Colostrum provides essential nutrients and immunologically active factors that are beneficial to newborns.Our previous work demonstrated that milk contains large amounts of miRNA that is largely stored in milk-derived...Colostrum provides essential nutrients and immunologically active factors that are beneficial to newborns.Our previous work demonstrated that milk contains large amounts of miRNA that is largely stored in milk-derived microvesicles(MVs).In the present study,we found that the MVs from colostrum contain signifi cantly higher levels of several immune-related miRNAs.We hypothesized that the colostrum MVs may transfer the immune-related miR-NAs into cells,which contribute to its immune modulatory feature.We isolated colostrum MVs by ultracentrifugation and demonstrated several immune modulation features associated with miRNAs.We also provide evidence that the physical structure of milk-derived MVs is essential for transfer miRNAs and following immune modulation effect.Moreover,we found that colostrum powder-derived MVs also contains higher levels of immune-related miRNAs that display similar immune modulation effects.Taken together,these results show that MV-containing immunerelated miRNAs may be a novel mechanism by which co-lostrum modulates body immune response.展开更多
Extracellular vesicles （EVs） are membrane-bound complexes secreted from cells under both physiological and pathological conditions. They contain proteins, nucleic acids and lipids and act as messengers for cell-cell...Extracellular vesicles （EVs） are membrane-bound complexes secreted from cells under both physiological and pathological conditions. They contain proteins, nucleic acids and lipids and act as messengers for cell-cell communication and signalling, particularly between immune cells. EV research is a rapidly evolving and expanding field, and it appears that all biological fluids contain very large numbers of EVs; they are produced from all cells that have been studied to date, and are known to have roles in several reproductive processes. This review analyses the evidence for the role of EVs throughout human reproduction, starting with the paternal and maternal gametes, followed by the establishment and continuation of successful pregnancies, with specific focus, where possible, on the interaction of EVs with the maternal immune system. Importantly, variations within the EV populations are identified in various reproductive disorders, such as pre-term labour and pre-eclampsia.展开更多
Extracellular vesicles,including exosomes and microvesicles,play a fundamental role in the activity of the nervous system,participating in signal transmission between neurons and providing the interaction of central n...Extracellular vesicles,including exosomes and microvesicles,play a fundamental role in the activity of the nervous system,participating in signal transmission between neurons and providing the interaction of central nervous system with all body systems.In many neurodegenerative diseases,neurons pack toxic substances into vesicles and release them into the extracellular space,which leads to the spread of misfolded neurotoxic proteins.The contents of neuron-derived extracellular vesicles may indicate pathological changes in the central nervous system,and the analysis of extracellular vesicle molecular content contributes to the development of non-invasive methods for the diagnosis of many central nervous system diseases.Extracellular vesicles of neuronal origin can be isolated from various biological fluids due to their ability to cross the blood-brain barrier.Today,the diagnostic potential of almost all toxic proteins involved in nervous system disease pathogenesis,specificallyα-synuclein,tau protein,superoxide dismutase 1,FUS,leucine-rich repeat kinase 2,as well as some synaptic proteins,has been well evidenced.Special attention is paid to extracellular RNAs mostly associated with extracellular vesicles,which are important in the onset and development of many neurodegenerative diseases.Depending on parental cell type,extracellular vesicles may have different therapeutic properties,including neuroprotective,regenerative,and anti-inflammatory.Due to nano size,biosafety,ability to cross the blood-brain barrier,possibility of targeted delivery and the lack of an immune response,extracellular vesicles are a promising vehicle for the delivery of therapeutic substances for the treatment of neurodegenerative diseases and drug delivery to the brain.This review describes modern approaches of diagnosis and treatment of central nervous system diseases using extracellular vesicles.展开更多
