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Objective:To observe the effect of lipopolysaccharide (LPS) induced conditioned medium of alveolar epithelial cells on the inflammatory response and cell damage of vascular endothelial cells, and explore its mechanism.Methods:The LPS induced type Ⅱ alveolar epithelial cells (A549) conditioned medium was used as a stimulus to induce human umbilical vein endothelial cells (HUVEC) damage. The cell counting kit-8 (CCK-8) was used to detect the effect of 0% (blank group), 12.5%, 25%, 50%, 75% and 100% A549 cell conditioned medium cultured for 6, 12, 24 and 48 hours on the cell viability of HUVEC. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of inflammatory factors [interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α)] and vasoactive substances [vascular endothelial growth factor (VEGF), endothelin-1 (ET-1)] in the supernatant. Phalloidin staining was used to observe the effects of A549 cells conditioned medium on cell morphology. The expressions of protein kinase B/nuclear factor-κB (AKT/NF-κB) pathway in HUVEC induced by conditioned medium was detected by Western blotting.Results:Compared with the blank group, A549 cells conditioned medium with concentrations of 12.5%, 25%, and 50% had no significant effects on cell viability of HUVEC after 6, 12, and 24 hours, but the activity of HUVEC decreased significantly after 48 hours. Therefore, 12.5%, 25%, 50% A549 cell conditioned medium stimulated for 24 hours was selected as the induction condition for follow-up experiments. Compared with the blank group, the level of IL-6 was significantly increased in 12.5% and 50% conditioned medium groups (ng/L: 2?438.95±64.89, 3?036.41±96.69 vs. 1?736.75±20.99, both P < 0.05), the level of TNF-α was significantly increased in 12.5% and 25% conditioned medium groups (ng/L: 174.08±11.09, 81.37±8.17 vs. 50.03±0.26, both P < 0.01), the levels of VEGF and ET-1 were significantly increased in 12.5%, 25% and 50% conditioned medium groups [VEGF (ng/L): 173.60±41.44, 192.49±12.38, 318.89±27.90 vs. 66.68±19.65; ET-1 (ng/L): 54.88±1.37, 36.69±0.29, 24.07±0.73 vs. 10.67±0.25, all P < 0.01]. Phalloidin staining showed that HUVEC induced by 25% A549 cells conditioned medium were irregular in shape, uneven in size, disordered in arrangement, widened in gap, dense and unclear in microfilament structure and serrated in cell membrane. Furthermore, the average fluorescence intensity of 25% conditioned medium group significantly increased compared to the blank group (67?205.60±3?430.40 vs. 56?272.67±7?650.95, P < 0.05). Western blotting showed that compared with the blank group, the expression of HUVEC cells phosphonated inhibitor α of NF-κB (p-IκBα) was significantly decreased in the 12.5%, 25%, and 50% conditioned medium groups (p-IκBα/IκBα: 0.38±0.08, 0.67±0.12, 0.31±0.07 vs. 1.00±0.00, all P < 0.01), the expressions of phosphonated-AKT (p-AKT) and VEGF were significantly increased (p-AKT/AKT: 1.50±0.18, 1.42±0.27, 1.61±0.14 vs. 1.00±0.00, VEGF/GAPDH: 1.37±0.10, 1.53±0.22, 1.40±0.12 vs. 1.00±0.00, all P < 0.05), the expression of phosphonated NF-κB p65 (p-P65) was significantly increased in the 25% conditioned medium group (p-P65/P65: 1.45±0.14 vs. 1.00±0.00, P < 0.05). Conclusion:LPS induced conditional culture medium of alveolar epithelial cells induced endothelial cell damage via activating AKT/NF-κB pathway.
