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In this manuscript, a simple method combining atomic layer deposition and magnetron sputtering is developed to fabricate high-performance Pd/SnO2 film patterns applied for micro-electro-mechanical systems (MEMS) H2 sensing chips. SnO2 film is first accurately deposited in the central areas of MEMS micro hotplate arrays by a mask-assistant method, leading the patterns with wafer-level high consistency in thickness. The grain size and density of Pd nanoparticles modified on the surface of the SnO2 film are further regulated to obtain an optimized sensing performance. The resulting MEMS H2 sensing chips show a wide detection range from 0.5 to 500 ppm, high resolution, and good repeatability. Based on the experiments and density functional theory calculations, a sensing enhancement mechanism is also proposed: a certain amount of Pd nanoparticles modified on the SnO2 surface could bring stronger H2 adsorption followed by dissociation, diffusion, and reaction with surface adsorbed oxygen species. Obviously, the method provided here is quite simple and effective for the manufacturing of MEMS H2 sensing chips with high consistency and optimized performance, which may also find broad applications in other MEMS chip technologies.
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BACKGROUND: Ovarian carcinoma (OC) is one of the most common malignancies of the female reproductive organs, with a low survival rate primarily due to the lack of effective methods for early diagnosis and prognosis. OBJECTIVE: In this article, our motivation is to explore the lncRNA-related network mechanisms involved in the pathogenesis of OC. METHODS: Public lncRNAs and mRNA expression datasets for OC were collected from the Gene Expression Omnibus (GEO) database. By integrated bioinformatics analysis, we constructed a UCA1-miRNA-mRNA network. We studied lncRNA-related molecular modulation mechanism in ovarian cancer cells based on MTT assay, dual luciferase reporter gene assays, quantitative realtime PCR, and western blotting. RESULTS: UCA1 was higher in ovarian tumor tissues and cells than normal tissues and cells. It was demonstrated in this study that knockdown of UCA1 inhibited ovarian cancer cell viability, which a miR-99b-3p inhibitor could reverse in vitro. Further, UCA1 was shown to regulate the expression of SRPK1 by directly binding to miR-99b-3p. CONCLUSION: These results suggest that UCA1 functions as an oncogene in ovarian cancer. Inhibition of UCA1/miR-99b-3p/SRPK1 axis may become a novel target for treating ovarian cancer.
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MicroRNAs , Neoplasias Ovarianas , RNA Longo não Codificante , Feminino , Humanos , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Regulação Neoplásica da Expressão Gênica , Proliferação de Células , Linhagem Celular Tumoral , MicroRNAs/genética , MicroRNAs/metabolismo , Neoplasias Ovarianas/genética , RNA Mensageiro , Proteínas Serina-Treonina QuinasesRESUMO
Bioartificial liver (BAL) support system has been proposed as potential treatment method for end-stage liver diseases. We described an improved BAL system based on a choanoid fluidized bed bioreactor containing alginate-chitosan encapsulated primary porcine hepatocytes. The feasibility, safety, and efficiency of this device were estimated using an allogeneic fulminant hepatic failure (FHF) model. FHF was induced with intravenous administration of D-galactosamine. Thirty FHF pigs were divided into three groups: (1) an FHF group which was only given intensive care; (2) a sham BAL group which was treated with the BAL system with empty encapsulation, and (3) a BAL group which was treated with the BAL system containing encapsulated freshly isolated primary porcine hepatocytes. The survival times and biochemical parameters of these animals were measured, and properties of the encapsulations and hepatocytes before and after perfusion were also evaluated. Compared to the two control groups, the BAL-treated group had prolonged the survival time and decreased the blood lactate levels, blood glucose, and amino acids remained stable. No obvious ruptured beads or statistical decline in viability or function of encapsulated hepatocytes were observed. This new fluidized bed BAL system is safe and efficient. It may represent a feasible alternative in the treatment of liver failure.