BACKGROUND Endothelial activation plays an important role in sepsis-mediated inflammation,but the triggering factors have not been fully elucidated.Microvesicles carrying mitochondrial content(mitoMVs)have been implic...BACKGROUND Endothelial activation plays an important role in sepsis-mediated inflammation,but the triggering factors have not been fully elucidated.Microvesicles carrying mitochondrial content(mitoMVs)have been implicated in several diseases and shown to induce endothelial activation.AIM To explore whether mitoMVs constitute a subset of MVs isolated from plasma of patients with sepsis and contribute to endothelial activation.METHODS MVs were isolated from human plasma and characterized by confocal microscopy and flow cytometry.Proinflammatory cytokines,including interleukin(IL)-6,IL-8 and tumour necrosis factor(TNF)-α,and soluble vascular cell adhesion molecule(sVCAM)-1 were detected by ELISA.Human umbilical vein endothelial cells(HUVECs)were stimulated with the circulating MVs to evaluate their effect on endothelial activation.RESULTS MitoMVs were observed in plasma from patients with sepsis.Compared with those in healthy controls,expression of MVs,mitoMVs,proinflammatory cytokines and sVCAM-1 was increased.The number of mitoMVs was positively associated with TNF-αand sVCAM-1.In vitro,compared with MVs isolated from the plasma of healthy controls,MVs isolated from the plasma of patients with sepsis induced expression of OAS2,RSAD2,and CXCL10 in HUVECs.MitoMVs were taken up by HUVECs,and sonication of MVs significantly reduced the uptake of mitoMVs by HUVECs and expression of the above three type I IFNdependent genes.CONCLUSION MitoMVs are increased in the plasma of patients with sepsis,which induces elevated expression of type I IFN-dependent genes.This suggests that circulating mitoMVs activate the type I IFN signalling pathway in endothelial cells and lead to endothelial activation.展开更多
AIM:To develop protocols for isolation of exosomes and characterization of their RNA content.METHODS:Exosomes were extracted from He La cell culture media and human blood serum using the Total exosome isolation(from c...AIM:To develop protocols for isolation of exosomes and characterization of their RNA content.METHODS:Exosomes were extracted from He La cell culture media and human blood serum using the Total exosome isolation(from cell culture media)reagent,and Total exosome isolation(from serum)reagent respectively.Identity and purity of the exosomes was confirmed by Nanosight?analysis,electron microscopy,and Western blots for CD63 marker.Exosomal RNA cargo was recovered with the Total exosome RNA and protein isolation kit.Finally,RNA was profiled using Bioanalyzer and quantitative reverse transcriptionpolymerase chain reaction(q RT-PCR)methodology.RESULTS:Here we describe a novel approach for robust and scalable isolation of exosomes from cell culture media and serum,with subsequent isolation and analysis of RNA residing within these vesicles.The isolation procedure is completed in a fraction of the time,compared to the current standard protocols utilizing ultracentrifugation,and allows to recover fully intact exosomes in higher yields.Exosomes were found tocontain a very diverse RNA cargo,primarily short sequences 20-200 nt(such as mi RNA and fragments of m RNA),however longer RNA species were detected as well,including full-length 18S and 28S r RNA.CONCLUSION:We have successfully developed a set of reagents and a workflow allowing fast and efficient extraction of exosomes,followed by isolation of RNA and its analysis by q RT-PCR and other techniques.展开更多
Cell-derived microvesicles(MVs) are secreted from almost all kinds of mammalian cells into the extracellular space, and play crucial roles in intercellular communication and transporting biomolecules between cells. Ho...Cell-derived microvesicles(MVs) are secreted from almost all kinds of mammalian cells into the extracellular space, and play crucial roles in intercellular communication and transporting biomolecules between cells. However, there is a great challenge for visualizing and monitoring of MVs’ bio-behaviors due to the limitations of existing labeling methods. Herein, we report the first paradigm of designer cell-self-implemented labeling of MVs secreted from living mammalian MCF-7 cells in situ using the intracellular-synthesized fluorescent quantum dots(QDs) during the formation of MVs. By elaborately coupling intracellular biochemical reactions and metabolism pathways, the MCF-7 cells can be illuminated brightly by intracellular-biosynthesized fluorescent CdSe QDs. Simultaneously, intracellular-synthesized QDs can be in situ encapsulated by the secreted MVs budding from the plasma membrane of the fluorescing cells to label the MVs with an efficiency of up to 89.9%. The whole labeling process skillfully combines designer precise cell-tuned intricate synthesis of CdSe QDs with mild in-situ labeling via cell-selfimplementation just after feeding the cell with suitable chemicals, which is structure-or function-nondestructive and much more straightforward and milder than those by chemical conjugation or indirect encapsulation with conventional fluorogenic labels.展开更多