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BACKGROUND: Increasing evidence suggests a double-faceted role of alpha-synuclein (α-syn) following infection by a variety of viruses, including SARS-CoV-2. Although α-syn accumulation is known to contribute to cell toxicity and the development and/or exacerbation of neuropathological manifestations, it is also a key to sustaining anti-viral innate immunity. Consistently with α-syn aggregation as a hallmark of Parkinson's disease, most studies investigating the biological function of α-syn focused on neural cells, while reports on the role of α-syn in periphery are limited, especially in SARS-CoV-2 infection. RESULTS: Results herein obtained by real time qPCR, immunofluorescence and western blot indicate that α-syn upregulation in peripheral cells occurs as a Type-I Interferon (IFN)-related response against SARS-CoV-2 infection. Noteworthy, this effect mostly involves α-syn multimers, and the dynamic α-syn multimer:monomer ratio. Administration of excess α-syn monomers promoted SARS-CoV-2 replication along with downregulation of IFN-Stimulated Genes (ISGs) in epithelial lung cells, which was associated with reduced α-syn multimers and α-syn multimer:monomer ratio. These effects were prevented by combined administration of IFN-ß, which hindered virus replication and upregulated ISGs, meanwhile increasing both α-syn multimers and α-syn multimer:monomer ratio in the absence of cell toxicity. Finally, in endothelial cells displaying abortive SARS-CoV-2 replication, α-syn multimers, and multimer:monomer ratio were not reduced following exposure to the virus and exogenous α-syn, suggesting that only productive viral infection impairs α-syn multimerization and multimer:monomer equilibrium. CONCLUSIONS: Our study provides novel insights into the biology of α-syn, showing that its dynamic conformations are implicated in the innate immune response against SARS-CoV-2 infection in peripheral cells. In particular, our results suggest that promotion of non-toxic α-syn multimers likely occurs as a Type-I IFN-related biological response which partakes in the suppression of viral replication. Further studies are needed to replicate our findings in neuronal cells as well as animal models, and to ascertain the nature of such α-syn conformations.
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Humans , Interferon Type I , alpha-Synuclein , SARS-CoV-2 , COVID-19 , Virus Replication , Cell Line , Endothelial CellsABSTRACT
Chronic obstructive pulmonary disease ( COPD ) major chronic disease threatening public health with complex pathological mechanisms. The change of the cell microenvironment of the lung is an important part of the pathophysiology of COPD. Cell culture technology is an important method to investigate the pathological mechanism of COPD and evaluate the pharmacological effect of medicine. Here we introduce the composition of the cell microenvironment of the lung, the change of the cell microenvironment in the pathological process of COPD, and summarize the application of in vitro model mimics cell microenvironment of COPD in the study of mechanism. In addition, we aim to put forward the ideas of the in vitro model establishment of cell microenvironment of COPD.
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Objective To investigate the isolation and culture of porcine bone marrow mesenchymal stem cell (BMSC) with α-1, 3-galactosyltransferase (GGTA1) gene knockout (GTKO), GTKO/ human CD46 (hCD46) insertion and cytidine monopho-N-acetylneuraminic acid hydroxylase (CMAH)/GGTA1 gene knockout (Neu5GC/Gal), and the protective effect of co-culture with porcine islets on islet cells. Methods Bone marrow was extracted from different transgenic pigs modified with GTKO, GTKO/hCD46 and Neu5GC/Gal. Porcine BMSC were isolated by the whole bone marrow adherent method and then cultured. The morphology of BMSC was observed and the surface markers of BMSC were identified by flow cytometry. Meantime, the multi-directional differentiation induced by BMSC was observed, and the labeling and tracing of BMSC were realized by green fluorescent protein (GFP) transfection. The porcine BMSC transfected with GFP were co-cultured with porcine islet cells. Morphological changes of porcine islet cells were observed, and compared with those in the porcine islet cell alone culture group. Results BMSC derived from pigs were spindle-shaped in vitro, expressing biomarkers of CD29, CD44, CD73, CD90, CD105 and CD166 rather than CD34 and CD45. These cells were able to differentiate into adipocytes, osteoblasts and chondrocytes. Porcine BMSC with GFP transfection could be labeled and traced, which could be stably expressed in the daughter cells after cell division. Porcine BMSC exerted certain protective effect on islet cells. Conclusions GFP-labeled porcine BMSC modified with GTKO, GTKO/hCD46 and Neu5GC/Gal are successfully established, which exert certain protective effect upon islet cells.
