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1.
Nature ; 579(7798): 260-264, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32132711

RESUMO

The production of pore-forming toxins that disrupt the plasma membrane of host cells is a common virulence strategy for bacterial pathogens such as methicillin-resistant Staphylococcus aureus (MRSA)1-3. It is unclear, however, whether host species possess innate immune mechanisms that can neutralize pore-forming toxins during infection. We previously showed that the autophagy protein ATG16L1 is necessary for protection against MRSA strains encoding α-toxin4-a pore-forming toxin that binds the metalloprotease ADAM10 on the surface of a broad range of target cells and tissues2,5,6. Autophagy typically involves the targeting of cytosolic material to the lysosome for degradation. Here we demonstrate that ATG16L1 and other ATG proteins mediate protection against α-toxin through the release of ADAM10 on exosomes-extracellular vesicles of endosomal origin. Bacterial DNA and CpG DNA induce the secretion of ADAM10-bearing exosomes from human cells as well as in mice. Transferred exosomes protect host cells in vitro by serving as scavengers that can bind multiple toxins, and improve the survival of mice infected with MRSA in vivo. These findings indicate that ATG proteins mediate a previously unknown form of defence in response to infection, facilitating the release of exosomes that serve as decoys for bacterially produced toxins.


Assuntos
Proteínas Relacionadas à Autofagia/metabolismo , Toxinas Bacterianas/metabolismo , Exossomos/metabolismo , Células A549 , Proteína ADAM10/metabolismo , Animais , Toxinas Bacterianas/farmacologia , Sobrevivência Celular/efeitos dos fármacos , DNA Bacteriano/farmacologia , Exossomos/efeitos dos fármacos , Exossomos/ultraestrutura , Feminino , Células HEK293 , Humanos , Masculino , Staphylococcus aureus Resistente à Meticilina/patogenicidade , Staphylococcus aureus Resistente à Meticilina/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Infecções Estafilocócicas/mortalidade
2.
Muscle Nerve ; 61(3): 401-407, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31889318

RESUMO

BACKGROUND: Exosomal miRNA expression for myasthenia gravis (MG) has not been studied. METHODS: Plasma samples from 92 patients with MG and 49 age-matched healthy controls (HCs) were screened (by means of deep sequencing and quantitative real-time polymerase chain reaction) for differentially expressed exosomal microRNA (miRNAs). miR-106a-5p was chosen to further verify because it is reportedly involved in MG pathogenesis. Spearman's correlation analysis was used to assess correlations between candidate miRNAs and patient quantitative MG scores (QMGSs). Area under the curve (AUC) of the receiver operating characteristic analysis was used to evaluate the diagnostic accuracy of the identified miRNAs for MG. RESULTS: miR-106a-5p levels were significantly decreased in MG patients compared with HCs, and were associated with patient QMGS. The AUC values for hsa-miR-106a-5p were 0.728 and 0.813 in ocular and generalized MG patients, respectively. CONCLUSIONS: Exosomal miR-106a-5p was expressed differently in different types of MG and was associated with MG severity.


Assuntos
MicroRNAs/sangue , Miastenia Gravis/sangue , Miastenia Gravis/diagnóstico , Adulto , Exossomos/metabolismo , Exossomos/ultraestrutura , Feminino , Humanos , Masculino , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e Especificidade , Índice de Gravidade de Doença
3.
PLoS One ; 15(1): e0227949, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31999742

RESUMO

Extracellular vesicles (EVs) are membrane-enclosed vesicles which play important role for cell communication and physiology. EVs are found in many human biological fluids, including blood, breast milk, urine, cerebrospinal fluid (CSF), ejaculate, saliva etc. These nano-sized vesicles contain proteins, mRNAs, microRNAs, non-coding RNAs and lipids that are derived from producing cells. EVs deliver complex sets of biological information to recipient cells thereby modulating their behaviors by their molecular cargo. In this way EVs are involved in the pathological development and progression of many human disorders, including neurodegenerative diseases. In this study EVs purified by ultracentrifugation from CSF of patients with Parkinson's disease (PD) and individuals of the comparison group were characterized using nanoparticle tracking analysis, flow cytometry and cryo-electron microscopy. Vesicular size and the presence of exosomal marker CD9 on the surface provided evidence that most of the EVs were exosome-like vesicles. Cryo-electron microscopy allowed us to visualize a large spectrum of extracellular vesicles of various size and morphology with lipid bilayers and vesicular internal structures. Thus, we described the diversity and new characteristics of the vesicles from CSF suggesting that subpopulations of EVs with different and specific functions may exist.


