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BACKGROUND: Status epilepticus (SE) is a life-threatening neurological disorder. The hippocampus, as an important area of the brain that regulates cognitive function, is usually damaged after SE, and cognitive deficits often result from hippocampal neurons lost after SE. Fyn, a non-receptor Src family of tyrosine kinases, is potentially associated with the onset of seizure. Saracatinib, a Fyn inhibitor, suppresses epileptogenesis and reduces epileptiform spikes. However, whether saracatinib inhibits cognitive deficits after SE is still unknown. METHODS: In the present study, a pilocarpine-induced SE mouse model was used to answer this question by using the Morris water maze and normal object recognition behavioral tests. RESULTS: We found that saracatinib inhibited the loss in cognitive function following SE. Furthermore, we found that the number of hippocampal neurons in the saracatinib treatment group was increased, when compared to the SE group. CONCLUSIONS: These results showed that saracatinib can improve cognitive functions by reducing the loss of hippocampal neurons after SE, suggesting that Fyn dysfunction is involved in cognitive deficits after SE, and that the inhibition of Fyn is a possible treatment to improve cognitive function in SE patients.
Assuntos
Benzodioxóis/farmacologia , Cognição/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-fyn/antagonistas & inibidores , Quinazolinas/farmacologia , Estado Epiléptico , Animais , Disfunção Cognitiva/etiologia , Inibidores Enzimáticos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Estado Epiléptico/complicações , Estado Epiléptico/fisiopatologiaRESUMO
Exosomes have been considered as promising biomarkers for cancer diagnosis due to their abundant information from originating cells. However, sensitive and reliable detection of exosomes is still facing technically challenges due to the lack of a sensing platform with high sensitivity and reproducibility. To address the challenges, here we propose a portable surface plasmon resonance (SPR) sensing of exosomes with a three-layer Au mirror/SiO2 spacer/Au nanohole sensor, fabricated by an economical polystyrene nanosphere self-assembly method. The SiO2 spacer can act as an optical cavity and induce mode hybridization, leading to excellent optimization of both sensitivity and full width at half maximum compared with normal single layer Au nanohole sensors. When modified with CD63 or EpCAM aptamers, a detection of limit (LOD) of as low as 600 particles/µL was achieved. The sensors showed good capability to distinguish between non-tumor derived L02 exosomes and tumor derived HepG2 exosomes. Additionally, high reproducibility was also achieved in detection of artificial serum samples with RSD as low as 2%, making it feasible for clinical applications. This mode hybridization plasmonic sensor provides an effective approach to optimize the detection sensitivity of exosomes, pushing SPR sensing one step further towards cancer diagnosis.
Assuntos
Exossomos , Ouro , Limite de Detecção , Dióxido de Silício , Ressonância de Plasmônio de Superfície , Exossomos/química , Humanos , Ouro/química , Dióxido de Silício/química , Aptâmeros de Nucleotídeos/química , Molécula de Adesão da Célula Epitelial , Tetraspanina 30 , Células Hep G2 , Técnicas Biossensoriais/métodos , Técnicas Biossensoriais/instrumentação , Reprodutibilidade dos Testes , Desenho de Equipamento , Nanosferas/química , Hibridização de Ácido Nucleico , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/análiseRESUMO
BACKGROUND: The significance of p28GANK in gliomas remains unknown. This study aims to clarify the clinical significance of p28GANK in human gliomas. METHODS: The expression of p28GANK in 138 gliomas and 50 matched para-cancerous tissues was detected by immunohistochemical staining, and statistical analyses were performed to test the correlation of p28GANK with clinical parameters. To investigate the effects of p28GANK down-regulation on the growth of cells both in vitro and in vivo, an siRNA targeting p28GANK was transfected into U251 cells. RESULTS: P28GANK expression was significantly higher in tumor specimens than in matched para-cancerous tissues. Over-expressed p28GANK significantly correlated with high karnofsky performance score (KPS), advanced WHO grade and poor overall survival of the patients. Univariate analysis showed that WHO grade and KPS also correlated with the survival of patients, and multivariate analysis suggested that KPS and p28GANK expression were two independent prognostic factors. Moreover, p28GANK gene silencing decreased the malignant growth of U251 cells both in vitro and in vivo. CONCLUSIONS: Increased expression of p28GANK is correlated with poor clinical outcomes in glioma patients. The down-regulation of p28GANK significantly inhibited cell proliferation, indicating that p28GANK might be a potential therapeutic target for glioma treatment.
