RESUMO
A simple and wash-free POCT platform based on microcapillary was developed, using breast cancer cell-derived exosomes as a model. This method adopted the "one suction and one extrusion" mode. The hybridized complex of epithelial cell adhesion molecule (EpCAM) aptamer and complementary DNA-horseradish peroxidase conjugate (CDNA-HRP) was pre-modified on the microcapillary's inner surface. "One suction" meant inhaling the sample into the functionalized microcapillary. The exosomes could specifically bind with the EpCAM aptamer on the microcapillary's inner wall, and then the CDNA-HRP complex was released. "One extrusion" referred to squeezing the shedding CDNA-HRP into the 3,3',5,5'-tetramethylbenzidine (TMB)/H2O2 solution, and then the enzyme-catalyzed reaction would occur to make the solution yellow using sulfuric acid as the terminator. Therefore, exosome detection could be realized. The limit of detection was 2.69 × 104 particles mL-1 and the signal value had excellent linearity in the concentration range from 2.75 × 104 to 2.75 × 108 particlesâ mL-1 exosomes. In addition, the wash-free POCT platform also displayed a favorable reproducibility (RSD = 2.9%) in exosome detection. This method could effectively differentiate breast cancer patients from healthy donors. This work provided an easy-to-operate method for detecting cancer-derived exosomes without complex cleaning steps, which is expected to be applied to breast cancer screening.
Assuntos
Neoplasias da Mama , Exossomos , Humanos , Feminino , Neoplasias da Mama/diagnóstico , DNA Complementar/metabolismo , Exossomos/metabolismo , Peróxido de Hidrogênio/metabolismo , Molécula de Adesão da Célula Epitelial/metabolismo , Reprodutibilidade dos Testes , Sucção , Peroxidase do Rábano Silvestre/metabolismoRESUMO
Exosomes, as a non-invasive biomarker, perform an important role in breast cancer screening and prognosis monitoring. However, establishing a simple, sensitive, and reliable exosome analysis technique remains challenging. Herein, a one-step multiplex analysis electrochemical aptasensor based on a multi-probe recognition strategy was constructed to analyze breast cancer exosomes. HER2-positive breast cancer cell (SK-BR-3) exosomes were selected as the model targets and three aptamers including CD63, HER2 and EpCAM aptamers were used as the capture units. Methylene blue (MB) functionalized HER2 aptamer and ferrocene (Fc) functionalized EpCAM aptamer, which were modified on gold nanoparticles (Au NPs), i.e. MB-HER2-Au NPs and Fc-EpCAM-Au NPs, were used as signal units. When the mixture of target exosomes, MB-HER2-Au NPs and Fc-EpCAM-Au NPs were added on the CD63 aptamer modified gold electrode, two Au NPs modified by MB and Fc could be specifically captured on the electrode by the recognition of three aptamers with target exosomes. Then one-step multiplex analysis of exosomes was achieved by detecting two independent electrochemical signals. This strategy can not only distinguish breast cancer exosomes from other exosomes (including normal exosomes and other tumor exosomes) but also HER2-positive breast cancer exosomes and HER2-negative breast cancer exosomes. Besides, it had high sensitivity and can detect SK-BR-3 exosomes with a concentration as low as 3.4 × 103 particles mL-1. Crucially, this method can be applicable to the examination of exosomes in complicated samples, which is anticipated to afford assistance for the screening and prognosis of breast cancer.
Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , Neoplasias da Mama , Exossomos , Nanopartículas Metálicas , Humanos , Feminino , Neoplasias da Mama/diagnóstico , Ouro , Molécula de Adesão da Célula Epitelial , Técnicas Eletroquímicas/métodos , Técnicas Biossensoriais/métodosRESUMO
Combined detection of multiple markers related to the same disease could improve the accuracy of disease diagnosis. However, the abundance levels of multiple markers of the same disease varied widely in real samples, making it difficult for the traditional detection method to meet the requirements of a wide detection range. Herein, three kinds of cardiac biomarkers, cardiac troponin I (cTnI), myoglobin (Myo), and C-reaction protein (CRP), which were from the pM level to the µM level in real samples, were selected as model targets. Valency-controlled signal probes based on DNA tetrahedron nanostructures (DTNs) and platinum nanoparticles (PtNPs) were constructed for tunable cardiac biomarker detection. PtNPs with high horseradish peroxidase-like activity and stability served as signal molecules, and DTNs with unique spatial structure and sequence specificity were used for precisely controlling the number of connected PtNPs. By controlling the number of PtNPs connected to DTNs, monovalent, bivalent, and trivalent signal probes were obtained and were used for the detection of cardiac markers in different concentration ranges. The limit of detection of cTnI, Myo, and CRP was 3.0 pM, 0.4 nM, and 6.7 nM, respectively. Furthermore, it performed satisfactorily for the detection of cardiac markers in 10% human serum. It was anticipated that the design of valency-controlled signal probes based on DTNs and nanozymes could be extended to the construction of other multi-target detection platforms, thus providing a basis for the development of a new precision medical detection platform.
Assuntos
Nanopartículas Metálicas , Humanos , Nanopartículas Metálicas/química , Platina/química , DNA , Troponina I , MioglobinaRESUMO
Gold nanoparticles (Au NPs) has been widely used to develop label-free colorimetric biosensors. Since the lyophilization process of Au NPs might cause various stresses and lead to irreversible aggregation, Au NPs were usually preserved in an aqueous suspension, which was inconvenienced for transportation and storage. In addition, the potential adsorption interaction between target and Au NPs was often ignored, which may lead to false-signal for Au NPs based colorimetric strategy. Herein, polydopamine-coated gold nanoparticles (Au@PDA NPs) freeze-dried powder was prepared with the assistance of polyvinylpyrrolidone (PVP) (i.e. Au@PDA-PVP NPs) or polyethylene glycol (PEG) (i.e. Au@PDA-PEG NPs). After freeze-dried powder of Au@PDA nanoparticles was redissolved, not only their spectral properties can still be maintained, but also the Au@PDA nanoparticles have nice monodispersity. Besides, the freeze-dried powder has long-term stability and could be stored for at least nine months. Since kanamycin, an aminoglycoside antibiotic, can be absorbed on the surface of Au NPs and induce easily the false signal, it was difficult to be detected using conventional Au NPs-based colorimetric method. Thus, kanamycin was chosen as the model target, a simple, sensitive and label-free colorimetric sensor was established. Given that the adsorption between kanamycin and Au@PDA-PVP NPs was effectively avoided, the possibility of false-positive signal was also reduced. The detection limit of kanamycin was 0.22 nM (S/N = 3), which was met the requirements for the detection of kanamycin residues in milk. This work not only provided an effective and facile way to prepare the nanomaterial lyophilized powder, but also expanded the application of the Au NPs based colorimetric method.
Assuntos
Técnicas Biossensoriais , Nanopartículas Metálicas , Adsorção , Colorimetria , Ouro/química , Canamicina , Nanopartículas Metálicas/química , Polímeros/química , PósRESUMO
A microcapillary-based multicolor assay was developed for proteins quantification in serum sample with the assistance of manual centrifugal platform. The proposed assay only required the operation of "one suction and one extrusion" to realize the target detection. Myoglobin (Myo), a biomarker in the early stage of acute myocardial infarction (AMI), was chosen as the model target. The microcapillary was first modified with polydopamine (PDA), then Myo aptamer was immobilized on the PDA modified microcapillary and hybridized with glucose oxidase (Gox) functionalized DNA probe (DNA-Gox). The step "one suction" referred to the inhalation of the sample into the functionalized microcapillary. Then the target Myo in the sample could bind to the Myo aptamer on the microcapillary so that DNA-Gox complexes were released from the microcapillary into solution. Through the step "one extrusion", the DNA-Gox complexes in the solution could catalyze glucose to generate hydrogen peroxide, and then the etching of gold nanorods (AuNRs) was initiated, causing a color change from brown to yellow. According to the color change based on the etching of AuNRs, as low as 0.1 nM Myo was detected with naked eyes. Combined with the manual centrifugal platform, even the Myo in the serum samples could be detected without power supply. It was expected to build a universal and adaptable sensing platform for different targets more quickly and efficiently.