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BACKGROUND:Stem cell transplantation is a new way to prevent and cure intervertebral disc degeneration.However,whether the transplanted stem cells can survive,proliferate,differentiate,and restore the function of nucleus pulposus cells after transplantation,is the key and difficult point to overcome. OBJECTIVE:To explore the effects of Bushenhuoxue decoction on survival,proliferation,and nucleus pulposus-like differentiation of adipose-derived stem cells. METHODS:A Transwell chamber was used to construct a co-culture model of human adipose-derived stem cells and human degenerative nucleus pulposus cells.The experiment was divided into control group,model group,drug-containing serum group,and drug-free serum group.Except for the control group,the co-culture system of other groups was treated with 50 μmol/L tert-butyl hydrogen peroxide for 24 hours.The drug-containing serum group and drug-free serum group were treated with DMEM low-glucose complete culture medium containing drug-containing serum of Bushenhuoxue decoction or drug-free serum with 20％volume fraction for 48 hours.The sublayer adipose-derived stem cells were taken.Toluidine blue staining was used to detect proteoglycan synthesis levels.Real-time PCR method was used to detect mRNA expression of type Ⅱ collagen,proteoglycan and SRY-box transcription factor 9.The protein expression of SOX9 was detected by western blot assay.Lactate dehydrogenase assay was used to detect cytotoxicity.Flow cytometry was used to detect reactive oxygen species,and β-galactosidase staining was used to detect cell senescence. RESULTS AND CONCLUSION:(1)Compared with the control group,the proportion of necrotic cells in the model group increased;toluidine blue staining became lighter,and the expression levels of type Ⅱ collagen,proteoglycan,SOX9 mRNA and SOX9 protein decreased(P<0.05).Compared with the model group,the drug-containing serum of Bushenhuoxue decoction could significantly reduce cell injury and promote the expression of type Ⅱ collagen,proteoglycan,SOX9 mRNA,and SOX9 protein(P<0.05),but the improvement in the drug-free serum group was not significant(P>0.05).(2)Compared with the control group,the contents of cytotoxicity,reactive oxygen species,and cell senescence in the model group were significantly increased.Compared with the model group,the microenvironment of the coculture system was significantly improved by drug-containing serum of Bushenhuoxue decoction(P<0.05),while drug-free serum had no significant effect on the microenvironment of the co-culture system(P>0.05).(3)The results show that Bushenhuoxue decoction can promote the survival,proliferation,and nucleus pulposus-like differentiation of adipose-derived stem cells.
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BACKGROUND:In the co-culture environment of mesenchymal stem cells and macrophages,mesenchymal stem cells can promote the polarization of macrophages into anti-inflammatory macrophages to reduce inflammation,and macrophages can promote the osteogenic differentiation of mesenchymal stem cells.The co-culture of both plays an important role in regulating the immune system and promoting tissue regeneration. OBJECTIVE:To summarize the methods,influencing factors and possible mechanisms of co-culture between mesenchymal stem cells and macrophages,and to provide theoretical basis and experimental methods for the application of co-culture of mesenchymal stem cells and macrophages in tissue engineering. METHODS:The first author searched the relevant articles published from January 1970 to September 2023 in PubMed and CNK by computer from January to September 2023.The Chinese and English key words were"mesenchymal stem cells,macrophages,co-culture".Finally,63 articles were included and analyzed. RESULTS AND CONCLUSION:(1)In vitro co-culture of mesenchymal stem cells and macrophages can be divided into direct contact co-culture and indirect contact co-culture according to the model,and two-dimensional cell co-culture and three-dimensional cell co-culture according to the dimension.(2)The co-culture of mesenchymal stem cells and macrophages can promote the polarization of macrophages towards M2 type and enhance the osteogenic effect of mesenchymal stem cells.(3)In the co-culture model,the methods of co-culture,the proportion and time of co-culture,the phenotype of macrophages,and the cell source and conditions all affected the immune regulation of macrophages and the osteogenesis of mesenchymal stem cells.(4)Cell interaction in co-culture may regulate the immune function of macrophages,proliferation,migration and osteogenesis of mesenchymal stem cells through cell-secreted soluble factors,extracellular vesicles,cell-cell contact,and metabolic pathways.(5)Mesenchymal stem cells and macrophages can enhance cardiac function after acute myocardial infarction,promote epithelial wound healing,reduce lung inflammation,improve renal function,and accelerate bone repair.(6)There are still some problems in co-culture of mesenchymal stem cells and macrophages,such as the selection of co-culture conditions,the maintenance of good cell state and interaction of co-cultured cells.(7)The co-culture of mesenchymal stem cells and macrophages can improve the local inflammatory microenvironment and promote tissue regeneration and repair,so it will have a broad application prospect in tissue engineering.
