentage. The enzyme-linked immunosorbent assay (ELISA) was used to determine the content of tumor necrosis factor alpha (TNF-α), macrophage inflammatory protein-2 (MIP-2) and interleukin-10 (IL-10) in the supernatant. Western blot was used to examine the expression levels of phosphorylated AKT (p-AKT) and total AKT protein. The results showed that pretreatment of RLE-6TN cells or co-culture of RLE-6TN and NR 8383 cells with 32 and 128 µg/mL Sal for 1 h, followed by continuous culture for 24 h, significantly increased the cell proliferation (P < 0.05). Compared with control group, 32 and 128 µg/mL Sal pretreatment significantly increased the ratio of p-AKT/AKT in RLE-6TN cells (P < 0.05). Pretreatment of 32 µg/mL Sal not only inhibited the secretion of TNF-α and MIP-2 by NR 8383 cells induced by LPS (P < 0.05), but also enhanced the inhibitory effect of RLE-6TN and NR 8383 cells co-culture on the secretion of TNF-α and MIP-2 by NR 8383 cells induced by LPS (P < 0.05). In addition, 32 µg/mL Sal pretreatment promoted LPS-induced IL-10 secretion by NR 8383 cells (P < 0.05), and enhanced the promoting effect of co-culture of RLE-6TN and NR 8383 cells on the IL-10 secretion by LPS-induced NR 8383 cells (P < 0.05). In conclusion, Sal may directly inhibit LPS-induced inflammatory activation of AM (NR 8383), promote the proliferation of AEC II (RLE-6TN) through PI3K/AKT signaling pathway, and enhance the regulatory effect of AEC II on LPS-induced inflammatory activation of AM.


Subject(s)
Alveolar Epithelial Cells , Metabolism , Animals , Cell Line , Chemokine CXCL2 , Metabolism , Coculture Techniques , Glucosides , Pharmacology , Interleukin-10 , Metabolism , Lipopolysaccharides , Macrophages, Alveolar , Metabolism , Phenols , Pharmacology , Phosphatidylinositol 3-Kinases , Metabolism , Proto-Oncogene Proteins c-akt , Metabolism , Rats , Signal Transduction , Tumor Necrosis Factor-alpha , Metabolism
12.
Article in English | WPRIM | ID: wpr-776892

ABSTRACT

Antifungal drug resistance is a significant clinical problem, and antifungal agents that can evade resistance are urgently needed. In infective niches, resistant organisms often co-existed with sensitive ones, or a subpopulation of antibiotic-susceptible organisms may evolve into resistant ones during antibiotic treatment and eventually dominate the whole population. In this study, we established a co-culture assay in which an azole-resistant Candida albicans strain was mixed with a susceptible strain labeled with green fluorescent protein to mimic in vivo conditions and screen for antifungal drugs. Fluconazole was used as a positive control to verify the validity of this co-culture assay. Five natural molecules exhibited antifungal activity against both susceptible and resistant C. albicans. Two of these compounds, retigeric acid B (RAB) and riccardin D (RD), preferentially inhibited C. albicans strains in which the efflux pump MDR1 was activated. This selectivity was attributed to greater intracellular accumulation of the drugs in the resistant strains. Changes in sterol and lipid compositions were observed in the resistant strains compared to the susceptible strain, and might increase cell permeability to RAB and RD. In addition, RAB and RD interfered with the sterol pathway, further aggregating the decrease in ergosterol in the sterol synthesis pathway in the MDR1-activated strains. Our findings here provide an alternative for combating resistant pathogenic fungi.


Subject(s)
ATP-Binding Cassette Transporters , Genetics , Metabolism , Antifungal Agents , Chemistry , Metabolism , Pharmacology , Azoles , Pharmacology , Biosynthetic Pathways , Genetics , Candida albicans , Chemistry , Metabolism , Cell Membrane , Chemistry , Metabolism , Coculture Techniques , Drug Resistance, Fungal , Ergosterol , Metabolism , Fungal Proteins , Genetics , Metabolism , Lipids , Chemistry , Molecular Structure , Permeability , Phenyl Ethers , Chemistry , Metabolism , Pharmacology , Sterols , Chemistry , Metabolism , Stilbenes , Chemistry , Metabolism , Pharmacology , Triterpenes , Chemistry , Metabolism , Pharmacology
13.
Article in English | WPRIM | ID: wpr-739659