Assuntos
Técnicas Biossensoriais , Nanotubos , Glucose Oxidase , Ouro , Testes ImediatosRESUMO
Nanozymes have drawn increasing attention with their broad applications but most nanozymes lack enzyme-like molecular structures, resulting in weak selectivity and low activity. Bioinspired molecular assembly provides an extremely promising strategy to mimic natural enzyme processes and develop function enhanced architectures. Herein, a new bioinspired molecular assembly strategy based on human serum albumin@polydopamine/Fe nanocomposites (HSA@PDA/Fe NCs) was proposed, in which Fe(iii)/Fe(ii) were anchored on HSA supported on PDA. HSA@PDA/Fe NCs with iron as the active center and HSA@PDA as the skeleton showed excellent peroxidase-like activity, which was nearly 1000 times higher than that of free Fe(iii). This may be attributed to the phenomenon that the cycle of quinones and the hydroxyl group on the nanocomposite surface greatly accelerate the conversion of Fe(iii)/Fe(ii) in acidic microenvironments. Systematic experimental studies illustrated that its activity was mainly affected by the metal active center, followed by the polymeric ligand, while the protein framework has little effect on its activity. Meanwhile, even after freeze-thaw and thermal cycle tests, it also showed excellent catalytic stability. Besides, a colorimetric assay based on HSA@PDA/Fe NCs was developed for detection of H2O2in vitro and in situ detection of H2O2 generated from live cells. This work will facilitate the developments on theoretical analysis, rational design and practical applications of nanozymes based on bioinspired molecular assemblies.
Assuntos
Peróxido de Hidrogênio , Nanocompostos , Catálise , Compostos Férricos , Humanos , FerroRESUMO
A photothermal and fluorescent dual-mode assay for sensitive organophosphate pesticides (Ops) determination is reported based on alkaline phosphatase (ALP)-inhibition-induced formation of polydopamine (PDA) nanoparticles. In the presence of ALP, ascorbic acid 2-phosphate (AAP) can be catalyzed to produce ascorbic acid (AA). AA can reduce MnO2 nanosheets, further inhibiting the oxidation of dopamine (DA). Ops as an inhibitor for ALP activity prevents the formation of AA and the reduction of MnO2 nanosheets. Eventually, the formation of PDA nanoparticles is promoted. The inhibitory effect of Ops on ALP activity causes obvious changes of photothermal signals and fluorescence signal at 495 nm. The detection limit (LOD) of dimethoate is 0.1 µM. The method displays excellent sensing capability for the dimethoate assay in real water with good recoveries of 99.4-107.6%. Graphical abstract A photothermal and fluorescent dual-mode biosensor for sensitive Ops detection was reported based on alkaline phosphatase (ALP)-inhibition-induced formation of polydopamine (PDA) nanoparticles. The dual-mode method significantly improved the accuracy and reliability of the results.