Background:Trastuzumab is a first-line targeted therapy for human epidermal growth factor receptor-2(HER2)-positive gastric cancer.However,the inevitable occurrence of acquired trastuzumab resistance limits the drug b...Background:Trastuzumab is a first-line targeted therapy for human epidermal growth factor receptor-2(HER2)-positive gastric cancer.However,the inevitable occurrence of acquired trastuzumab resistance limits the drug benefit,and there is currently no effective reversal measure.Existing researches on the mechanism of trastuzumab resistance mainly focused on tumor cells themselves,while the understanding of the mechanisms of environment-mediated drug resistance is relatively lacking.This study aimed to further explore the mechanisms of trastuzumab resistance to identify strategies to promote survival in these patients.Methods:Trastuzumab-sensitive and trastuzumab-resistant HER2-positive tumor tissues and cells were collected for transcriptome sequencing.Bioinformatics were used to analyze cell subtypes,metabolic pathways,and molecular signaling pathways.Changes in microenvironmental indicators(such as macrophage,angiogenesis,and metabolism)were verified by immunofluorescence(IF)and immunohistochemical(IHC)analyses.Finally,a multi-scale agent-based model(ABM)was constructed.The effects of combination treatment were further validated in nude mice to verify these effects predicted by the ABM.Results:Based on transcriptome sequencing,molecular biology,and in vivo experiments,we found that the level of glutamine metabolism in trastuzumabresistant HER2-positive cells was increased,and glutaminase 1(GLS1)was significantly overexpressed.Meanwhile,tumor-derived GLS1 microvesicles drove M2macrophage polarization.Furthermore,angiogenesis promoted trastuzumab resistance.IHC showed high glutamine metabolism,M2 macrophage polarization,and angiogenesis in trastuzumab-resistant HER2-positive tumor tissues from patients and nudemice.Mechanistically,the cell division cycle 42(CDC42)promoted GLS1 expression in tumor cells by activating nuclear factor kappa-B(NF-κB)p65 and drove GLS1microvesicle secretion through IQmotif-containing GTPase-activating protein 1(IQGAP1).Based on the ABM and in vivo experiments,we confirmed that the combi展开更多
BACKGROUND Pulmonary fibrosis is one of the main reasons for the high mortality rate among acute respiratory distress syndrome(ARDS)patients.Mesenchymal stromal cell-derived microvesicles(MSC-MVs)have been shown to ex...BACKGROUND Pulmonary fibrosis is one of the main reasons for the high mortality rate among acute respiratory distress syndrome(ARDS)patients.Mesenchymal stromal cell-derived microvesicles(MSC-MVs)have been shown to exert antifibrotic effects in lung diseases.AIM To investigate the effects and mechanisms of MSC-MVs on pulmonary fibrosis in ARDS mouse models.METHODS MSC-MVs with low hepatocyte growth factor(HGF)expression(siHGF-MSC-MVs)were obtained via lentivirus transfection and used to establish the ARDS pulmonary fibrosis mouse model.Following intubation,respiratory mechanics-related indicators were measured via an experimental small animal lung function tester.Homing of MSC-MVs in lung tissues was investigated by near-infrared live imaging.Immunohistochemical,western blotting,ELISA and other methods were used to detect expression of pulmonary fibrosis-related proteins and to compare effects on pulmonary fibrosis and fibrosis-related indicators.RESULTS The MSC-MVs gradually migrated and homed to damaged lung tissues in the ARDS model mice.Treatment with MSC-MVs significantly reduced lung injury and pulmonary fibrosis scores.However,low expression of HGF(siHGF-MSC-MVs)significantly inhibited the effects of MSC-MVs(P<0.05).Compared with the ARDS pulmonary fibrosis group,the MSC-MVs group exhibited suppressed expression of type I collagen antigen,type III collagen antigen,and the proteins transforming growth factor-βandα-smooth muscle actin,whereas the siHGF-MVs group exhibited significantly increased expression of these proteins.In addition,pulmonary compliance and the pressure of oxygen/oxygen inhalation ratio were significantly lower in the MSC-MVs group,and the effects of the MSC-MVs were significantly inhibited by low HGF expression(all P<0.05).CONCLUSION MSC-MVs improved lung ventilation functions and inhibited pulmonary fibrosis in ARDS mice partly via HGF mRNA transfer.展开更多
基金supported by grants from the National Natural Science Foundation of China(Grant Nos.30988003,31071232,3100032331000478 and 90608010)the National Basic Research Program(973 Program)(No.2011CB504803).