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Biomarcadores Tumorais/metabolismo , Neoplasias Encefálicas/mortalidade , Proliferação de Células , Glioma/mortalidade , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Adolescente , Adulto , Idoso , Animais , Western Blotting , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Estudos de Casos e Controles , Criança , Feminino , Glioma/metabolismo , Glioma/patologia , Humanos , Técnicas Imunoenzimáticas , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Gradação de Tumores , Prognóstico , Complexo de Endopeptidases do Proteassoma/genética , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , RNA Interferente Pequeno/genética , Taxa de Sobrevida , Células Tumorais Cultivadas , Regulação para Cima , Adulto JovemRESUMO
Purpose: Valproate (VPA) resistance was reported to be an important predictor of intractable epilepsy. We conducted this study to identify candidate biomarkers in peripheral blood correlated with VPA resistance. Methods: The microarray dataset (GSE143272) was downloaded from the Gene Expression Omnibus database. Weighted gene co-expression network analysis (WGCNA) was performed to construct co-expression modules and obtain the most prominent module associated with VPA resistance. Differentially expressed genes (DEGs) between VPA-responsive and VPA-resistant patients were obtained using the "Limma" package in R. The intersections between the most prominent module and DEGs were identified as target genes. Metascape was performed to discover the possible involved pathways of the target genes. GeneCards database was used to know the function of each target gene. Results: All genes in the GSE143272 were divided into 24 different modules. Among these modules, the darkred module showed a pivotal correlation with VPA resistance. A total of 70 DEGs between VPA-responsive and VPA-resistant patients were identified. After taking the intersection, 25 target genes were obtained. The 25 target genes were significantly enriched in T cell receptor recognition, T cell receptor signaling pathway, regulation of T cell activation, cytokine-cytokine receptor interaction, and in utero embryonic development. Half of the target genes (CD3D, CD3G, CXCR3, CXCR6, GATA3, GZMK, IL7R, LIME1, SIRPG, THEMIS, TRAT1, and ZNF683) were directly involved in the T cell development, migration, and activation signaling pathway. Conclusion: We identified 25 target genes prominently associated with VPA resistance, which could be potential candidate biomarkers for epilepsy resistance in peripheral blood. The peripheral blood T cells may play a crucial role in VPA resistance. Those genes and pathways might become therapeutic targets with clinical usefulness in the future.
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BACKGROUND: It is reported that the osteogenesis in bone marrow mesenchymal stem cells (BMSCs) can alleviate osteoporosis progression. It has been found that Kae can promote the osteogenesis in BMSCs. However, the mechanism by which Kae mediates the osteogenesis in BMSCs is largely unknown. METHODS: RBMSCs were collected from rats. The cytotoxicity of Kae was detected by CCK-8 assay. The osteogenic calcification in rBMSCs was measured by alizarin red staining, and ALP staining was performed to test the ALP activity in rBMSCs. The binding relationship between SOX2 and miR-124-3p was explored by dual luciferase report assay and Chromatin Immunoprecipitation (ChIP). RT-qPCR and western blot were performed to assess mRNA and protein levels, respectively. RESULTS: Kae (10 µM) significantly increased the calcification, ALP activity, SOX2 level, activated PI3K/Akt/mTOR signaling and inhibited miR-124-3p level in rBMSCs, while knockdown of SOX2 reversed this phenomenon. Meanwhile, SOX2 suppressed the transcription of miR-124-3p, and SOX2 promoted the osteogenic differentiation in rBMSCs via regulation of miR-124-3p. MiR-124-3p could inactivate PI3K/Akt/mTOR to inhibit the osteogenic differentiation. CONCLUSION: Kae significantly promoted the osteogenesis in rBMSCs via mediation of SOX2/miR-124-3p/PI3K/Akt/mTOR axis. Thus, our study might shed new lights in exploring new methods against osteoporosis.