Assuntos
Indóis/química , Nanopartículas/química , Compostos Organofosforados/análise , Praguicidas/análise , Processos Fotoquímicos , Polímeros/química , Espectrometria de Fluorescência/métodos , Limite de Detecção , Compostos de Manganês/química , Óxidos/química , Reprodutibilidade dos Testes , Poluentes Químicos da Água/análiseRESUMO
Ischaemia-reperfusion injury (IRI) is closely related to cardiovascular disease (CVD), which is the leading cause of death and disability. Exosomes appear to be involved in several diseases, including CVD. However, the role of mesenchymal stem cell (MSC)-derived exosomes in IRI remains unclear. miRNA expression levels in exosomes from rat normal MSCs or hypoxia-reoxygenation (H/R) MSCs were determined by qPCR and the two significantly upregulated miRNAs were selected for further investigation. Rat cardiomyoblasts (H9c2) were transfected with either miRNA mimics or scramble controls, followed by H/R induction. The effects of miRNA overexpression and exosome administration on H/R damage were then investigated. H/R increased Faslg, suppressed ß-catenin, inhibited cell proliferation and migration, and stimulated apoptosis and reactive oxygen species (ROS) production. miR-149 or Let-7c mimics or exosomes reversed H/R-induced damage. The luciferase reporter assay proved the targeted regulation of Faslg by both miR149 and Let-7c. Inhibition of ß-catenin suppressed cell migration, proliferation, and ΔΨm but increased apoptosis and ROS. Overall, bone marrow MSC-derived exosomes protected rat cardiomyoblasts from H/R injury via the miR-149/Let-7c/Faslg axis.
Assuntos
Exossomos/metabolismo , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Movimento Celular , Proliferação de Células , Humanos , Masculino , Ratos , TransfecçãoRESUMO
Given that a split aptamer provides a chance for the development of a sandwich assay for targets with only one aptamer, it has received extensive attention in biosensing. However, due to the lack of binding mechanisms and reliable methods, there were still a few split aptamers that bind to proteins. In this work, cardiac biomarker myoglobin (Myo) was selected as a model, a new strategy of engineering split aptamers was explored with atomic force spectroscopy (AFM), and split aptamers against target protein could be achieved by choosing the optimal binding probability between split aptamers and target. Then, the obtained split aptamers were designed for Myo detection based on dynamic light scattering (DLS). The results demonstrated that the obtained split aptamers could be used to detect targets in human serum. The strategy of engineering split aptamers has the advantages of being intuitive and reliable and could be a general strategy for obtaining split aptamers.
Assuntos
Aptâmeros de Nucleotídeos/genética , Aptâmeros de Nucleotídeos/metabolismo , Engenharia Genética , Mioglobina/metabolismo , Humanos , Microscopia de Força Atômica , Ligação ProteicaRESUMO
Conventional imaging ellipsometry-based biosensing faces the challenges of poor sensitivity and narrow dynamic range, especially for some small molecules such as microRNA. Given that detection of various exosomal miRNAs with tunable range could provide high-precision disease information and improve the accuracy of diagnosis, a sensitive imaging ellipsometry sensor was introduced to improve sensitivity with a tunable detection range by terminus-regulated DNA hydrogelation. Tetrahedron DNA probes with complementary sequence to the target miRNA were used as biorecognition elements to form DNA hydrogelation. This DNA hydrogelation was formed by template-independent and isothermal amplification on the Au film. Due to its high dielectric constant, DNA hydrogelation structure could be used for improving the sensitivity of imaging ellipsometry significantly. Importantly, by changing the cycle of the DNA hydrogelation amplification, this strategy showed a tunable detection range from fM to nM for miRNA with a limit of detection of 0.2 fM for let-7a, 10 fM for miR-375, and 40 pM for miR-21. Furthermore, it also performed satisfactorily for the miRNA sensing in 50% human serum and 50% human plasma. This DNA hydrogelation-enhanced imaging ellipsometry could broaden the applications of conventional imaging ellipsometry in biosensing and provide a sensitive method for sensing miRNAs at different abundances.