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Diesel exhaust (DE) is an important pollution source widely existing in the living and production environment, which is closely related to the health of the public and occupational groups. The International Agency for Research on Cancer has classified DE as a Group 1 carcinogen. Considering the negative health impacts on the respiratory system due to DE exposure in vitro, it is crucial to apply reliable test systems allowing accurate assessment of the biological effects of DE. The exposure technology of respiratory system in vitro is considered as one of the feasible measures to implement the 3R (reduce, refine, and replace) principle in animal experiments. Compared with the traditional submerged culture in vitro models, the air-liquid interface (ALI) exposure technology has the advantages including fewer influencing factors, easier exposure condition control, and shorter exposure cycle. ALI has become an important tool to study molecular events associated with physiology and pathology of respiratory system, and action modes and interactions of different cell types. Also, ALI has been increasingly widely used because it can simulate the actual processes of human respiratory system cells and/or tissues to DE exposure. This review was intended to introduce the development and advantages of ALI exposure technology, and further summarized the application progress of ALI exposure technology in studying the respiratory toxicity induced by DE exposure in vitro, so as to provide new ideas and pathways for the use of ALI exposure technology in the study of biomarkers and mechanisms of respiratory toxicity associated with DE exposure, and provide basic data to screen and promote biomarkers for exposed populations.
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A BBB co-culture cell model consisting of rat brain microvascular endothelial cells (BMEC) and astrocytes (AS) was established to study the effect of Angelica dahurica coumarins on the transport behavior of puerarin across blood-brain barrier (BBB) in vitro and in vivo. The barrier function of this model was evaluated by measuring the transendothelial resistance, phenol red permeability and BBB related protein expression. The permeability assay and western blot methods were performed to study the effects of Angelica dahurica coumarins on the BBB permeability and the expression of BBB related protein. The animal experiment protocols in this study were approved by the Animal Ethics Committee of Xi'an Jiaotong University (Animal Ethics No.: 2021-1329). The results showed that the established BMEC/AS co-culture model could be used to evaluate drug transport across BBB in vitro. After combined with Angelica dahurica coumarins, the transport capacity of puerarin was significantly increased in vitro and in vivo. Additionally, Angelica dahurica coumarins enhanced BBB permeability and inhibited the protein expression of P-glycoprotein (P-gp), zonula occludens-1 (ZO-1) and occludin. Angelica dahurica coumarins might increase BBB permeability by inhibiting the expression of P-gp and tight junction protein, thereby increasing the content of puerarin in brain tissue.
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A new siderophore chelate (1) and 8 known compounds were identified from the liquid co-cultures of the marine-derived Streptomyces sp. IMB18-531 and Cladosporium sp. IMB19-099 by a combination of chromatography methods, including C18 reversed-phase medium pressure chromatography, gel column chromatography and HPLC. Their structures were determined by spectroscopic analysis and chemical methods as aluminioxamine E (1), desferrioxamine E (2), ferrioxamine E (3), terragine E (4), capsimicin (5), cyclo(L-prolinyl-L-tyrosine) (6), anthranilic acid (7), (Z)-14-methylpentadec-9-enoic acid (8), and (Z)-hexadec-8-enoic acid (9). Compound 2 showed inhibitory activities against the expression of liver fibrosis related genes COL1A1, MMP2, and TIMP2. Compounds 5, 8, and 9 displayed antibacterial activities against methicillin-resistant Staphylococcus aureus, S. epidermidis and Bacillus subtilis, with MICs of 16-64 μg·mL-1. Compound 5 showed cytotoxicities against human pancreatic cancer MIA Paca-2 and human colon cancer HT-29 cell lines with IC50 of 2.9 and 6.3 μmol·L-1, respectively.