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Reatores Biológicos , Falência Hepática Aguda/terapia , Fígado Artificial , Alginatos/química , Análise de Variância , Animais , Estudos de Viabilidade , Galactosamina/farmacologia , Ácido Glucurônico/química , Hepatócitos/citologia , Hepatócitos/metabolismo , Ácidos Hexurônicos/química , Estimativa de Kaplan-Meier , Falência Hepática Aguda/induzido quimicamente , Masculino , Suínos , Porco MiniaturaRESUMO
BACKGROUND: The bioartificial liver is anticipated to be a promising alternative choice for patients with liver failure. Toxic substances which accumulate in the patients' plasma exert deleterious effects on hepatocytes in the bioreactor, and potentially reduce the efficacy of bioartificial liver devices. This study was designed to investigate the effects of plasma from patients with acute on chronic liver failure (AoCLF) on immortalized human hepatocytes in terms of cytochrome P450 gene expression, drug metabolism activity and detoxification capability. METHODS: Immortalized human hepatocytes (HepLi-2 cells) were cultured in medium containing fetal calf serum or human plasma from three patients with AoCLF. The cytochrome P450 (CYP3A5, CYP2E1, CYP3A4) expression, drug metabolism activity and detoxification capability of HepLi-2 cells were assessed by RT-PCR, lidocaine clearance and ammonia elimination assay. RESULTS: After incubation in medium containing AoCLF plasma for 24 hours, the cytochrome P450 mRNA expression of HepLi-2 cells was not significantly decreased compared with control culture. Ammonia elimination and lidocaine clearance assay showed that the ability of ammonia removal and drug metabolism remained stable. CONCLUSIONS: Immortalized human hepatocytes can be exposed to AoCLF plasma for at least 24 hours with no significant reduction in the function of cytochrome P450. HepLi-2 cells appear to be effective in metabolism and detoxification and can be potentially used in the development of bioartificial liver.
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Proteínas Sanguíneas/toxicidade , Sistema Enzimático do Citocromo P-450/genética , Doença Hepática Terminal/sangue , Hepatócitos , Falência Hepática Aguda/sangue , Fígado Artificial , Amônia/metabolismo , Anestésicos Locais/farmacocinética , Linhagem Celular Transformada , Doença Hepática Terminal/terapia , Estudos de Viabilidade , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/fisiologia , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/fisiologia , Humanos , Técnicas In Vitro , Inativação Metabólica/fisiologia , Lidocaína/farmacocinética , Falência Hepática Aguda/terapiaRESUMO
BACKGROUND/AIMS: Microencapsulated hepatocytes have been proposed as promising bioactive agents for packed-bed or fluidized-bed bioartificial liver assist devices (BLaDs) and for hepatocyte transplantation because of the potential advantages they offer of high mass transport rate and an optimal microenvironment for hepatocyte culture. We developed a large-scale and high-production alginate-chitosan (AC) microcapsule roller bottle culture system for the encapsulation of hepLL immortalized human hepatocytes. In this study, the efficacy of upscaling encapsulated hepLL cells production with roller bottle cultivation was evaluated in vitro. METHODS: Microencapsulated hepLL cells were grown at high yield in large-scale roller bottles, with free cells cultured in roller bottle spinners serving as controls. The mechanical stability and the permeability of the AC microcapsules were investigated, and the growth, metabolism and functions of the encapsulated hepLL cells were evaluated as compared to free cells. RESULTS: The microcapsules withstood well the shear stress induced by high agitation rates. The microcapsules were permeable to albumin, but prevented the release of immunoglobulins. Culture in roller bottles of immortalized human hepatocytes immobilized in the AC microcapsules improved cell growth, albumin synthesis, ammonia elimination and lidocaine clearance as compared with free cells cultured in roller bottles. CONCLUSIONS: Encapsulated hepLL cells may be cultured on a large scale in roller bottles. This makes them possible candidates for use in cell-based liver assist therapies.