Assuntos
Microscopia Crioeletrônica , Exossomos/ultraestrutura , Vesículas Extracelulares/ultraestrutura , Doença de Parkinson/líquido cefalorraquidiano , Idoso , Animais , Biomarcadores/líquido cefalorraquidiano , Líquido Cefalorraquidiano/diagnóstico por imagem , Exossomos/química , Vesículas Extracelulares/química , Feminino , Citometria de Fluxo , Humanos , Bicamadas Lipídicas/líquido cefalorraquidiano , Masculino , MicroRNAs/química , MicroRNAs/isolamento & purificação , Pessoa de Meia-Idade , Nanopartículas/química , Doença de Parkinson/patologia , Tetraspanina 29/líquido cefalorraquidiano
4.
Int J Nanomedicine ; 14: 8603-8610, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31802872

RESUMO

Purpose: The primary goal of the present study was to develop the nano-drug consisting of doxorubicin and exosome derived from mesenchymal stem cells, and to explore its effect on osteosarcoma in vitro. Methods: The exosomes were isolated from bone marrow MSCs (BM-MSCs) by an Exosome Isolation Kit. The exosome-loaded doxorubicin (Exo-Dox) was prepared by mixing exosome with Dox-HCl, desalinizing with triethylamine and then dialyzing against PBS overnight. The nanoparticle tracking analysis (NTA) and transmission electron microscope (TEM) were used to characterize of the exosome and Exo-Dox. The cytotoxicity of Exo-Dox was determined by CCK-8 assay. Further, the cellular uptake of different drugs was analyzed using inverted fluorescence microscope and flow cytometry. Results: The typical exosome structures can be observed by TEM. After loading with doxorubicin, its size is larger than free exosome. Compared with the free Dox, the prepared Exo-Dox showed enhanced cellular uptake efficiency and anti-tumor effect in osteosarcoma MG63 cell line but low cytotoxicity in myocardial H9C2 cell line. Conclusion: The prepared Exo-Dox could be used as an excellent chemotherapeutic drug for treatment of osteosarcoma in vitro. Considering the tumor-homing feature of BM-MSCs, the Exo-Dox may be a good candidate for targeted osteosarcoma treatment in future study.


Assuntos
Doxorrubicina/uso terapêutico , Exossomos/metabolismo , Células-Tronco Mesenquimais/metabolismo , Nanopartículas/química , Osteossarcoma/tratamento farmacológico , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/patologia , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/farmacologia , Liberação Controlada de Fármacos , Exossomos/ultraestrutura , Humanos , Camundongos , Nanopartículas/ultraestrutura , Osteossarcoma/patologia , Ratos
5.
Parasit Vectors ; 12(1): 467, 2019 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-31597577

RESUMO

BACKGROUND: Pathogenic protozoans use extracellular vesicles (EVs) for intercellular communication and host manipulation. Acanthamoeba castellanii is a free-living protozoan that may cause severe keratitis and fatal granulomatous encephalitis. Although several secreted molecules have been shown to play crucial roles in the pathogenesis of Acanthamoeba, the functions and components of parasite-derived EVs are far from understood. METHODS: Purified EVs from A. castellanii were confirmed by electron microscopy and nanoparticle tracking analysis. The functional roles of parasite-derived EVs in the cytotoxicity to and immune response of host cells were examined. The protein composition in EVs from A. castellanii was identified and quantified by LC-MS/MS analysis. RESULTS: EVs from A. castellanii fused with rat glioma C6 cells. The parasite-derived EVs induced an immune response from human THP-1 cells and a cytotoxic effect in C6 cells. Quantitative proteomic analysis identified a total of 130 proteins in EVs. Among the identified proteins, hydrolases (50.2%) and oxidoreductases (31.7%) were the largest protein families in EVs. Furthermore, aminopeptidase activities were confirmed in EVs from A. castellanii. CONCLUSIONS: The proteomic profiling and functional characterization of EVs from A. castellanii provide an in-depth understanding of the molecules packaged into EVs and their potential mechanisms mediating the pathogenesis of this parasite.


Assuntos
Acanthamoeba castellanii/fisiologia , Exossomos/química , Exossomos/fisiologia , Proteômica , Ceratite por Acanthamoeba/parasitologia , Acanthamoeba castellanii/patogenicidade , Acanthamoeba castellanii/ultraestrutura , Aminopeptidases/análise , Animais , Infecções Protozoárias do Sistema Nervoso Central/parasitologia , Meios de Cultura , DNA Complementar/biossíntese , Exossomos/imunologia , Exossomos/ultraestrutura , Humanos , Microscopia Eletrônica de Transmissão , Neuroglia/parasitologia , RNA de Protozoário/genética , RNA de Protozoário/isolamento & purificação , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células THP-1/imunologia , Células THP-1/parasitologia
6.
Int J Mol Sci ; 20(20)2019 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-31600881