ABSTRACT

Coculture with adipose-derived stem cells (ADSCs) can stimulate proliferation and migration of melanocytes. To enhance outcomes of skin disorders caused by melanocyte loss or death, mixed transplantation with ADSCs has been suggested. However, role of cocultured ADSCs in proliferation and migration of melanocytes remains unclear. This study determined the effect of ADSCs on production of growth factors and expression levels of intergrins in primary culture of adult human melanocytes with or without ADSCs and in nude mice grafted with such melanocytes. Higher amounts of growth factors for melanocytes, such as bFGF and SCF were produced and released from ADSCs by coculturing with melanocytes. Relative levels of integrins β1, α5, and α6 as well as adhesion to fibronectin and laminin were increased in melanocytes cocultured with ADSCs. Such increases were inhibited by neutralization of bFGF or SCF. Relative levels of bFGF, SCF and integrins were increased in nude mice skin after grafting with melanocyte+ADSC cocultures. Collectively, these results indicate that ADSCs can stimulate proliferation and migration of melanocytes by increasing expression of integrins in melanocytes through upregulation of production/release of melanocyte growth factors such as bFGF and SCF.


Subject(s)
Adult , Animals , Coculture Techniques , Extracellular Matrix , Fibronectins , Humans , Integrins , Intercellular Signaling Peptides and Proteins , Laminin , Melanocytes , Mice , Mice, Nude , Skin , Stem Cells , Transplants , Up-Regulation
14.
Article in English | WPRIM | ID: wpr-761919

ABSTRACT

BACKGROUND: Brown adipocytes have thermogenic characteristics in neonates and elicit anti-inflammatory responses. We postulated that thermogenic brown adipocytes produce distinctive intercellular effects in a hypobaric state. The purpose of this study is to analyze the correlation between brown adipocyte and regulatory T cell (T(reg)) expression under intermittent hypobaric conditions. METHODS: Brown and white adipocytes were harvested from the interscapular and flank areas of C57BL6 mice, respectively. Adipocytes were cultured with syngeneic splenocytes after isolation and differentiation. Intermittent hypobaric conditions were generated using cyclic negative pressure application for 48 h in both groups of adipocytes. Expression levels of T(regs) (CD4 + CD25 + Foxp3 + T cells), cytokines [tumor necrosis factor-α (TNF-α) and interleukin-10 (IL-10), and the programmed death-ligand 1 (PD-L1)] co-inhibitory ligand were examined. RESULTS: Splenocytes, cultured with brown and white adipocytes, exhibited comparable T(reg) expression in a normobaric state. Under hypobaric conditions, brown adipocytes maintained a subset of T(regs). However, a decrease in T(regs) was found in the white adipocyte group. TNF-α levels increased in both groups under hypobaric conditions. In the brown adipocyte group, anti-inflammatory IL-10 expression increased significantly; meanwhile, IL-10 expression decreased in the white adipocyte group. PD-L1 levels increased more significantly in brown adipocytes than in white adipocytes under hypobaric conditions. CONCLUSION: Both brown and white adipocytes support T(reg) expression when they are cultured with splenocytes. Of note, brown adipocytes maintained T(reg) expression in intermittent hypobaric conditions. Anti-inflammatory cytokines and co-inhibitory ligands mediate the immunomodulatory effects of brown adipocytes under altered atmospheric conditions. Brown adipocytes showed the feasibility as a source of adjustment in physical stresses.


Subject(s)
Adipocytes , Adipocytes, Brown , Adipocytes, White , Animals , Coculture Techniques , Cytokines , Humans , Infant, Newborn , Interleukin-10 , Ligands , Mice , Necrosis
15.
Article in English | WPRIM | ID: wpr-758941

ABSTRACT

Porcine endogenous retroviruses (PERVs) integrate into germline DNA as proviral genome that enables vertical transmission from parents to their offspring. The provirus usually survives as part of the host genome rather than as an infectious agent, but may become pathogenic if it crosses species barriers. Therefore, replication-competent PERV should be controlled through selective breeding or knockout technologies. Two microRNAs (miRNAs), dual LTR1 and LTR2, were selected to inhibit the expression of PERV in primary porcine kidney cells. The inhibition efficiency of the miRNAs was compared based on their inhibition of different PERV regions, specifically long terminal repeats (LTRs), gag, pol, and env. Gene expression was quantified using real-time polymerase chain reaction and the C-type reverse transcriptase (RT) activity was determined. The messenger RNA (mRNA) expression of the PERV LTR and env regions was determined in HeLa cells co-cultured with primary porcine kidney cells. The mRNA expression of the LTR, gag, pol, and env regions of PERV was dramatically inhibited by dual miRNA from 24 to 144 h after transfection, with the highest inhibition observed for the LTR and pol regions at 120 h. Additionally, the RT activity of PERV in the co-culture experiment of porcine and human cells was reduced by 84.4% at the sixth passage. The dual LTR 1+2 miRNA efficiently silences PERV in primary porcine kidney cells.