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Hepatócitos/citologia , Fígado Artificial , Alginatos , Cápsulas , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Quitosana , Ácido Glucurônico , Ácidos Hexurônicos , Humanos , Teste de Materiais , Membranas Artificiais , Estresse MecânicoRESUMO
Bioartificial livers may act as a promising therapy for fulminant hepatic failure (FHF) with better accessibility and less injury compared to orthotopic liver transplantation. This study aims to evaluate the efficacy and safety of a fluidized bed bioartificial liver (FBBAL) and to explore its therapeutic mechanisms based on metabolomics. FHF was induced by D-galactosamine. Eighteen hours later, pigs were treated with an FBBAL containing encapsulated primary porcine hepatocytes (B group), with a sham FBBAL (containing cell-free capsules, S group) or with only intensive care (C group) for 6 h. Serum samples were assayed using ultra-performance liquid chromatography-mass spectrometry. The difference in survival time (51.6 ± 7.9 h vs. 49.3 ± 6.6 h) and serum metabolome was negligible between the S and C groups, whereas FBBAL treatment significantly prolonged survival time (70.4 ± 11.5h, P < 0.01) and perturbed the serum metabolome, resulting in a marked decrease in phosphatidylcholines, lysophosphatidylcholines, sphingomyelinase, and fatty acids and an increase in conjugated bile acids. The FBBAL exhibits some liver functions and may exert its therapeutic effect by altering the serum metabolome of FHF pigs. Moreover, alginate-chitosan capsules have less influence on serum metabolites. Nevertheless, the alterations were not universally beneficial, revealing that much should be done to improve the FBBAL.
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Transplante de Células/métodos , Falência Hepática Aguda/terapia , Fígado Artificial , Metabolômica , Animais , Transplante de Células/efeitos adversos , Células Imobilizadas/metabolismo , Cromatografia Líquida , Modelos Animais de Doenças , Falência Hepática Aguda/induzido quimicamente , Espectrometria de Massas , Soro/química , Análise de Sobrevida , Suínos , Resultado do TratamentoRESUMO
PURPOSE: Bioartificial liver assist devices (BLADs) are expected to bridge liver failure patients to liver transplantation, but porcine endogenous retroviruses (PERVs) still pose a potential risk in pig-to-human xenotransplantation and thereby limit the use of bioartificial liver therapy. In our lab, fluidized-bed BLADs based on microencapsulated primary porcine hepatocytes have been successfully used to treat liver failure pigs. We detected the risk of PERVs transmission of microencapsulated primary porcine hepatocytes-the key component of fluidized-bed BLADs, to evaluate the biosafety of this device for further clinical applications. METHODS: Microencapsulated primary porcine hepatocytes (cell diameter = 300 µm) were cultured in Dulbecco's modified Eagles medium (DMEM). Microencapsulated cell culture supernatants were collected at 6, 12, 24 and 72 h. HEK-293 were cocultured with these supernatants, and the cocultured cells were harvested every 7 days. RT-PCR was used to detect PERVs transmission. RT-qPCR was used to get the number of virus copies. PK-15 was used as the positive control whereas HepG2 was used as the negative control. RESULTS: PERV was detected in all supernatants, and the viral load of the supernatants increased with time. Moreover, cocultured 293 cells were positive for PERV-specific sequences. CONCLUSION: The kind of fluidized-bed BLADs based on microencapsulated primary porcine hepatocytes have risk of PERVs transmission. Further extensive pre-clinical study focused on biosafety is warranted.