RESUMO

Despite the different strategies used to treat ovarian cancer, around 70% of women/patients eventually fail to respond to the therapy. Cancer stem cells (CSCs) play a role in the treatment failure due to their chemoresistant properties. This capacity to resist chemotherapy allows CSCs to interact with different components of the tumor microenvironment, such as mesenchymal stem cells (MSCs), and thus contribute to tumorigenic processes. Although the participation of MSCs in tumor progression is well understood, it remains unclear how CSCs induce the pro-tumorigenic activity of MSCs in response to chemotherapy. Small extracellular vesicles, including exosomes, represent one possible way to modulate any type of cell. Therefore, in this study, we evaluate if small extracellular vesicle (sEV) derived from ovarian cancer spheroids (OCS), which are enriched in CSCs, can modify the activity of MSCs to a pro-tumorigenic phenotype. We show that sEV released by OCS in response to cisplatin induce an increase in the migration pattern of bone marrow MSCs (BM-MSCs) and the secretion interleukin-6 (IL-6), interleukin-8 (IL-8), and vascular endothelial growth factor A (VEGFA). Moreover, the factors secreted by BM-MSCs induce angiogenesis in endothelial cells and the migration of low-invasive ovarian cancer cells. These findings suggest that cisplatin could modulate the cargo of sEV released by CSCs, and these exosomes can further induce the pro-tumorigenic activity of MSCs.


Assuntos
Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/metabolismo , Cisplatino/farmacologia , Vesículas Extracelulares/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Neoplasias Ovarianas/etiologia , Neoplasias Ovarianas/metabolismo , Linhagem Celular Tumoral , Citocinas/metabolismo , Exossomos/metabolismo , Exossomos/ultraestrutura , Vesículas Extracelulares/ultraestrutura , Feminino , Expressão Gênica , Humanos , Metaloproteases/genética , Metaloproteases/metabolismo , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Neoplasias Ovarianas/patologia , Esferoides Celulares , Microambiente Tumoral
7.
Mol Cell Biochem ; 462(1-2): 97-105, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31473882

RESUMO

Our previous study shows that high Chloride intracellular channel 1 (CLIC1) expression can efficiently enhance invasion and migration of gastric cancer (GC) cells in vitro. Growing evidences have found that exosomes are involved in chemotherapy resistance in several cancers including GC. We aimed to evaluate the effect of the exosome-mediated transfer of CLIC1 in the vincristine-resistance of GC. The effect of exosome-mediated transfer of CLIC1 on the development of resistance to vincristine in GC cell line SGC-7901 and the potential underlying mechanisms were investigated by Cell Counting Kit-8 (CCK8), RT-PCR, and Western blotting. Exosomes were isolated from cell supernatants by differential ultracentrifugation. Comparing with SGC-7901, the expression level of CLIC1 is higher in vincristine­resistant cell line SGC-7901/VCR (P < 0.05). After silencing the expression of CLIC1 by RNA interference, the half inhibition concentration (IC50) to vincristine decreased significantly in SGC-7901/VCR, and the expression of CLIC1 decreased significantly in exosomes from SGC-7901/VCR. After 48 h co-culturing with exosomes from SGC-7901/VCR, the IC50 to vincristine in SGC-7901 increased significantly, and the expression of CLIC1, P-gp, and Bcl-2 were significantly up-regulated. CLIC1 was closely associated with the resistance to vincristine in GC, and exosome-mediated transfer of CLIC1 could induce the development of resistance to vincristine in vitro. The possible mechanism was related to up-regulated P-gp and Bcl-2. However, in vivo study was needed to confirm the results in future.


Assuntos
Canais de Cloreto/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Exossomos/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Vincristina/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Exossomos/ultraestrutura , Inativação Gênica/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Neoplasias Gástricas/ultraestrutura
8.
Mol Cell Biochem ; 462(1-2): 115-122, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31473883

RESUMO

Rasal2, a Ras-GTPase-activating protein (RasGAP), is a tumor suppressor in Luminal B breast cancer, frequently metastatic and recurrent. Exosomes (Exos) are small membrane vesicles secreted by various cell types, including tumor cells, recognized as vehicles for cell-to-cell communication. Our study aimed to investigate whether Rasal2 regulates breast cancer cell growth via affecting this process. In this paper, we described that Rasal2 knockout (KO) in MCF-7 cells enhanced exosomal release and increased autophagy-related proteins in exosomal fraction, while attenuated by exosome release inhibitor GW4869. Moreover, MCF-7 cells with chloroquine (CQ) treatment boosted Rasal2 KO-induced secretory autophagy. In addition, we presented that exosomes derived from KO MCF-7 cells (KO-exo) significantly promoted breast cancer cell proliferation compared to those from MCF-7 cells transfected with an empty crispr-cas9 plasmid serving as controls (sgNT-exo); however, exosomes purified from KO MCF-7 cells co-cultured with 3-methyladenine ((3-MA + KO)-exo)/CQ ((CQ + KO)-exo) dramatically inhibited/facilitated MCF-7 cell proliferation in contrast to KO-exo group, separately. In conclusion, our findings revealed a new mechanism of Rasal2 in the regulation of breast cancer cell proliferation via autophagy-exo-mediated pathway.