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BACKGROUND: Duchenne muscular dystrophy (DMD) is an X-linked lethal genetic disorder for which there is no effective treatment. Previous studies have shown that stem cell transplantation into mdx mice can promote muscle regeneration and improve muscle function, however, the specific molecular mechanisms remain unclear. DMD suffers varying degrees of hypoxic damage during disease progression. This study aimed to investigate whether induced pluripotent stem cells (iPSCs) have protective effects against hypoxia-induced skeletal muscle injury. RESULTS: In this study, we co-cultured iPSCs with C2C12 myoblasts using a Transwell nested system and placed them in a DG250 anaerobic workstation for oxygen deprivation for 24 h. We found that iPSCs reduced the levels of lactate dehydrogenase and reactive oxygen species and downregulated the mRNA and protein levels of BAX/BCL2 and LC3II/ LC3I in hypoxia-induced C2C12 myoblasts. Meanwhile, iPSCs decreased the mRNA and protein levels of atrogin-1 and MuRF-1 and increased myotube width. Furthermore, iPSCs downregulated the phosphorylation of AMPKA and ULK1 in C2C12 myotubes exposed to hypoxic damage. CONCLUSIONS: Our study showed that iPSCs enhanced the resistance of C2C12 myoblasts to hypoxia and inhibited apoptosis and autophagy in the presence of oxidative stress. Further, iPSCs improved hypoxia-induced autophagy and atrophy of C2C12 myotubes through the AMPK/ULK1 pathway. This study may provide a new theoretical basis for the treatment of muscular dystrophy in stem cells.
Subject(s)
Animals , Mice , AMP-Activated Protein Kinases/metabolism , Induced Pluripotent Stem Cells , Atrophy/metabolism , Atrophy/pathology , Autophagy , RNA, Messenger/metabolism , Mice, Inbred mdx , Muscle, Skeletal/metabolism , Muscle Fibers, Skeletal/metabolism , Hypoxia/metabolismABSTRACT
Bibenzyls, a kind of important plant polyphenols, have attracted growing attention for their broad and remarkable pharmacological activities. However, due to the low abundance in nature, uncontrollable and environmentally unfriendly chemical synthesis processes, these compounds are not readily accessible. Herein, one high-yield bibenzyl backbone-producing Escherichia coli strain was constructed by using a highly active and substrate-promiscuous bibenzyl synthase identified from Dendrobium officinale in combination with starter and extender biosynthetic enzymes. Three types of efficiently post-modifying modular strains were engineered by employing methyltransferases, prenyltransferase, and glycosyltransferase with high activity and substrate tolerance together with their corresponding donor biosynthetic modules. Structurally different bibenzyl derivatives were tandemly and/or divergently synthesized by co-culture engineering in various combination modes. Especially, a prenylated bibenzyl derivative ( 12) was found to be an antioxidant that exhibited potent neuroprotective activity in the cellular and rat models of ischemia stroke. RNA-seq, quantitative RT-PCR, and Western-blot analysis demonstrated that 12 could up-regulate the expression level of an apoptosis-inducing factor, mitochondria associated 3 (Aifm3), suggesting that Aifm3 might be a new target in ischemic stroke therapy. This study provides a flexible plug-and-play strategy for the easy-to-implement synthesis of structurally diverse bibenzyls through a modular co-culture engineering pipeline for drug discovery.
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Macrophages are important cells of the immune system. Tumor-associated macrophages are enriched macrophages near tumor cells or tissues. Their role is mainly to promote the construction of tumor inflammatory microenvironment and inhibit tumor immune response. Cell co-culture system is a symbiotic culture system formed by mimicking the internal environment of the body in vitro. The co-culture condition is relatively consistent with the environment in vivo, enabling better information exchange and material exchange between cells, which is a supplement to the monolayer cell culture and animal experiments. Tumor-associated macrophages and tumor cells co-exist in the tumor microenvironment. Thus, constructing a co-culture system for tumor-associated macrophages and tumor cells would be conducive to studying the antitumor effect of tumor-associated macrophages and developing new immunotherapy drugs. The co-culture system would provide a new direction for treating malignant tumors. This article mainly reviewed the co-culture patterns of macrophages and the antitumor effects of different phenotypes of macrophages, and highlighted the importance of using immunotherapy to treat malignant tumors in the tumor microenvironment.
