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Objective:To study the role and possible molecular mechanism of neural precursor cell-expressed developmentally down-regulated gene 4-like ( NEDD4 L) in the replication of hepatitis B virus (HBV). Methods:Small interfering RNA (siRNA) targeting NEDD4 L, plasmid expressing NEDD4 L with hemagglutinin(HA) C-terminal tag (pcDNA3.1- NEDD4 L-HA), plasmid expressing 1.3×HBV genome (pGEM-HBV1.3) and poly (dAT: dAT) were respectively transfected into HepG2 cells using Lipofectamine2000. HepG2.2.15 cells, a cell line that can stably express HBV, were used as control. The mRNA levels of NEDD4 L, interferon (IFN)-α, IFN-β, interferon-stimulated gene 56 ( ISG56), myxovirus resistance protein A ( MxA), oligoadenylate synthetase ( OAS), and the levels of HBV DNA or 3.5 kb HBV RNA were detected by real-time fluorescent quantitative polymerase chain reaction (qRT-PCR). Western blot was used to detect the silence and over-expression of NEDD4 L, and the protein levels of the related signaling molecules. The amount of IFN-β in the cellular supernatant was measured by enzyme linked immunosorbent assay (ELISA). Student t test was used for comparison of continuous data between groups. Results:The levels of NEDD4L mRNA and protein in HepG2.2.15 cells were 10.53±0.47 and 4.17±0.43, respectively, which were both statistically higher than those in HepG2 cells (1.00±0.05, t=3.27, P=0.008 and 1.26±0.25, t=1.68, P=0.030, respectively). In HepG2 cells with knockdown of NEDD4 L, the expression level of HBV DNA in cellular supernatant was 0.32±0.09, which was statistically lower than that in the control (1.00±0.05, t=-0.93, P=0.020), and the expression level of 3.5 kb HBV RNA was 0.49±0.11, which was statistically lower than that in the control (1.00±0.05, t=-0.68, P=0.040), while the mRNA levels of IFN-β and downstream effector molecules ( ISG56, MxA and OAS) were all significantly increased compared with the control ( t=4.66, 9.38, 7.29 and 7.01, respectively, all P<0.01). With poly (dAT: dAT) treatment and vesicular stomatitis virus (VSV) stimulation, the levels of IFN-β in HepG2 cells with knockdown of NEDD4 L were (776.41±115.49) ng/L and (961.21±130.19) ng/L, respectively, which were both statistically higher than those of the control group ((320.15± 56.05) ng/L, t=2.43, P=0.020; (440.17±67.82) ng/L, t=2.85, P=0.030, respectively). With poly (dAT: dAT) treatment and VSV stimulation, the levels of IFN-β in HepG2 cells with overexpression of NEDD4 L were (156.18±26.47) ng/L and (176.67±34.51) ng/L, respectively, which were both statistically lower than those of the control group ((320.38±49.39) ng/L, t=-2.03, P=0.040; (440.59±68.83) ng/L, t=-1.93, P=0.030, respectively). Western blot showed that the replication of HBV reduced the protein level of melanoma differentiation-associated protein 5 (MDA5), a key molecule in upstream of IFN-β, but the down-regulation was not obvious in cells with the knockdown of NEDD4 L. Conclusion:The replication of HBV could promote the up-regulation of NEDD4L protein and subsequently reduce the protein level of MDA5, thereby inhibiting the production of IFN-β, which facilitates HBV to escape the innate immune response.
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Medical immunology is an important frontier and bridge subject to connect basic medicine and clinical medicine, and its knowledge is abstract and difficult to understand. Micro-course is the new teaching resource with the education information. Its design, development and research have become a hot topic in network learning and mobile learning. In this article, it is elaborated that teaching design is the soul of the quality for micro-course and because micro class software is simple and easy to learn, it is a positive guarantee for the civilization and popularization of micro class, and besides, PPT is the important material in the micro-course. The article also explores the knowledge system of the fragmented micro lessons in series, and analyzes the potential value of micro teaching in the course teaching and personnel training.