Assuntos
Autofagia , Neoplasias da Mama/patologia , Proteínas Ativadoras de GTPase/metabolismo , Autofagia/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cloroquina/farmacologia , Exossomos/efeitos dos fármacos , Exossomos/metabolismo , Exossomos/ultraestrutura , Feminino , Humanos , Células MCF-7
9.
Mol Med Rep ; 20(3): 2549-2562, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31524256

RESUMO

Post­operative cognitive dysfunction (POCD) is a complication of the central nervous system characterized by mental disorders, anxiety, personality changes and impaired memory. POCD occurs frequently after coronary artery bypass grafting (CABG) and can severely affect quality of life for patients. To date, the development of POCD biomarkers remains a challenge. Alterations in the expression of non­coding RNAs from brain tissue and peripheral blood have been linked to POCD. The present study aimed to detect the differential circular RNAs (circRNAs) in plasma exosomes of patients with POCD after CABG. The relative expression levels of circRNAs were analyzed using circRNA microarray analysis in the plasma exosomes of patients with POCD. Differentially altered circRNAs (P<0.05, fold change >1.5) were validated by reverse transcription­quantitative PCR in the plasma exosomes of patients with POCD. The target genes of the microRNAs were predicted using bioinformatics analysis. The functions and signaling pathways of these target genes were investigated by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes analyses. The microarray results indicated that the levels of nine circRNAs in patients with POCD were higher than those in the control subjects; and six circRNAs were at a lower level than those in control subjects. The RT­qPCR results from patients with POCD showed that only circRNA_089763 of the 15 circRNAs identified was significantly increased compared with control subjects. circRNA target gene prediction and functional annotation analysis showed significant enrichment in several GO terms and pathways associated with POCD. The present study provides evidence for the abnormal expression of POCD­induced circRNA_089763 in human plasma exosomes, as well as the involvement of POCD.


Assuntos
Disfunção Cognitiva/sangue , Disfunção Cognitiva/etiologia , Ponte de Artéria Coronária , Exossomos , Expressão Gênica , /sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores , Biologia Computacional/métodos , Ponte de Artéria Coronária/efeitos adversos , Exossomos/metabolismo , Exossomos/ultraestrutura , Feminino , Perfilação da Expressão Gênica , Ontologia Genética , Humanos , Masculino , Pessoa de Meia-Idade , Período Pós-Operatório
10.
Toxicol Lett ; 316: 73-84, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31513886

RESUMO

In the liver microenvironment, interactions among diverse types of hepatic cells are involved in liver fibrosis. In fibrotic tissues, exosomes act as transporters in intercellular communication. Long non-coding RNAs (lncRNAs) are involved in the activation of hepatic stellate cells (HSCs), which are participants in liver fibrosis. However, the functions of exosomal lncRNAs in liver fibrosis induced by arsenite are undefined. The purposes of the present study were (a) to determine if lncRNAs secreted from human hepatic (L-02) cells exposed to arsenite are shuttled to hepatic stellate LX-2 cells and (b) to establish their effects on LX-2 cells. In mice, MALAT1 was overexpressed in the progression of liver fibrosis induced by arsenite as well as in L-02 cells exposed to arsenite. Co-cultures with arsenite-treated L-02 cells induced the activation of LX-2 cells and overexpression of MALAT1. Arsenite-treated L-02 cells transported MALAT1 into LX-2 cells. Downregulation of MALAT1, which reduced the MALAT1 levels in exosomes derived from arsenite-treated L-02 cells, inhibited the activation of LX-2 cells. Additionally, exosomal MALAT1 derived from arsenite-treated L-02 cells promoted the activation of LX-2 cells via microRNA-26b regulation of COL1A2. Furthermore, circulating exosomal MALAT1 was up-regulated in people exposed to arsenite. In sum, exosomes derived from arsenite-treated hepatic cells transferred MALAT1 to HSCs, which induced their activation. These findings support the concept that, during liver fibrosis induced by arsenite, exosomal lncRNAs are involved in cell-cell communication.