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MicroRNAs , Osteoporose , Animais , Diferenciação Celular/genética , Células Cultivadas , Quempferóis , MicroRNAs/genética , MicroRNAs/metabolismo , Osteogênese/genética , Osteoporose/metabolismo , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Ratos , Serina-Treonina Quinases TORRESUMO
This study aimed to investigate the effect of Fyn gene silencing on the apoptosis of oligodendrocytes (OLs) in epileptic model in vitro and the involved mechanism. Primary oligodendrocyte pro-genitor cells (OPCs) were separated from rats and differentiated to OLs. Immunofluorescent labeling showed positive expression of A2B5 in OPCs and Olig2 in OLs, suggesting the successful separation of OPCs and OLs. Three Fyn siRNAs (si-Fyn) and Fyn siRNA negative control (NC) were transfected into OLs. Western blot showed that among three si-Fyn groups, si-Fyn3 caused the lowest Fyn expression, so si-Fyn3 was chosen for following experiment. Cells were divided into four groups: Control, Model, NC and si-Fyn. In the Model group, cells were cultured in Mg-free extracellular fluid for 3 h. The morphology of control cells was normal. However, the migration of neurons, the aggregation of cell bodies and the "grid-like" changes of neural networks were observed in the model cells. OLs apoptosis in various groups was assessed by flow cytometry. Expression of Fyn, ERK1/2 and phosphorylated ERK1/2 (p-ERK1/2) in OLs of various groups was evaluated by western blot. Compared with the Control group, the apoptotic rates, the Fyn expression and p-ERK1/2/ERK1/2 ratio in the Model and NC groups increased significantly (p < .05). However, the apoptotic rate, the Fyn expression and p-ERK1/2/ERK1/2 ratio in the si-Fyn group were remarkably smaller than those in the Model group (p < .05). In conclusion, Fyn gene silencing reduced the apoptosis of OLs through inhibiting the phosphorylation of ERK1/2 in epileptic model.
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Apoptose/genética , Epilepsia/genética , Inativação Gênica , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Oligodendroglia/patologia , Proteínas Proto-Oncogênicas c-fyn/genética , Animais , Epilepsia/patologia , Regulação Enzimológica da Expressão Gênica/genética , Fosforilação/genética , Proteínas Proto-Oncogênicas c-fyn/deficiência , Ratos , Ratos Sprague-DawleyRESUMO
OBJECTIVE: Spinal muscular atrophy (SMA) is an autosomal recessive neurodegenerative disease. It is characterized by selective loss of spinal cord motor neurons leading to muscle atrophy and is the result of mutation or deletion of the survival motor neuron (SMN) gene. Currently, there are no effective therapies for this disease. Stem cell therapy is a new prospect for SMA patients. This study aimed to investigate whether mesenchymal stem cells (MSCs) can be differentiated into neuron-like cells (NLCs) in SMA patients in order to provide a basis for stem cell therapy for SMA. METHODS: SMA was definitively diagnosed using polymerase chain reaction-restriction fragment length polymerphhism (PCR-RFLP). Two children without SMN1 gene deletion were used as controls. MSCs were isolated and purified from SMA patients and controls, and induced into NLCs by bFGF and baicalin. The NLCs were identified by immunofluourescence staining with NSE and NF monoclonal antibodies. RESULTS: SMA patients showed the deletion of SMN1 exon 7. The morphous and proliferative speed of MSCs between SMA patients and controls were similar. After 6-day induction, MSCs of the two groups displayed similar morphology to that of neurons, with long processes forming extensive networks. NSE and NF, the neuronal markers, were detected in the differentiated NLCs of the two groups. CONCLUSIONS: SMN1 deletion appears not to affect the proliferation and differentiation of MSCs. MSCs of SMA patients can be differentiated into NLCs.
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Células-Tronco Mesenquimais/citologia , Atrofia Muscular Espinal/patologia , Neurônios/citologia , Adolescente , Diferenciação Celular , Proliferação de Células , Criança , Feminino , Humanos , MasculinoRESUMO
OBJECTIVE: To detect the difference of SMN2 mRNA expression between the neuron-like cells (NLCs) derived from patients with spinal muscular atrophy (SMA). METHODS: Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed to diagnose the patients with SMA and the controls. Mesenchymal stem cells (MSCs) were induced into neuron-like cells which become the models of neurons. The transcripts of SMN2 were testified with RT-PCR combined with sequencing. RESULTS: Two bands (266 bp and 212 bp) were found in the gel picture of RT-PCR and the band of 266 bp was the full length transcript (fl-SMN mRNA). The band of 212 bp was testified by sequencing to be deletion of the exon 7, which was the production of alternative splicing (skipping exon 7, SMNDelta7 mRNA). The expression of fl-SMN mRNA accounted for 23.2% of the total SMN mRNA in the SMA patients, being much lower than the rate in the controls (82.0%). In contrast, 76.8% of SMN gene transcripts was SMNDelta7 mRNA in the SMA patients, being much higher than the rate of 18% in the controls. CONCLUSIONS: Alternative splicing exists in SMN2 gene transcription, that is, exon 7 is skipped during the processing of SMN2 mRNA in the NLCs of SMA patients, which is one of the reasons of the full-length SMN protein lacking.