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@#Introduction: Breast cancer is the most common cancer in women and the world’s second leading cause of death in women, after lung cancer. Calreticulin (CRT), an endoplasmic reticulum (ER) multipurpose protein, has been proposed as a potential biomarker for breast cancer. However, reports on the correlation between CRT expression and cell invasiveness in breast cancer micro-tissues are scarce. Thus, in the current study, we analyzed the potential correlation between CRT and invasiveness of breast cancer in a biological scaffold-based 3D co-culture system. Methods: MCF7, MDA-MB-231 and MCF-10A breast cell lines were co-cultured in a 3-dimensional (3D) system with MRC-5 lung fibroblast cell line in the cell density ratio of 3:1. Thereafter, calreticulin gene and protein expression levels were determined based on quantitative reverse transcription-polymerase chain reaction (RT-qPCR) and immunohistochemistry, respectively. Moreover, via RT-qPCR analysis, the gene expression levels of calreticulin-related candidate metastasis genes in breast cancer micro-tissues were carried out. Results: The results showed occasional foci of lumen-like morphology in the non-cancerous breast micro-tissues and the formation of solid clusters for breast cancer micro-tissues. Moreover, immunohistochemistry results revealed protein expression of calreticulin in non-cancerous and cancerous breast micro-tissues with cytoplasmic and nucleic acid localizations. It was found that PCMT1 and ER-α genes were significantly downregulated (p < 0.01) in invasive breast cancer micro-tissues. Conclusion: This study suggests that CRT and CRT-related candidate metastasis genes may potentially serve as prognostic biomarkers in invasive breast carcinoma.
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RESUMEN El cultivo de Phaseolus vulgaris L. se realiza en varias partes del mundo. La transformación genética vía Agrobacterium tumefaciens constituye una alternativa para la obtención de cultivares con tolerancia o resistencia a condiciones ambientales desfavorables de esta especie. Sin embargo, la condición cultivar dependiente y la baja regeneración del tejido transformado en los protocolos establecidos para P. vulgaris, han limitado el empleo de esta tecnología a un grupo reducido de cultivares. El objetivo de este trabajo fue regenerar brotes a partir de tejido transformado de P. vulgaris cv. 'ICA Pijao' y 'BAT 93'. Se determinó el efecto de la concentración de la suspensión bacteriana, la infiltración por vacío, el periodo de co-cultivo sobre la regeneración de brotes. Además, se determinó la efectividad de la geneticina y el glufosinato de amonio durante la etapa de selección en la regeneración de brotes a partir de explantes inoculados con A. tumefaciens. Los explantes V NC-1 inoculados con una suspensión bacteriana ajustada a una densidad óptica de 0,1, sin infiltración con vacío y mantenidos por tres días en co-cultivo presentaron la mejor respuesta de regeneración. La geneticina y el glufosinato de amonio fueron efectivos para la selección de brotes. La regeneración de brotes a partir de tejido transformado vía A. tumefaciens en P. vulgaris cv. 'ICA Pijao' y 'BAT 93', no descrita en la literatura científica, resulta importante para su posterior aplicación en programas de mejoramiento genéticos de esta especie.
ABSTRACT The cultivation of Phaseolus vulgaris L. is widespread in the world. The genetic transformation via Agrobacterium tumefaciens, is an alternative to obtain cultivars with tolerance or resistance to adverse environmental conditions for this species. However, the cultivar-dependent condition and the low regeneration of the transformed tissue in the protocols established for P. vulgaris have limited the use of this technology to a small group of cultivars. The aim of this work was to regenerate shoots from transformed tissue in P. vulgaris cv. 'ICA Pijao' and 'BAT 93'. The effect of the concentration of the bacterial suspension, the infiltration by vacuum, the period of co-cultivation on the regeneration of shoots was determined. In addition, the effectiveness of geneticin and glufosinate-ammonium was determined during the selection stage in the regeneration of shoots from explants inoculated with A. tumefaciens. The explants V NC-1 inoculated with a bacterial suspension adjusted to an optical density of 0.1, without infiltration with vacuum and maintained for three days in co-culture presented the best regeneration response. Geneticin and glufosinate ammonium were effective in the selection of shoots. The regeneration of shoots from transformed tissue by A. tumefaciens in P. vulgaris cv. 'ICA Pijao' and 'BAT 93', not described in the scientific literature, is important for its subsequent application in breeding programs of this species.