Assuntos
Arsenitos , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Exossomos/metabolismo , Células Estreladas do Fígado/metabolismo , Cirrose Hepática Experimental/metabolismo , Fígado/metabolismo , MicroRNAs/metabolismo , RNA Longo não Codificante/metabolismo , Compostos de Sódio , Animais , Linhagem Celular , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Doença Hepática Induzida por Substâncias e Drogas/genética , Doença Hepática Induzida por Substâncias e Drogas/patologia , Técnicas de Cocultura , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Exossomos/genética , Exossomos/ultraestrutura , Regulação da Expressão Gênica , Células Estreladas do Fígado/ultraestrutura , Humanos , Fígado/ultraestrutura , Cirrose Hepática Experimental/induzido quimicamente , Cirrose Hepática Experimental/genética , Cirrose Hepática Experimental/patologia , Masculino , Camundongos , MicroRNAs/genética , RNA Longo não Codificante/genética , Transdução de Sinais
11.
Int J Mol Sci ; 20(18)2019 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-31514388

RESUMO

The thyroid is a major component of the endocrine system and its pathology can cause serious diseases, e.g., papillary carcinoma (PC). However, the carcinogenic mechanisms are poorly understood and clinical useful biomarkers are scarce. Therefore, we determined if there are quantitative patterns of molecular chaperones in the tumor tissue and circulating exosomes that may be useful in diagnosis and provide clues on their participation in carcinogenesis. Hsp27, Hsp60, Hsp70, and Hsp90 were quantified by immunohistochemistry in PC, benign goiter (BG), and normal peritumoral tissue (PT). The same chaperones were assessed in plasma exosomes from PC and BG patients before and after ablative surgery, using Western blotting. Hsp27, Hsp60, and Hsp90 were increased in PC in comparison with PT and BG but no differences were found for Hsp70. Similarly, exosomal levels of Hsp27, Hsp60, and Hsp90 were higher in PC than in BG, and those in PC were higher before ablative surgery than after it. Hsp27, Hsp60, and Hsp90 show distinctive quantitative patterns in thyroid tissue and circulating exosomes in PC as compared with BG, suggesting some implication in the carcinogenesis of these chaperones and indicating their potential as biomarkers for clinical applications.


Assuntos
Exossomos/metabolismo , Proteínas de Choque Térmico/metabolismo , Glândula Tireoide/imunologia , Glândula Tireoide/patologia , Carcinoma Papilar/imunologia , Carcinoma Papilar/metabolismo , Carcinoma Papilar/patologia , Exossomos/ultraestrutura , Feminino , Bócio/metabolismo , Bócio/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Glândula Tireoide/metabolismo
12.
Nat Commun ; 10(1): 4136, 2019 09 12.
Artigo em Inglês | MEDLINE | ID: mdl-31515491

RESUMO

Astroglia play active and diverse roles in modulating neuronal/synaptic functions in the CNS. How these astroglial functions are regulated, especially by neuronal signals, remains largely unknown. Exosomes, a major type of extracellular vesicles (EVs) that originate from endosomal intraluminal vesicles (ILVs), have emerged as a new intercellular communication process. By generating cell-type-specific ILVs/exosome reporter (CD63-GFPf/f) mice and immuno-EM/confocal image analysis, we found that neuronal CD63-GFP+ ILVs are primarily localized in soma and dendrites, but not in axonal terminals in vitro and in vivo. Secreted neuronal exosomes contain a subset of microRNAs (miRs) that is distinct from the miR profile of neurons. These miRs, especially the neuron-specific miR-124-3p, are potentially internalized into astrocytes. MiR-124-3p further up-regulates the predominant glutamate transporter GLT1 by suppressing GLT1-inhibiting miRs. Our findings suggest a previously undescribed neuronal exosomal miR-mediated genetic regulation of astrocyte functions, potentially opening a new frontier in understanding CNS intercellular communication.


Assuntos
Astrócitos/metabolismo , Comunicação Celular , Sistema Nervoso Central/metabolismo , Exossomos/metabolismo , Genes Reporter , Neurônios/metabolismo , Animais , Astrócitos/ultraestrutura , Transportador 2 de Aminoácido Excitatório/metabolismo , Exossomos/ultraestrutura , Proteínas de Fluorescência Verde/metabolismo , Humanos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , MicroRNAs/metabolismo , Modelos Biológicos , Neurônios/ultraestrutura
13.
Molecules ; 24(16)2019 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-31416240

RESUMO

Exosomes contain different functional bimolecular characteristics related to physiological or pathological processes and are now recognized as new biomarkers in different human cancers. Rapid detection and classification of cancer-related exosomes might be helpful in the rapid screening of patients that may have cancer. Here, we report a surface enhanced Raman scattering technology for rapid and label-free exosomal detection (Exo-SERS) to aid in the discrimination of different cancer cells based on specific Raman phenotypes and multivariate statistical analysis. The results demonstrated that exosomes derived from both tumor cells and normal cells exhibit special, unique Raman phenotypes. Using the Exo-SERS method, the cancer cells were accurately discriminated from normal cells, and subtle molecular changes between the different cell types could be detected with high sensitive. This research provides a rapid, label-free and non-destructive manner for detecting and discriminating between cancer types.