文摘Colostrum provides essential nutrients and immunologically active factors that are beneficial to newborns.Our previous work demonstrated that milk contains large amounts of miRNA that is largely stored in milk-derived microvesicles(MVs).In the present study,we found that the MVs from colostrum contain signifi cantly higher levels of several immune-related miRNAs.We hypothesized that the colostrum MVs may transfer the immune-related miR-NAs into cells,which contribute to its immune modulatory feature.We isolated colostrum MVs by ultracentrifugation and demonstrated several immune modulation features associated with miRNAs.We also provide evidence that the physical structure of milk-derived MVs is essential for transfer miRNAs and following immune modulation effect.Moreover,we found that colostrum powder-derived MVs also contains higher levels of immune-related miRNAs that display similar immune modulation effects.Taken together,these results show that MV-containing immunerelated miRNAs may be a novel mechanism by which co-lostrum modulates body immune response.
文摘Extracellular vesicles （EVs） are membrane-bound complexes secreted from cells under both physiological and pathological conditions. They contain proteins, nucleic acids and lipids and act as messengers for cell-cell communication and signalling, particularly between immune cells. EV research is a rapidly evolving and expanding field, and it appears that all biological fluids contain very large numbers of EVs; they are produced from all cells that have been studied to date, and are known to have roles in several reproductive processes. This review analyses the evidence for the role of EVs throughout human reproduction, starting with the paternal and maternal gametes, followed by the establishment and continuation of successful pregnancies, with specific focus, where possible, on the interaction of EVs with the maternal immune system. Importantly, variations within the EV populations are identified in various reproductive disorders, such as pre-term labour and pre-eclampsia.
基金financially supported by the Russian Government Program of Competitive Growth of Kazan Federal Universitysupported by state assignment 20.5175.2017/6.7 of the Ministry of Education and Science of Russian Federationthe President of the Russian Federation grant НШ-3076.2018.4
文摘Extracellular vesicles,including exosomes and microvesicles,play a fundamental role in the activity of the nervous system,participating in signal transmission between neurons and providing the interaction of central nervous system with all body systems.In many neurodegenerative diseases,neurons pack toxic substances into vesicles and release them into the extracellular space,which leads to the spread of misfolded neurotoxic proteins.The contents of neuron-derived extracellular vesicles may indicate pathological changes in the central nervous system,and the analysis of extracellular vesicle molecular content contributes to the development of non-invasive methods for the diagnosis of many central nervous system diseases.Extracellular vesicles of neuronal origin can be isolated from various biological fluids due to their ability to cross the blood-brain barrier.Today,the diagnostic potential of almost all toxic proteins involved in nervous system disease pathogenesis,specificallyα-synuclein,tau protein,superoxide dismutase 1,FUS,leucine-rich repeat kinase 2,as well as some synaptic proteins,has been well evidenced.Special attention is paid to extracellular RNAs mostly associated with extracellular vesicles,which are important in the onset and development of many neurodegenerative diseases.Depending on parental cell type,extracellular vesicles may have different therapeutic properties,including neuroprotective,regenerative,and anti-inflammatory.Due to nano size,biosafety,ability to cross the blood-brain barrier,possibility of targeted delivery and the lack of an immune response,extracellular vesicles are a promising vehicle for the delivery of therapeutic substances for the treatment of neurodegenerative diseases and drug delivery to the brain.This review describes modern approaches of diagnosis and treatment of central nervous system diseases using extracellular vesicles.