Assuntos
Técnicas Biossensoriais/métodos , DNA/química , Nanopartículas Metálicas/química , MicroRNAs/genética , HumanosRESUMO
A novel surface plasmon resonance (SPR) strategy is introduced for the specific determination of exosomes based on aptamer recognition and polydopamine-functionalized gold nanoparticle (Au@PDA NP)-assisted signal amplification. Exosomes derived from hepatic carcinoma SMMC-7721 were selected as the model target. SMMC-7721 exosomes can be specifically captured by the aptamer ZY-sls that was complementary to the DNA tetrahedron probes (DTPs), and then the CD63 aptamer-linked Au@PDA NPs recognized SMMC-7721 exosomes for signal amplification. The DTPs were modified on a Au film for preventing Au deposition on the surface during the introduction of HAuCl4, and PDA coated on the AuNPs was used to reduce HAuCl4 in situ without any reductant assistance. It results in a further enhanced SPR signal. The assay can clearly distinguish SMMC-7721 exosomes from others (HepG2 exosomes, Bel-7404 exosomes, L02 exosomes, MCF-7 exosomes, and SW480 exosomes, respectively). SMMC-7721 exosomes are specifically determined as low as 5.6 × 105 particles mL-1. The method has successfully achieved specific determination of SMMC-7721 exosomes even in 50% of human serum without any pretreatment. Graphical abstract A novel surface plasmon resonance (SPR) strategy was introduced for the specific determination of exosomes based on aptamer recognition and polydopamine functionalized gold nanoparticles (Au@PDA NPs). The SPR signal was improved using the Au@PDA NPs assisted amplification.
Assuntos
Aptâmeros de Nucleotídeos/química , Exossomos/química , Indóis/química , Nanopartículas Metálicas/química , Polímeros/química , Linhagem Celular Tumoral , Ouro/química , Humanos , Ressonância de Plasmônio de SuperfícieRESUMO
Prostate cancer (PCa) is the most prevalent cancer among men and the second leading cause of tumor-associated deaths worldwide, with increasing incidence rates over the last 10 years. Recently, miR-195 was reported to be hypermethylated at its promoter CpG island and down-regulated in hepatocellular carcinoma. However, the function of miR-195 and the underlying mechanisms in PCa remain unknown. Here, we report that a significant down-regulation of microRNA-195 (miR-195) in PCa tissues and cell lines was associated with promoter methylation status. Overexpression of miR-195 significantly suppressed cell proliferation, migration, invasion and epithelial-mesenchymal transition (increased E-cadherin and decreased N-cadherin) in PCa cells. We further demonstrated that transfection with a miR-195 inhibitor reversed the inhibitory effect of the DNA methyltransferase inhibitor 5-azacytidine on the proliferation, migration and invasion ability of PCa cells. In summary, our findings suggest that miR-195 may function as a crucial tumor suppressor in PCa.
Assuntos
Movimento Celular/genética , Proliferação de Células/genética , Desmetilação/efeitos dos fármacos , MicroRNAs/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Transdução de Sinais/genética , Apoptose/efeitos dos fármacos , Apoptose/genética , Azacitidina/farmacologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ilhas de CpG , Metilação de DNA/efeitos dos fármacos , Regulação para Baixo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Genes Supressores de Tumor , Humanos , Masculino , MicroRNAs/genética , Invasividade Neoplásica/genética , Células PC-3 , Regiões Promotoras Genéticas , Transdução de Sinais/efeitos dos fármacos , TransfecçãoRESUMO
Developing a strategy of modulating ß-amyloid (Aß) aggregation with low cost, easy synthesis, high efficiency, and biosafety is significant and a challenge for Alzheimer's disease (AD) therapy. Herein, DNA aptamer (Aß-Apt) against Aß42 obtained by in vitro selection was developed as a potent inhibitor of Aß42 aggregation for the first time. Indeed, the Aß42 monomer fibrillation was inhibited completely by Aß-Apt. Notably, the inhibition effect of Aß-Apt on the Aß42 oligomer aggregation was more obvious than that on the Aß42 monomer aggregation. It was presumed that the distinguishing effect may be attributed to different binding behaviors of Aß-Apt with Aß42 monomer and Aß42 oligomer. Surface plasmon resonance analysis demonstrated that Aß-Apt specifically recognized Aß42 monomer and Aß42 oligomer. Furthermore, the binding affinity of Aß-Apt with Aß42 oligomer was larger than that of Aß-Apt with Aß42 monomer. This work provided a promising platform with high efficiency for manipulating Aß aggregation.