Assuntos
Biomarcadores Tumorais , Exossomos/química , Neoplasias/diagnóstico , Análise Espectral Raman , Linhagem Celular Tumoral , Exossomos/classificação , Exossomos/ultraestrutura , Ouro/química , Humanos , Nanopartículas Metálicas/química , Nanopartículas Metálicas/ultraestrutura , Análise Multivariada , Fenótipo , Análise Espectral Raman/métodos
14.
Int J Nanomedicine ; 14: 5679-5690, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31413568

RESUMO

Background: Exosomes are natural nanovesicles with unique characteristics, such as long circulating half-life, the intrinsic ability to target tissues, biocompatibility, and minimal or no inherent systemic toxicity. Mesenchymal stem cells produce large amounts of exosomes with regenerative properties and more stability in human plasma. TUBO breast cancer cell lines overexpress rat HER2/neu protein. Methods: Targeted exosomes were isolated from transduced bone marrow mesenchymal stem cells. Doxorubicin was encapsulated into exosomes by electroporation. Flow cytometry was used to assess the attachment of exosomes to the target cells. The in vitro cytotoxicity effect of targeted doxorubicin-loaded exosomes on TUBO cells was determined using MTT assay. Selective delivery of doxorubicin to tumor tissues was analyzed by measuring the auto-fluorescence of doxorubicin by in vivo imaging system. Moreover, tumor growth inhibition and body weight were monitored following injection of free doxorubicin, and targeted and untargeted doxorubicin-loaded exosomes in a TUBO breast cancer model. Finally, mouse tissues were examined for the presence of intrinsic fluorescence of doxorubicin. Results: Flow cytometry results revealed significant differences in binding of targeted exosomes to HER2-positive (46.05%) and HER2-negative (13.9%) cells. The results of MTT assay showed that cytotoxicity of targeted doxorubicin-loaded exosomes was higher than free doxorubicin at 72 hours. Selective distribution of targeted doxorubicin-loaded exosomes in the target tissues of the murine breast cancer model suggested specific delivery of doxorubicin by targeted exosomes, rather than untargeted exosomes. Free doxorubicin and untargeted doxorubicin-loaded exosomes showed insignificant effects, whereas targeted doxorubicin-loaded exosomes reduced the tumor growth rate. Conclusion: Herein, we report efficient delivery of targeted doxorubicin-loaded exosomes in vitro, corroborated with a significant reduction of murine breast cancer model tumor growth rate.


Assuntos
Doxorrubicina/farmacologia , Sistemas de Liberação de Medicamentos , Receptor ErbB-2/metabolismo , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Exossomos/metabolismo , Exossomos/ultraestrutura , Feminino , Células HEK293 , Humanos , Células-Tronco Mesenquimais/metabolismo , Camundongos Nus , Ratos , Distribuição Tecidual/efeitos dos fármacos
15.
Biomed Res Int ; 2019: 3612020, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31467883

RESUMO

Background: Severe community-acquired pneumonia (SCAP) requiring intensive care unit (ICU) treatment commonly causes acute respiratory distress syndrome (ARDS) with high mortality. This study was aimed at evaluating whether microRNAs (miRNAs) in circulating exosomes have the predictive values for patients at risk of developing ARDS due to SCAP. Methods: ARDS/ALI-relevant miRNAs were obtained by literature search. Exosomes in serum were isolated by ultracentrifugation method and identified by Transmission Electron Microscopy. Then the miR profiling in the exosomes using real-time PCR was analyzed in SCAP patients with or without ARDS. Moreover, multivariate Cox proportional regression analysis was performed to estimate the odds ratio of miRNA for the occurrence of ARDS and prognosis. The receiver operating characteristics (ROC) curves were calculated to discriminate ARDS cases. Finally, the Kaplan-Meier curve using log-rank method was performed to test the equality for survival distributions with different miRNA classifiers. Results: A total of 53 SCAP patients were finally recruited. Ten miRNAs were picked out. Further, a subset of exosomal miRNAs, including the miR-146a, miR-27a, miR-126, and miR-155 in ARDS group exhibited significantly elevated levels than those in non-ARDS group. The combined expression of miR-126, miR-27a, miR-146a, and miR-155 predicted ARDS with an area under the curve of 0.909 (95 % CI 0.815 -1). Only miR-126 was selected to have potential to predict the 28-day mortality (OR=1.002, P=0.024) with its median value classifier. Conclusions: The altered levels of circulating exosomal microRNAs may be useful biologic confirmation of ARDS in patients with SCAP.