文摘BACKGROUND Endothelial activation plays an important role in sepsis-mediated inflammation,but the triggering factors have not been fully elucidated.Microvesicles carrying mitochondrial content(mitoMVs)have been implicated in several diseases and shown to induce endothelial activation.AIM To explore whether mitoMVs constitute a subset of MVs isolated from plasma of patients with sepsis and contribute to endothelial activation.METHODS MVs were isolated from human plasma and characterized by confocal microscopy and flow cytometry.Proinflammatory cytokines,including interleukin(IL)-6,IL-8 and tumour necrosis factor(TNF)-α,and soluble vascular cell adhesion molecule(sVCAM)-1 were detected by ELISA.Human umbilical vein endothelial cells(HUVECs)were stimulated with the circulating MVs to evaluate their effect on endothelial activation.RESULTS MitoMVs were observed in plasma from patients with sepsis.Compared with those in healthy controls,expression of MVs,mitoMVs,proinflammatory cytokines and sVCAM-1 was increased.The number of mitoMVs was positively associated with TNF-αand sVCAM-1.In vitro,compared with MVs isolated from the plasma of healthy controls,MVs isolated from the plasma of patients with sepsis induced expression of OAS2,RSAD2,and CXCL10 in HUVECs.MitoMVs were taken up by HUVECs,and sonication of MVs significantly reduced the uptake of mitoMVs by HUVECs and expression of the above three type I IFNdependent genes.CONCLUSION MitoMVs are increased in the plasma of patients with sepsis,which induces elevated expression of type I IFN-dependent genes.This suggests that circulating mitoMVs activate the type I IFN signalling pathway in endothelial cells and lead to endothelial activation.
文摘AIM:To develop protocols for isolation of exosomes and characterization of their RNA content.METHODS:Exosomes were extracted from He La cell culture media and human blood serum using the Total exosome isolation(from cell culture media)reagent,and Total exosome isolation(from serum)reagent respectively.Identity and purity of the exosomes was confirmed by Nanosight?analysis,electron microscopy,and Western blots for CD63 marker.Exosomal RNA cargo was recovered with the Total exosome RNA and protein isolation kit.Finally,RNA was profiled using Bioanalyzer and quantitative reverse transcriptionpolymerase chain reaction(q RT-PCR)methodology.RESULTS:Here we describe a novel approach for robust and scalable isolation of exosomes from cell culture media and serum,with subsequent isolation and analysis of RNA residing within these vesicles.The isolation procedure is completed in a fraction of the time,compared to the current standard protocols utilizing ultracentrifugation,and allows to recover fully intact exosomes in higher yields.Exosomes were found tocontain a very diverse RNA cargo,primarily short sequences 20-200 nt(such as mi RNA and fragments of m RNA),however longer RNA species were detected as well,including full-length 18S and 28S r RNA.CONCLUSION:We have successfully developed a set of reagents and a workflow allowing fast and efficient extraction of exosomes,followed by isolation of RNA and its analysis by q RT-PCR and other techniques.
基金the National Natural Science Foundation of China(21535005,91859123,21705111).
文摘Cell-derived microvesicles(MVs) are secreted from almost all kinds of mammalian cells into the extracellular space, and play crucial roles in intercellular communication and transporting biomolecules between cells. However, there is a great challenge for visualizing and monitoring of MVs’ bio-behaviors due to the limitations of existing labeling methods. Herein, we report the first paradigm of designer cell-self-implemented labeling of MVs secreted from living mammalian MCF-7 cells in situ using the intracellular-synthesized fluorescent quantum dots(QDs) during the formation of MVs. By elaborately coupling intracellular biochemical reactions and metabolism pathways, the MCF-7 cells can be illuminated brightly by intracellular-biosynthesized fluorescent CdSe QDs. Simultaneously, intracellular-synthesized QDs can be in situ encapsulated by the secreted MVs budding from the plasma membrane of the fluorescing cells to label the MVs with an efficiency of up to 89.9%. The whole labeling process skillfully combines designer precise cell-tuned intricate synthesis of CdSe QDs with mild in-situ labeling via cell-selfimplementation just after feeding the cell with suitable chemicals, which is structure-or function-nondestructive and much more straightforward and milder than those by chemical conjugation or indirect encapsulation with conventional fluorogenic labels.