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Alphavirus replicons, in which structural protein genes are replaced by heterologous genes, express high levels of the heterologous proteins. On the basis of the potencies of replicons to self-replicate and express foreign proteins and the remarkable intercellular transport property of VP22, a novel alphavirus Semliki Forest virus (SFV) replicon system of VP22 fused with a model antigen, hemagglutinin (HA), of the human-avian H5N1 influenza virus, was explored in this study. Further, replicon particles expressing HA, VP22, and enhanced green fluorescent protein (EGFP) individually were used as controls. By flow cytometry based on the analysis of transfection efficiency, SFV-EGFP replicon particle titer was 1.13 x 10(7)transducing units (TU)/ml. The titers of SFV-HA, SFV-VP22 and SFV-VP22-HA replicon particles, which were titrated by using SFV-EGFP replicon particles, were 1.42 x 10(7), 3.23 x 10(7), and 1.01 x 10(7)TU/ml, respectively. HA and VP22-HA expression was observed in SFV-HA- and SFV-VP22-HA-transfected BHK-21 cells, respectively. Immunofluorescence staining revealed that the fluorescence intensity in the SFV-VP22-HA-transfected BHK-21 cells was more than that in the SFV-HA-transfected BHK-21 cells. Both SFV-VP22-HA and SFV-HA replicon particles presented a promising approach for developing vaccines against human-avian influenza. VP22-HA fusion protein with similar trafficking properties may also enhance vaccine potency.
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Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Virus da Influenza A Subtipo H5N1/fisiologia , Replicon/fisiologia , Vírus da Floresta de Semliki/fisiologia , Animais , Apoptose/fisiologia , Linhagem Celular , Cricetinae , Proteínas de Fluorescência Verde/metabolismo , Virus da Influenza A Subtipo H5N1/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Replicon/genética , Vírus da Floresta de Semliki/genética , TransfecçãoRESUMO
A modular novel bioartificial liver support system was designed and constructed in order to simplify tedious operation of artificial liver treatment and to improve the applicability in the system. The design ideas, structure composition, system function, and etc, were described in detail. In this system, the variety of the therapy modes could be conveniently connected by the interface of modular structure. Industrial control computer was used as the main control platform, and physical of control parameters such as pressure, pump speed, dissolved oxygen, temperature, and etc, were transmitted into computer, then according to the instruction, process of the treatment was accomplished by the executing units implemented by main control system. Touch screen of human-computer interface was adopted, which made the system better operational and more comfortable. The system has passed the spot function test, and all indexes can meet requirements for the clinical treatment requested. It has the character such as modular design, systematic distribution, building-block structure, and etc, which supports a great novel operation platform for artificial therapy.
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Fígado Artificial , Algoritmos , Engenharia Biomédica/métodos , Técnicas de Cultura de Células , Computadores , Desenho de Equipamento , Hepatócitos/metabolismo , Humanos , Oxigênio/metabolismo , Software , Temperatura , Fatores de Tempo , Engenharia Tecidual/métodos , Interface Usuário-ComputadorRESUMO
Several approaches are being taken worldwide to develop vaccines against H5N1 viruses; most of them, however, pose both practical and immunological challenges. One potential strategy for improving the immunogenicity of vaccines involves the use of alphavirus replicons and VP22, a herpes simplex type 1 (HSV-1) protein. In this study, we analysed the antigenic peptides and homogeneity of the HA sequences (human isolates of the H5N1 subtype, from 1997 to 2003) and explored a novel alphavirus replicon system of VP22 fused with HA, to assess whether the immunogenicity of an HA-based replicon vaccine could be induced and augmented via fusion with VP22. Further, replicon particles expressing VP22, and enhanced green fluorescent protein (EGFP) were individually used as controls. Cellular immune responses in mice immunised with replicons were evaluated by identifying specific intracellular cytokine production with flow cytometry (FCM). Animal-based experimentation indicated that both the IL-4 expression of CD4(+) T cells and the IFN-gamma expression of CD8(+) T cells were significantly increased in mice immunised with VPR-HA and VPR-VP22/HA. A dose titration effect vis-à-vis both IL-4 expression and IFN-gamma expression were observed in VPR-HA- and VPR-VP22/HA-vaccinated mice. Our results revealed that both VPR-VP22/HA and VPR-HA replicon particles presented a promising approach for developing vaccines against human-avian influenza, and VP22 could enhance the immunogenicity of the HA antigens to which it is fused.