Assuntos
Infecções Comunitárias Adquiridas/sangue , MicroRNAs/sangue , Pneumonia/sangue , Síndrome do Desconforto Respiratório do Adulto/sangue , MicroRNA Circulante/sangue , MicroRNA Circulante/genética , Infecções Comunitárias Adquiridas/genética , Infecções Comunitárias Adquiridas/patologia , Exossomos/genética , Exossomos/ultraestrutura , Feminino , Humanos , Unidades de Terapia Intensiva , Estimativa de Kaplan-Meier , Masculino , MicroRNAs/genética , Microscopia Eletrônica de Transmissão , Pessoa de Meia-Idade , Pneumonia/genética , Pneumonia/patologia , Prognóstico , Síndrome do Desconforto Respiratório do Adulto/genética , Síndrome do Desconforto Respiratório do Adulto/patologia
16.
Cells ; 8(7)2019 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-31311206

RESUMO

The use of extracellular vesicles, specifically exosomes, as carriers of biomarkers in extracellular spaces has been well demonstrated. Despite their promising potential, the use of exosomes in the clinical setting is restricted due to the lack of standardization in exosome isolation and analysis methods. The purpose of this review is to not only introduce the different types of extracellular vesicles but also to summarize their differences and similarities, and discuss different methods of exosome isolation and analysis currently used. A thorough understanding of the isolation and analysis methods currently being used could lead to some standardization in the field of exosomal research, allowing the use of exosomes in the clinical setting to become a reality.


Assuntos
Fracionamento Celular/métodos , Exossomos/química , Animais , Exossomos/metabolismo , Exossomos/ultraestrutura , Humanos , Proteômica/métodos
17.
Biomed Res Int ; 2019: 2045915, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31312654

RESUMO

Purpose: To elucidate the microRNAs existent in exosomes derived from stored red blood cell (RBC) unit and their potential function. Materials and Methods: Exosomes were isolated from the supernatant derived from stored RBC units by sequential centrifugation. Isolated exosomes were characterized by TEM (transmission electron microscopy), western blotting, and DLS (dynamic light scattering). MicroRNA (miRNA) microarray was performed to detect the expression of miRNAs in 3 exosome samples. Results revealed miRNAs that were simultaneously expressed in the 3 exosome samples and were previously reported to exist in mature RBCs. Functions and potential pathways of some detected miRNAs were illustrated by bioinformatic analysis. Validation of the top 3 abundant miRNAs was carried out by qRT-PCR (quantitative reverse transcription-polymerase chain reaction). Results: TEM and DLS revealed the mean size of the exosomes (RBC-derived) as 64.08 nm. These exosomes exhibited higher abundance of short RNA than the long RNA. 78 miRNAs were simultaneously detected in 3 exosome samples and mature RBCs. Several biological processes might be impacted by these miRNAs, through their target gene(s) enriched in a particular signalling pathway. The top 3 (abundant) miRNAs detected were as follows: miR-125b-5p, miR-4454, and miR-451a. qRT-PCR revealed higher abundance of miR-451a than others. Only miR-4454 and miR-451a abundance tended to increase with increasing storage time. Conclusion: Exosomes derived from stored RBC units possessed multiple miRNAs and, hence, could serve various functions. The function of exosomes (RBC-derived) might be implemented partly by the predominantly enriched miR-451a.


Assuntos
Exossomos/genética , MicroRNAs/genética , Transfusão de Sangue/tendências , Biologia Computacional , Difusão Dinâmica da Luz , Eritrócitos/imunologia , Eritrócitos/metabolismo , Exossomos/ultraestrutura , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Humanos , Imunomodulação/genética , MicroRNAs/classificação , Microscopia Eletrônica de Transmissão
18.
Biomed Pharmacother ; 117: 109109, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31229922