RESUMO
Understanding the binding of split aptamer/its target could become a breakthrough in the application of split aptamer. Herein, vascular endothelial growth factor (VEGF), a major biomarker of human diseases, was used as a model, and its interaction with split aptamer was explored with single molecule force spectroscopy (SMFS). SMFS demonstrated that the interaction force of split aptamer/VEGF165 was 169.44 ± 6.59 pN at the loading rate of 35.2 nN/s, and the binding probability of split aptamer/VEGF165 was dependent on the concentration of VEGF165 . On the basis of dynamic force spectroscopy results, one activation barrier in the dissociation process of split aptamer/VEGF165 complexes was revealed, which was similar to that of the intact aptamer/VEGF165 . Besides, the dissociation rate constant (koff ) of split aptamer/VEGF165 was close to that of intact aptamer/VEGF165 , and the interaction force of split aptamer/VEGF165 was higher than the force of intact aptamer/VEGF165 . It indicated that split aptamer also possessed high affinity with VEGF165 . The work can provide a new method for exploring the interaction of split aptamer/its targets at single-molecule level.
Assuntos
Microscopia de Força Atômica/métodos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Aptâmeros de Nucleotídeos/genética , Aptâmeros de Nucleotídeos/metabolismo , Humanos , Imagem Individual de MoléculaRESUMO
Exosomes have been demonstrated to be effective in the treatment of a variety of cardiac disorders. However, the effects of mesenchymal stem cell (MSC) exosomes on myocardial infarction is yet to be determined. The current study aimed to investigate the potential therapeutic effects of MSC exosomes on myocardial injuries that are caused by myocardial infarction. MSCs were isolated from rat bone marrow and were used for exosome enrichment using culture medium. Confirmation that MSCs and exosomes had been successfully extracted was performed using flow cytometry, electron microscopy and western blot analysis. A rat myocardial ischemia reperfusion (I/R) model was established by ligation of the left anterior descending coronary artery. Rat myocardial injuries were determined using 2,3,5-triphenyltetrazolium chloride, Masson and TUNEL staining. H9c2 cell proliferation, apoptosis and migration were analyzed using 5-ethynyl-2'-deoxyuridine, Hoechst staining, flow cytometry and Transwell assays. Marker gene expression was evaluated using reverse transcription-quantitative PCR, western blot analysis and immunofluorescence. Rat MSC exosomes were revealed to suppress myocardial injury and the myocardiocyte functions that were induced by I/R. The results also demonstrated decreased apoptotic protease activating factor-1 and increased autophagy-related protein 13 expression. The H9c2 cell proliferation and migration inhibition, as well as cell apoptosis during hypoxia-reoxygenation (H/R), were suppressed by rat MSC exosomes, with an alteration of the expression of apoptotic and autophagic genes also being demonstrated. The application of autophagy inhibitor 3-methyladenine significantly mitigated the effect of exosomes on H9c2 cell proliferation and apoptosis, which were induced by H/R. Rat MSC exosomes inhibited myocardial infarction pathogenesis, possibly by regulating autophagy.
RESUMO
The real application of DNA-functionalized gold nanoparticles (DNA-Au NPs) was limited by decreased stability and irreversible aggregation in high-ionic strength solutions and complex systems. Therefore, exploring a kind of DNA-Au NPs with excellent stability in high-ionic strength solutions and complex systems is challenging and significant. Herein, a novel universal bioconjugate strategy for constructing ultrastable DNA-Au NPs was designed based on the combination of polydopamine (PDA) shell and DNA linker. The obtained DNA-linked Au@polydopamine nanoparticles (DNA-Au@PDA NPs) showed colloidal stability in high-ionic strength solution and complex systems (such as human serum and cell culture supernatant). Moreover, the nanoparticles still maintained good dispersion after multiple freeze-thaw cycles. The high stability of DNA-Au@PDA NPs may be attributed to increasing the electrostatic and steric repulsions among nanoparticles through the effect of both PDA shell and DNA linker on Au@PDA NPs. For investigating the application of such nanoparticles, a highly sensitive assay for miRNA 141 detection was developed using DNA-Au@PDA NPs coupled with dynamic light scattering (DLS). Comparing with the regular DNA-Au NPs, DNA-Au@PDA NPs could detect as low as 50 pM miRNA 141 even in human whole serum. Taken together, the features of Bio/Nanointerface make the nanoparticle suitable for various applications in harsh biological and environmental conditions due to the stability. This work may provide a universal modification method for obtaining stable nanoparticles.