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To determine the possibility whether DNA vaccines pcDNA3/SjSDISP of Schistosoma japonicum to induce autoimmunity and immune tolerance in the vaccinated mice, the titer of the specific antibodies against SjSDISP and the production of the autoimmune antibodies, such as presence of anti-nuclear and anti-dsDNA antibodies in the vaccinated mice were detected by ELISA assay and the toxicity of the plasmid DNA was studied through the observation of the change in body weight and the pathological examination of the major organs in mice. It was found that the titer of the specific antibody against SjSDISP was 1∶400 as determined by ELISA, but no autoimmune antibody could detected. The difference of the body weight of mice between the experimental and the control groups was not significant. No abnormal results of histopathological examination were obtained in both groups. From these observations, it is clear that there is no evidence to show that DNA vaccine pcDNA3/SjSDISP can induce auto-immunity and immune tolerance in mice.
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AIM: To investigate that nicotine inhibits HMGB1 expression and release in RAW264.7 cells.METHODS: (1) RAW264.7 cells were cultured in 6 wells plate, treated with 250 μg/L LPS and 1 μmol/L or 10 μmol/L nicotine, in which the cells treated with or without 250 μg/L LPS were regarded as nicotine 1 group (N1), nicotine 2 group (N2), LPS group (LPS) and control group (C), respectively. HMGB1 protein in the cell culture media and in cell nuclear was examined by Western blotting and the cellular HMGB1 mRNA level was detected by RT-PCR. (2) Transfected with antisense RNA or sense RNA of α~7 subunit-containing nicotinic receptor (α~7nAChR), RAW264.7 cells were treated with 250 μg/L LPS and 10 μmol/L nicotine, HMGB1 protein in the culture media was also tested by Western blotting.RESULTS: (1) HMGB1 mRNA level in C group was low (1 659.20±121.05) and no significant statistical difference among groups of N1, N2 and LPS was observed (P>0.05). (2) Higher HMGB1 accumulation in the cell culture media was detected in LPS group (445.34±28.52) than that in C group. Compared to LPS group, both N1 and N2 groups distinctly attenuated HMGB1 accumulation in culture media (P<0.05). (3) Nuclear HMGB1 accumulation was lower in LPS group than that in C group, and two different nicotine concentrations markedly increased the nuclear HMGB1 accumulation compared to LPS group (P<0.05). (4) No significant difference of HMGB1 levels in culture media between antisense RNA group and LPS group was observed (P>0.05). In sense RNA group, however, HMGB1 level was observably reduced compared to antisense group (P<0.05).CONCLUSION: The present results suggest that nicotine dramatically inhibits RAW264.7 cell nuclear HMGB1 translocation and extracellular release, and this effect relies on α~7nAch receptor expression.
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OBJECTIVE@#To develop a rapid and simple immunoassay to detect antibodies in the sera of patients infect Schistosoma japonicum (S. japonicum).@*METHODS@#Soluble egg antigen (SEA) of S. japonicum conjugated with colloidal carbon in advance was used to react with the antibodies in the sera of patients with schistosomiasis. Then the carbon-antigen-antibody complex would be captured by SEA which had been absorbed on the nitrocellulose membrane and a gray band was shown.@*RESULTS@#A total of 137 sera samples from S. japonicum epidemic area were tested, and the consistency, sensitivity, and specificity of colloidal carbon dipstick assay were 98.54%, 98.99%, and 97.37%, respectively, compared with the IHA method. The gray scale of bands on the dipstick was curvilinear to serum titer which revealed that the assay could be used semi-quantitatively in serum analysis.@*CONCLUSION@#Colloidal carbon dipstick assay is not only rapid and simple, but also sensitive and specific for the detection of serum antibodies of schistosomiasis japonica. It will be a practical immunological assay for the diagnosis of schistosomiasis in the field testing.
Subject(s)
Animals , Humans , Antibodies, Helminth , Blood , Carbon , Chemistry , Colloids , Chemistry , Immunoassay , Methods , Schistosoma japonicum , Allergy and Immunology , Schistosomiasis japonica , Blood , Diagnosis , Sensitivity and SpecificityABSTRACT
0.05).Both HMGB1 mRNA expression and HMGB1 protein level were remarkably higher in LPS treatment group than that in control group(P0.05).CONCLUSION:RPM inhibits HMGB1 expression not only by directly suppressing STAT3 activation,but also by indirectly reducing TNF-? level.