RESUMO

Prostate carcinoma may develop into metastatic castration-resistant prostate carcinoma (mCRPC) after endocrine therapy. Exosomal microRNAs play an important role in the regulation of tumor microenvironment. Our study aimed to investigate the effect of exosomal miR-26a on tumor phenotype of prostate carcinoma. Low-grade prostate carcinoma cell line (LNCAP) and mCRPC cell line (PC-3) were treated as experimental subjects according to their miR-26a expressions. Wound healing, transwell and colony-forming unit assays were performed after miR-26a mimic/inhibitor transfection. Then, exosomes were isolated from LNCAP and PC-3 cells, and the levels of exosomal miR-26a were determined. After co-culture of LNCAP (PC-3) cells with PC-3 (LNCAP) exosomes, changes in malignant behaviors were measured. Moreover, LNCAP/PC-3 exosomes were injected into xenograft tumor mice to determine effects of the exosomes on tumorigenicity of LNCAP and PC-3 cells. MiR-26a showed a potently inhibitory effect on cell proliferation, migration and invasion of LNCAP and PC-3 cells. LNCAP exosomes had a higher miR-26a level, compared with PC-3 exosomes. Overexpression of miR-26a attenuated the enhanced malignant behavior of LNCAP cells induced by PC-3 exosomes, and miR-26a inhibition could reverse the inhibitory effects of LNCAP exosomes on PC-3 cells. Exosomal miR-26a could significantly alter the expressions of epithelial-mesenchymal transition (EMT)-related factors. Moreover, LNCAP exosomes suppressed the tumorigenicity of PC-3 cells, while PC-3 exosomes could promote the tumorigenicity of LNCAP cells. Our data suggest that exosomal miR-26a derived from prostate carcinoma cells had a suppressive effect on the metastasis and tumor growth of prostate carcinoma.


Assuntos
Exossomos/metabolismo , MicroRNAs/metabolismo , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Animais , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Transição Epitelial-Mesenquimal/genética , Exossomos/ultraestrutura , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/genética , Invasividade Neoplásica , Metástase Neoplásica
19.
Clin Lab ; 65(6)2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-31232015

RESUMO

BACKGROUND: Exosomes are potential markers for several diseases and health problems. Type 1 diabetes (T1D) has increasingly been reported in children and the world T1D has become the main type of diabetes in children. This study aimed to understand the function of urinary exosomal miRNAs in T1D in children. METHODS: We collected urinary samples from 30 healthy controls and 30 T1D in children. All exosomes were isolated with a combined centrifugation and were characterized by electron microscopy and western blot. The small RNA sequence was used to detect the urinary exosomal miRNAs, and miRNA markers were validated by real-time quantitative PCR. Two exosome diagnostic miRNAs were confirmed by receiver operating characteristic analyses. RESULTS: In this study, we found higher urinary exosomal miR-424 and miR-218 expression in T1D in children than in healthy controls. Urinary exosomal miR-424 and miR-218 showed better accuracy for T1D diagnose in children. CONCLUSIONS: Our results suggest that urinary exosomal miR-424 and miR-218 are biomarkers for T1D detection in children; miR-424 and miR-218 may be predictive of T1D prognosis in children.


Assuntos
Biomarcadores/metabolismo , Diabetes Mellitus Tipo 1/genética , Exossomos/genética , MicroRNAs/genética , Biomarcadores/urina , Criança , Diabetes Mellitus Tipo 1/diagnóstico , Diabetes Mellitus Tipo 1/urina , Exossomos/metabolismo , Exossomos/ultraestrutura , Feminino , Perfilação da Expressão Gênica/métodos , Hemoglobina A Glicada/análise , Humanos , Masculino , MicroRNAs/urina , Microscopia Eletrônica de Transmissão , Prognóstico , Curva ROC , Análise de Sequência de RNA/métodos
20.
Eur J Histochem ; 63(2)2019 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-31122004

RESUMO

Telocytes (TCs) are new interstitial cells, and they are involved in tissue regeneration, particularly in heart. Therefore, TCs are suggested to be a promising cell in regenerative medicine. However, the information of location structural characteristics and functions of TCs is still limited. In this study, cardiac TCs of the Chinese giant salamanders (Andrias davidianus) were identified by transmission electron microscopy. TCs were located in the interstitium between cardiomyocytes (CM). TCs possessed distinctive ultrastructural characteristics, including one to two very long and thin moniliform telopodes (Tps), emerging points from the cell body, caveolae, dichotomous branchings, labyrinthic systems, neighbouring exosomes and homo-cellular contacts between Tps. TCs/Tps were frequently observed in close proximity to cardiomyocytes. Moreover, Tps established hetero-cellular contacts with cardiomyocytes. Our results confirm the presence of TCs in the myocardium of the A. davidianus. This will help us to better understand roles of TCs in amphibian hearts.


Assuntos
Miocárdio/citologia , Telócitos/ultraestrutura , Animais , Cavéolas/ultraestrutura , Exossomos/ultraestrutura , Microscopia Eletrônica de Transmissão , Miócitos Cardíacos/citologia , Telopódios/ultraestrutura , Urodelos
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