Assuntos
DNA/química , Ouro/química , Nanopartículas Metálicas/química , MicroRNAs/análise , Humanos , Indóis/química , Teste de Materiais , Polímeros/químicaRESUMO
Long noncoding RNA (lncRNA) maternally expressed gene 3 (MEG3) plays an important role in protection of ischemia-reperfusion (I/R) injury in brain and liver. However, role of MEG3 in myocardial I/R injury remains unclear. Here, the role of MEG3 in protection of myocardial I/R injury and its association with microRNA-7-5p (miR-7-5p) was investigated using rat cardiac I/R model and myocardial I/R cell model. Our results showed that MEG3 was significantly up-regulated and miR-7-5p was significantly down-regulated after I/R. Following I/R, the levels of intact PARP and intact caspase-3 were reduced, while the cleaved fragments of PARP and caspase-3 were increased. TUNEL assay showed an increase in cardiomyocyte apoptosis after I/R. The levels of I/R-induced creatine kinase (CK) and lactate dehydrogenase (LDH) were inhibited by knockdown of MEG3 (siMEG3). SiMEG3 increased cell proliferation and inhibited cell apoptosis after I/R. In contrast, overexpression of MEG3 increased the I/R-induced CK and LDH activities and cell apoptosis and decreased cell proliferation. The dual-luciferase reporter system showed a direct binding of MEG3 to miR-7-5p. The level of miR-7-5p was negatively associated with the change in levels of MEG3 in H9c2 cells. The levels of intact RARP1 and caspase-3 were significantly increased by knockdown of MEG3. Co-transfection of miR-7-5p inhibitor with siMEG3 activates CK and LDH, significantly decreased cell proliferation, increased cell apoptosis, and decreased intact poly(ADP-ribose) polymerase 1 (PARP1) and caspase-3. In summary, down-regulation of MEG3 protects myocardial cells against I/R-induced apoptosis through miR-7-5p/PARP1 pathway, which might provide a new therapeutic target for treatment of myocardial I/R injury.
Assuntos
Regulação para Baixo , MicroRNAs/biossíntese , Traumatismo por Reperfusão Miocárdica/metabolismo , RNA Longo não Codificante/metabolismo , Animais , Linhagem Celular , Masculino , MicroRNAs/genética , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/patologia , RNA Longo não Codificante/genética , Ratos , Ratos Sprague-DawleyRESUMO
Alkaline phosphatase (ALP) is a significant biomarker in clinical diagnostics, and the abnormal level of ALP enzyme in serum is closely related to various diseases such as bone or liver cancer, bone metastases, and extrahepatic biliary obstruction. Herein a simple and portable photothermal biosensor was developed for sensitive detection of ALP enzyme based on the formation of polydopamine (PDA) nanoparticles using a thermometer or temperature discoloration sticker as readout. A MnO2 nanosheet was first prepared using a novel one-pot strategy which was operationally simple and not overly time-consuming. Then dopamine (DA) was quickly polymerized into PDA nanoparticles in the presence of the MnO2 nanosheet. When the model analyte ALP was present, the substrate 2-phospho-l-ascorbic acid trisodium salt (AAP) was catalytically hydrolyzed into l-ascorbic acid (AA), resulting in the inhibition of the formation of the PDA nanoparticles owing to the fact that the MnO2 nanosheet was reduced to Mn2+ by the generated AA. Thus, a portable biosensor based on the photothermal properties of PDA nanoparticles for ALP enzyme detection was established with a detection limit as low as 0.1 U/L (thermometer) and 1 U/L (temperature discoloration sticker). In addition, it also showed excellent sensing performance for the ALP assay in human serum. Such a simple, label-free, cost-effective, and sensitive assay could exhibit real potential application for ALP detection and early diagnosis, especially in developing countries or remote regions.