Subject(s)
Coculture Techniques , DNA , Endogenous Retroviruses , Gene Expression , Genome , HeLa Cells , Humans , Kidney , MicroRNAs , Parents , Proviruses , Real-Time Polymerase Chain Reaction , RNA, Messenger , RNA-Directed DNA Polymerase , Selective Breeding , Terminal Repeat Sequences , Transfection
16.
Article in English | WPRIM | ID: wpr-766105

ABSTRACT

PURPOSE: Several studies have shown that the oral cavity is a secondary location for Helicobacter pylori colonization and that H. pylori is associated with the severity of periodontitis. This study investigated whether H. pylori had an effect on the periodontium. We established an invasion model of a standard strain of H. pylori in human periodontal ligament fibroblasts (hPDLFs), and evaluated the effects of H. pylori on cell proliferation and cell cycle progression. METHODS: Different concentrations of H. pylori were used to infect hPDLFs, with 6 hours of co-culture. The multiplicity of infection in the low- and high-concentration groups was 10:1 and 100:1, respectively. The Cell Counting Kit-8 method and Ki-67 immunofluorescence were used to detect cell proliferation. Flow cytometry, quantitative real-time polymerase chain reaction, and western blots were used to detect cell cycle progression. In the high-concentration group, the invasion of H. pylori was observed by transmission electron microscopy. RESULTS: It was found that H. pylori invaded the fibroblasts, with cytoplasmic localization. Analyses of cell proliferation and flow cytometry showed that H. pylori inhibited the proliferation of periodontal fibroblasts by causing G2 phase arrest. The inhibition of proliferation and G2 phase arrest were more obvious in the high-concentration group. In the low-concentration group, the G2 phase regulatory factors cyclin dependent kinase 1 (CDK1) and cell division cycle 25C (Cdc25C) were upregulated, while cyclin B1 was inhibited. However, in the high-concentration group, cyclin B1 was upregulated and CDK1 was inhibited. Furthermore, the deactivated states of tyrosine phosphorylation of CDK1 (CDK1-Y15) and serine phosphorylation of Cdc25C (Cdc25C-S216) were upregulated after H. pylori infection. CONCLUSIONS: In our model, H. pylori inhibited the proliferation of hPDLFs and exerted an invasive effect, causing G2 phase arrest via the Cdc25C/CDK1/cyclin B1 signaling cascade. Its inhibitory effect on proliferation was stronger in the high-concentration group.


Subject(s)
Blotting, Western , CDC2 Protein Kinase , Cell Count , Cell Cycle , Cell Proliferation , Coculture Techniques , Colon , Cyclin B1 , Cytoplasm , Fibroblasts , Flow Cytometry , Fluorescent Antibody Technique , G2 Phase , Helicobacter pylori , Helicobacter , Humans , Methods , Microscopy, Electron, Transmission , Mouth , Periodontal Ligament , Periodontitis , Periodontium , Phosphorylation , Real-Time Polymerase Chain Reaction , Serine , Tyrosine
17.
Article in English | WPRIM | ID: wpr-786672

ABSTRACT

BACKGROUND: With the popularity of laparoscopic cholecystectomy, common bile duct injury has been reported more frequently. There is no perfect method for repairing porcine biliary segmental defects.METHODS: After the decellularization of human arterial blood vessels, the cells were cultured with GFP⁺ (carry green fluorescent protein) porcine bile duct epithelial cells. The growth and proliferation of porcine bile duct epithelial cells on the human acellular arterial matrix (HAAM) were observed by hematoxylin-eosin (HE) staining, electron microscopy, and immunofluorescence. Then, the recellularized human acellular arterial matrix (RHAAM) was used to repair biliary segmental defects in the pig. The feasibility of it was detected by magnetic resonance cholangiopancreatography, liver function and blood routine changes, HE staining, immunofluorescence, real-time quantitative PCR (RT-qPCR), and western blot.RESULTS: After 4 weeks (w) of co-culture of HAAM and GFP? porcine bile duct epithelial cells, GFP⁺ porcine bile duct epithelial cells grew stably, proliferated, and fused on HAAM. Bile was successfully drained into the duodenum without bile leakage or biliary obstruction. Immunofluorescence detection showed that GFP-positive bile duct cells could still be detected after GFP-containing bile duct cells were implanted into the acellular arterial matrix for 8 w. The implanted bile duct cells can successfully resist bile invasion and protect the acellular arterial matrix until the newborn bile duct is formed.CONCLUSION: The RHAAM can be used to repair biliary segmental defects in pigs, which provides a new idea for the clinical treatment of common bile duct injury.