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Objective:To investigate the effect of keloid fibroblasts on the polarization and expression of inflammatory factors of M0 macrophages and possible mechanisms, and provide theoretical basis for new targets for keloid therapy.Methods:Keloids, normal skin tissues and paraffin specimens from patients undergoing plastic surgery in the First Affiliated Hospital of Sun Yat-sen University from November 2020 to September 2021 were collected, and fibroblasts of keloids and normal skins were isolated and co-cultured with M0 cells formed form THP-1 by phorbol ester (PMA)-stimulation to detect the expression of macrophage polarization markers and cytokines. Besides, keloid fibroblasts were treated with exogenous tumor necrosis factor-α(TNF-α) to detect its effect on the proliferation and extracellular matrix expression.Results:Macrophages were dominated by CD163 + (M2) in keloid tissues. Moreover, M0 cells expressed more TNF-α when co-cultured with keloid fibroblasts, compared with those with normal skin fibroblasts, in which, the positive staining rates of TNF-α were 19.32% and 29.52% respectively by flow cytometry. Furthermore, the proliferation was promoted and the expression of extracellular matrix proteins (COL3A1 and FN1)and Vimentin were upregulated in keloid fibroblasts under TNF-α stimulation. However, there was no significant difference in the expression of polarization surface markers CD86 and CD163 in macrophages, when co-cultured with keloid fibroblasts or normal skin fibroblasts. Conclusions:Keloid fibroblasts promote the expression of TNF-α in macrophages, which in turn promotes the proliferation and extracellular matrix secretion of keloid fibroblasts.
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In recent years, the interaction mechanisms underpinning the synthetic microbial co-culture systems have gained increasing attention due to their potentials in various biotechnological applications. Exploration of the inter-species mechanisms underpinning the synthetic microbial co-culture system could contribute to a better understanding of the theoretical basis to further optimize the existing co-culture systems, and design new synthetic co-culture system for large-scale application. OMICS technologies such as genomics, transcriptomics, proteomics, and metabolomics could analyze the biological processes in a high throughput manner. Multi-omics analysis could achieve a "global view" of various members in the microbial co-culture systems, which presents opportunities in understanding synthetic microbial consortia better. This article summarizes recent advances in understanding the mechanisms of synthetic microbial co-culture systems using omics technologies, from the aspects of metabolic network, energy metabolism, signal transduction, membrane transport, stress response, community stability and structural rationality. All these findings could provide important theoretical basis for future application of the microbial co-culture systems with the aids of emerging biotechnologies such as synthetic biology and genome editing.
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Coculture Techniques , Genomics , Metabolomics , Proteomics , Synthetic BiologyABSTRACT
Intense utilization and mining of fossil fuels for energy production have resulted in environmental pollution and climate change. Compared to fossil fuels, microalgae is considered as a promising candidate for biodiesel production due to its fast growth rate, high lipid content and no occupying arable land. However, monocultural microalgae bear high cost of harvesting, and are prone to contamination, making them incompetent compared with traditional renewable energy sources. Co-culture system induces self-flocculation, which may reduce the cost of microalgae harvesting and the possibility of contamination. In addition, the productivity of lipid and high-value by-products are higher in co-culture system. Therefore, co-culture system represents an economic, energy saving, and efficient technology. This review aims to highlight the advances in the co-culture system, including the mechanisms of interactions between microalgae and other microorganisms, the factors affecting the lipid production of co-culture, and the potential applications of co-culture system. Finally, the prospects and challenges to algal co-culture systems were also discussed.
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Biofuels , Biomass , Coculture Techniques , Flocculation , MicroalgaeABSTRACT
Traditional methods of microbial synthesis usually rely on a single engineered strain to synthesize the target product through metabolic engineering. The key cofactors, precursors and energy are produced by the introduced complex synthetic pathways. This would increase the physiological burden of engineering strains, resulting in a decrease in the yield of target products. The modular co-culture engineering has become an attractive solution for effective heterologous biosynthesis, where product yield can be greatly improved. In the modular co-culture engineering, the coordination between the population of different modules is essential for increasing the production efficiency. This article summarized recent advances in the application of modular co-culture engineering and population control strategies.