基金National Natural Science Foundation of China,Grant/Award Number:82073325。
文摘Background:Trastuzumab is a first-line targeted therapy for human epidermal growth factor receptor-2(HER2)-positive gastric cancer.However,the inevitable occurrence of acquired trastuzumab resistance limits the drug benefit,and there is currently no effective reversal measure.Existing researches on the mechanism of trastuzumab resistance mainly focused on tumor cells themselves,while the understanding of the mechanisms of environment-mediated drug resistance is relatively lacking.This study aimed to further explore the mechanisms of trastuzumab resistance to identify strategies to promote survival in these patients.Methods:Trastuzumab-sensitive and trastuzumab-resistant HER2-positive tumor tissues and cells were collected for transcriptome sequencing.Bioinformatics were used to analyze cell subtypes,metabolic pathways,and molecular signaling pathways.Changes in microenvironmental indicators(such as macrophage,angiogenesis,and metabolism)were verified by immunofluorescence(IF)and immunohistochemical(IHC)analyses.Finally,a multi-scale agent-based model(ABM)was constructed.The effects of combination treatment were further validated in nude mice to verify these effects predicted by the ABM.Results:Based on transcriptome sequencing,molecular biology,and in vivo experiments,we found that the level of glutamine metabolism in trastuzumabresistant HER2-positive cells was increased,and glutaminase 1(GLS1)was significantly overexpressed.Meanwhile,tumor-derived GLS1 microvesicles drove M2macrophage polarization.Furthermore,angiogenesis promoted trastuzumab resistance.IHC showed high glutamine metabolism,M2 macrophage polarization,and angiogenesis in trastuzumab-resistant HER2-positive tumor tissues from patients and nudemice.Mechanistically,the cell division cycle 42(CDC42)promoted GLS1 expression in tumor cells by activating nuclear factor kappa-B(NF-κB)p65 and drove GLS1microvesicle secretion through IQmotif-containing GTPase-activating protein 1(IQGAP1).Based on the ABM and in vivo experiments,we confirmed that the combi
基金Research Project of Jiangsu Provincial Health Commission,No.Z2022008and Research Project of Yangzhou Health Commission,No.2023-2-27.
文摘BACKGROUND Pulmonary fibrosis is one of the main reasons for the high mortality rate among acute respiratory distress syndrome(ARDS)patients.Mesenchymal stromal cell-derived microvesicles(MSC-MVs)have been shown to exert antifibrotic effects in lung diseases.AIM To investigate the effects and mechanisms of MSC-MVs on pulmonary fibrosis in ARDS mouse models.METHODS MSC-MVs with low hepatocyte growth factor(HGF)expression(siHGF-MSC-MVs)were obtained via lentivirus transfection and used to establish the ARDS pulmonary fibrosis mouse model.Following intubation,respiratory mechanics-related indicators were measured via an experimental small animal lung function tester.Homing of MSC-MVs in lung tissues was investigated by near-infrared live imaging.Immunohistochemical,western blotting,ELISA and other methods were used to detect expression of pulmonary fibrosis-related proteins and to compare effects on pulmonary fibrosis and fibrosis-related indicators.RESULTS The MSC-MVs gradually migrated and homed to damaged lung tissues in the ARDS model mice.Treatment with MSC-MVs significantly reduced lung injury and pulmonary fibrosis scores.However,low expression of HGF(siHGF-MSC-MVs)significantly inhibited the effects of MSC-MVs(P<0.05).Compared with the ARDS pulmonary fibrosis group,the MSC-MVs group exhibited suppressed expression of type I collagen antigen,type III collagen antigen,and the proteins transforming growth factor-βandα-smooth muscle actin,whereas the siHGF-MVs group exhibited significantly increased expression of these proteins.In addition,pulmonary compliance and the pressure of oxygen/oxygen inhalation ratio were significantly lower in the MSC-MVs group,and the effects of the MSC-MVs were significantly inhibited by low HGF expression(all P<0.05).CONCLUSION MSC-MVs improved lung ventilation functions and inhibited pulmonary fibrosis in ARDS mice partly via HGF mRNA transfer.