Assuntos
Fosfatase Alcalina/metabolismo , Ensaios Enzimáticos/instrumentação , Sistemas Automatizados de Assistência Junto ao Leito , Temperatura , Termômetros , Animais , Bovinos , Cor , Indóis/química , Compostos de Manganês/química , Nanopartículas/química , Óxidos/química , Polímeros/químicaRESUMO
Sensitive detection of cancerous exosomes is critical to early diseases diagnosis and prognosis. Herein, a sensitive aptasensor was demonstrated for exosomes detection by surface plasmon resonance (SPR) with dual gold nanoparticle (AuNP)-assisted signal amplification. Dual nanoparticle amplification was achieved by controlled hybridization attachment of AuNPs resulting from electronic coupling between the Au film and AuNPs, as well as coupling effects in plasmonic nanostructures. By blocking the Au film surface with 11-Mercapto-1 -undecanol (MCU), nonspecific adsorption of AuNPs onto the SPR chip surface was suppressed and regeneration of the SPR sensor was realized. This method was highly sensitive and we have achieved the limit of detection (LOD) down to 5â¯×â¯103 exosomes/mL, which showed a 104-fold improvement in LOD compared to commercial ELISA. Moreover, the SPR sensor had the capability to differentiate the exosomes secreted by MCF-7 breast cancer cells and MCF-10A normal breast cells. Furthermore, the SPR sensor could effectively detect the exosomes in 30% fetal bovine serum. The work provides a sensitive and efficient quantification approach to detect cancerous exosomes and offers an avenue toward future diagnosis and comprehensive studies of exosomes.
Assuntos
Exossomos/química , Ressonância de Plasmônio de Superfície/métodos , Aptâmeros de Nucleotídeos/química , Ouro/química , Humanos , Células MCF-7 , Nanopartículas Metálicas/química , Nanopartículas Metálicas/ultraestruturaRESUMO
OBJECTIVES: Although androgen deprivation therapy remains the standard treatment for the initial therapy of advanced prostate cancer (PC), castration does not eliminate persistent intratumoral androgens within the prostate tumor microenvironment, which is capable of activating androgen receptor. Abiraterone effectively target adrenal and tumor androgen production in castration-resistant PC (CRPC). However, abiraterone-resistant CRPC is now common challenge in clinic via multiple mechanisms. METHODS: In this study, human CRPC cell line PC3 and androgen-sensitive cells LNCaP were used. The authors investigated the role of autophagy during the therapy of abiraterone in CRPC by analysis of transmission electron microscopy (TEM), Western blot and immunofluorescence assay. Cell cycle and apoptosis using flow cytometry analysis. RESULTS: The analysis of TEM showed more autophagic vesicles (AVs) in PC3 cell line than that in LNCaP cell line and indicated the high basic cellular autophagy in CRPC cell line PC3, which was confirmed by the upregulation of autophagy-related protein LC3, Atg5, and Beclin1. Interestingly, the treatment of abiraterone reduced the level of autophagic vesicles in two cell lines and inhibited the expressions of autophagic markers LC3, Atg5 and Beclin1 in parallel with decreased cell vitality and induced G2/M arrest in PC3 cell line and LNCaP cell line. Moreover, the addition of the autophagy inhibitor 3-methyladenine to the treatment of abiraterone inhibited the formation of AVs with downregulated autophagic markers, and inhibition of autophagy promoted the efficiency of cytotoxicity of abiraterone with further impaired cell vitality and G2/M arrest. CONCLUSION: These data suggested that inhibition of autophagy by its inhibitor benefits the treatment of abiraterone for CRPC patients.