Subject(s)
Bile , Bile Ducts , Blood Vessels , Blotting, Western , Cholangiopancreatography, Magnetic Resonance , Cholecystectomy, Laparoscopic , Coculture Techniques , Common Bile Duct , Duodenum , Epithelial Cells , Fluorescent Antibody Technique , Humans , Infant, Newborn , Liver , Methods , Microscopy, Electron , Polymerase Chain Reaction , Swine , Tissue Engineering
18.
Article in English | WPRIM | ID: wpr-786671

ABSTRACT

BACKGROUND: Macrophages have been known to have diverse roles either after tissue damage or during the wound healing process; however, their roles in flap wound healing are poorly understood. In this study, we aimed to evaluate how macrophages contribute to the flap wound regeneration.METHODS: A murine model of a pedicled flap was generated, and the time-course of the wound healing process was determined. Especially, the interface between the flap and the residual tissue was histopathologically evaluated. Using clodronate liposome, a macrophage-depleting agent, the functional role of macrophages in flap wound healing was investigated. Coculture of human keratinocyte cell line HaCaT and monocytic cell line THP-1 was performed to unveil relationship between the two cell types.RESULTS: Macrophage depletion significantly impaired flap wound healing process showing increased necrotic area after clodronate liposome administration. Interestingly, microscopic evaluation revealed that epithelial remodeling between the flap tissue and residual normal tissue did not occurred under the lack of macrophage infiltration. Coculture and scratch wound healing assays indicated that macrophages significantly affected the migration of keratinocytes.CONCLUSION: Macrophages play a critical role in the flap wound regeneration. Especially, epithelial remodeling at the flap margin is dependent on proper macrophage infiltration. These results implicate to support the cellular mechanisms of impaired flap wound healing.


Subject(s)
Cell Line , Clodronic Acid , Coculture Techniques , Humans , Keratinocytes , Liposomes , Macrophages , Regeneration , Surgical Flaps , Wound Healing , Wounds and Injuries
19.
Braz. j. microbiol ; 49(2): 362-369, Apr.-June 2018. graf
Article in English | LILACS | ID: biblio-889228

ABSTRACT

Abstract Aspergillus sp., Fusarium sp., and Ramularia sp. were endophytic fungi isolated from Rumex gmelini Turcz (RGT), all of these three strains could produce some similar bioactive secondary metabolites of their host. However the ability to produce active components degraded significantly after cultured these fungi alone for a long time, and were difficult to recover. In order to obtain more bioactive secondary metabolites, the co-culture of tissue culture seedlings of RGT and its endophytic fungi were established respectively, and RGT seedling was selected as producer. Among these fungi, Aspergillus sp. showed the most significant enhancement on bioactive components accumulation in RGT seedlings. When inoculated Aspergillus sp. spores into media of RGT seedlings that had taken root for 20 d, and made spore concentration in co-culture medium was 1 × 104 mL-1, after co-cultured for 12 d, the yield of chrysophaein, resveratrol, chrysophanol, emodin and physcion were 3.52-, 3.70-, 3.60-, 4.25-, 3.85-fold of the control group. The extreme value of musizin yield was 0.289 mg, which was not detected in the control groups. The results indicated that co-culture with endophytic fungi could significantly enhance bioactive secondary metabolites production of RGT seedlings.


Subject(s)
Humans , Adolescent , Ascomycota/metabolism , Rumex/metabolism , Rumex/microbiology , Endophytes/metabolism , Phytochemicals/metabolism , Ascomycota/isolation & purification , Ascomycota/growth & development , Time Factors , Coculture Techniques , Rumex/growth & development , Seedlings/growth & development , Seedlings/metabolism , Seedlings/microbiology , Endophytes/isolation & purification , Endophytes/growth & development
20.
Article in Chinese | WPRIM | ID: wpr-813117

ABSTRACT

To investigate the effect of ursolic acid on the invasion and migration of hepatocellular carcinoma (HCC) cells co-cultured with macrophages, and to explore the underlying mechanisms.
 Methods: The migration and invasion ability of HCC cells in the co-culture system with or without ursolic acid intervention were evaluated by transwell assay. The levels of epithelial-mesenchymal transition (EMT) markers E-cadherin, N-cadherin, and vimentin in HCC cells co-cultured with macrophages were detected by Western blot.
 Results: The migration and invasion ability and EMT were significantly enhanced when co-cultured with macrophages, and the expression of E-cadherin was significantly increased while N-cadherin and vimentin levels were significantly decreased. However, after ursolic acid treatment, the migration and invasion ability were significantly reduced, and the expression of E-cadherin was increased while N-cadherin and vimentin levels were decreased.
 Conclusion: Ursolic acid exerts inhibitory effect on the ability of migration, invasion, and EMT for HCC, which are enhanced by co-culturing with macrophages.


Subject(s)
Cadherins , Genetics , Carcinoma, Hepatocellular , Pathology , Cell Line, Tumor , Cell Movement , Coculture Techniques , Epithelial-Mesenchymal Transition , Gene Expression Regulation, Neoplastic , Humans , Liver Neoplasms , Pathology , Macrophages , Cell Biology , Neoplasm Invasiveness , Pathology , Triterpenes , Pharmacology
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