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Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/biossíntese , Atrofia Muscular Espinal/diagnóstico , Proteínas do Tecido Nervoso/biossíntese , Proteínas de Ligação a RNA/biossíntese , Adolescente , Criança , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Feminino , Humanos , Masculino , Neurônios Motores/metabolismo , Atrofia Muscular Espinal/genética , Proteínas do Tecido Nervoso/genética , RNA Mensageiro/biossíntese , Proteínas de Ligação a RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas do Complexo SMN , Proteína 2 de Sobrevivência do Neurônio MotorRESUMO
OBJECTIVE: The objective was to explore the effects of valproate (VPA) on the carotid artery intima-media thickness (CA-IMT) in epileptics. MATERIALS AND METHODS: A total of 30 epileptic patients treated with VPA was included as disease group, while 33 healthy people who matched general basic demographic details were the control group. The IMTs of the left and right carotids of the both groups were measured, and the average CA-IMT was calculated. The IMT-related risk factors were acquired for the univariate and multivariate analysis. RESULTS: The bilateral carotid and average CA-IMTs of the disease group were significantly higher than the control group (P < 0.001). The multivariant gradual regressive analysis screened out two CA-IMT-related factors, namely the disease duration and the drug administration duration were positively correlated with the average CA-IMT. The epileptic patients with disease course of more than 3 years had much higher average CA-IMT than that of the epileptics with ≤3 years disease (P < 0.001). The average CA-IMT of the patients with VPA-administration duration >1 year was also higher than that of the patients with VPA-administration duration <1 year, while the difference was not statistically significant (P = 0.196). CONCLUSIONS: The average CA-IMT of the epileptic patients treated with VPA was higher than that of healthy people.
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Anticonvulsivantes/efeitos adversos , Doenças das Artérias Carótidas/induzido quimicamente , Artéria Carótida Primitiva/efeitos dos fármacos , Espessura Intima-Media Carotídea , Epilepsia/tratamento farmacológico , Ultrassonografia Doppler em Cores , Ácido Valproico/efeitos adversos , Adolescente , Adulto , Biomarcadores/sangue , Doenças das Artérias Carótidas/sangue , Doenças das Artérias Carótidas/diagnóstico por imagem , Artéria Carótida Primitiva/diagnóstico por imagem , Estudos de Casos e Controles , Epilepsia/diagnóstico , Feminino , Humanos , Lipídeos/sangue , Masculino , Análise Multivariada , Fatores de Risco , Fatores de Tempo , Adulto JovemRESUMO
In the present study, we examined the proportions of Epstein-Barr virus-associated gastric carcinomas (EBV-GCs) in Guangzhou, southern China and Shenyang, northern China, two areas differing markedly in nasopharyngeal carcinoma (NPC) incidence. Using in situ hybridization assay, the presence of EBV-encoded small RNA (EBER) was examined in 198, and 180 gastric cancer cases in Guangzhou and Shenyang, respectively. The proportion of EBV-GC in Guangzhou (9%) was significantly higher than that in Shenyang (6%), and the odds ratio (OR) for Guangzhou, after adjusting for the effects of age, sex, and tumor subsite, was 2.7 (95% CI = 1.1-6.2) when Shenyang was taken as reference. There was a male predominance of EBV-GC, and the OR for male was 3.0 (95% CI = 1.2-7.3) when female was taken as reference. We observed a weak and negative age dependence in the proportion of EBV-GC (p-values for trend = 0.077). The EBV-GC was most commonly observed in the middle part of stomach in both series. The frequency of EBV-GCs was higher in cases with p53 overexpression than in cases without p53 expression (OR = 2.4, 95% CI = 1.0-5.8). Among p53-positive cases, the frequency of EBV-GC decreased as the proportion of p53-positive carcinoma cells increased (p for trend = 0.021). In conclusion, the present study suggested that the frequency of EBV-GC in Guangzhou, southern China, where NPC is the most common in the world, may be higher than that in other parts of China.