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Coculture Techniques , Metabolic Engineering , Population ControlABSTRACT
Objective @#To investigate the effect of overexpression of tyrosine kinase receptor 1 (Tie-1) in cervical cancer cells on the malignant biological behavior of tumor-related endothelial cells (TRECs) ．@*Methods @#Immuno- histochemical method was used to detect the expression of Tie-1 in cervical cancer cells ( CCCs) and TRECs of 96 patients with cervical cancer，and to analyze the correlation between the expression of Tie-1 in TRECs and clinico- pathological features and prognosis of patients．HeLa cells overexpressing Tie-1 (Hela-Tie1OE) were constructed， and HeLa-Tie1OE was co-cultured with human umbilical vein endothelial cells ( HUVECs) by Transwell cell co- culture method to obtain cervical cancer TRECs．Western blot was used to analyze Tie-1 protein expression．The migration，invasion and tubulogenesis of TRECs were detected by cell scratch assay，Transwell invasion and migra- tion assay and tubulogenesis assay. @*Results @#The expression of Tie-1 was positively correlated with CCCs and TRECs in 96 cervical cancer patients．The positive expression rate of Tie-1 in TRECs of patients with stage(FIGO) Ⅰ B2-ⅡA，tumor diameter ≥4 cm ，cervical muscle invasion depth ≥ 1 /2 full layer ，adenocarcinoma ，medium and low differentiation cervical cancer was higher than that of patients with Ⅰ A 1-Ⅰ B 1，tumor diameter＜4 cm， cervical muscle invasion depth ＜1 /2 full layer，squamous carcinoma，and high differentiation cervical cancer，re- spectively．The differences were statistically significant (P＜0. 05) ．The expression of Tie-1 in TRECs of cervical cancer patients had no significant correlation with age，lymph node metastasis and lymphatic space invasion．The positive expression of Tie-1 in cervical cancer TRECs was negatively correlated with 5-year progression-free survival time and overall survival time (P＜0. 05) ．The Tie-1 expression of TRECs obtained by co-culture of Hela-Tie1OE and HUVECs was up-regulated ，and the invasion ，migration and tubulogenesis of TRECs cells were enhanced. @*Conclusion @#The high expression of Tie-1 in TRECs of cervical cancer is related to FIGO stage，tumor diameter， degree of differentiation，depth of cervical muscular invasion，type of cervical cancer，and poor prognosis of pa- tients．Overexpression of Tie-1 can promote TRECs invasion，migration and tubulogenesis.
ABSTRACT
AIM: To explore the effect of Bcl-2 small molecule inhibitor ABT-737 on the growth and angiogenesis mimicry of SKOV3 cells in a co-culture system of Tumour-associated macrophages (TAMs) and human ovarian cancer cells SKOV3. METHODS: PMA and IL-4 was used to induce THP-1 cells into TAMs cells in vitro; MTT method was used to detect the cell survival rate of SKOV3 cells after 24 hours of treatment with different concentrations of ABT-737 culture medium; a co-culture system of SKOV3 cells and TAMs cells was established; the experimental groups were divided into control group, SKOV3+ABT-737 group (containing 5.0 μmol/L ABT-737 cultured cells), TAMs+SKOV3 group (SKOV3 cells co-cultured with TAMs cells), TAMs+SKOV3+ABT-737 group (SKOV3 cells Co-cultured with TAMs cells, and added ABT-737 containing 5.0 μmol/L), cells after 24 h was collected, MTT method was used to detect cell survival rate, EdU staining for cell proliferation, ranswell chamber experiment for cell migration and invasion, Flowcytometry for cell apoptosis, the vascular mimicry experiment for the ability of cells to form blood vessels, Western blot for the expression of vascular endothelial growth factor (VEGF), matrix metalloproteinase-2 (MMP-2) and MMP-9 in cells. RESULTS: THP-1 cells were successfully induced for TAMs cells; the survival rate of SKOV3 cells decreased under the action of ABT-737 (P<0.01); compared with the control group, the survival rate of SKOV3 cells in the SKOV3+ABT-737 group decreased, the number of EdU-labeled positive cells decreased, the number of cell migration and invasion also decreased, the rate of apoptosis increased, and the duct branches decreased, The protein expression of VEGF, MMP-2, MMP-9 decreased (P<0.01); Compared with the TAMs+SKOV3 group, the cell survival rate of the TAMs+SKOV3+ABT-737 group decreased, the number of EdU-labeled positive cells and the number of cell migration and invasion also decreased, the apoptosis rate increased, and the duct branches decreased. At the same time, the protein expression of VEGF, MMP-2, MMP-9 decreased (P<0.01). CONCLUSION: ABT-737 can inhibit SKOV3 cell proliferation, metastasis, apoptosis and angiogenesis in a co-culture system, and affect tumor progression.