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Objective To investigate the effect of lidocaine on Staphylococcus aureus exotoxin-stimulated peripheral blood mononuclear cells (PBMCs) from patients with atopic dermatitis (AD).Methods Peripheral blood samples were collected from 6 patients with AD,and PBMCs were isolated by a routine method.Then,the PBMCs were stimulated by the Staphylococcus aureus exotoxin toxic shock syndrome toxin-1 (TSST-1) in the absence or presence of lidocaine at varying concentrations.The 3H-TdR incorporation method was performed to detect the proliferation of monocytes,and enzyme-linked immunosorbent assay (ELISA) to quantify the levels of T helper type 1 (Th1) and Th2 cytokines released by PBMCs.Human HaCaT keratinocytes were co-cultured with lidocaine-and TSST-1-stimulated PBMCs from patients with AD for 72 hours,then,Western blot was conducted to examine the expression of filaggrin protein in HaCaT cells.Results TSST-1 (100 μg/L) significantly enhanced the proliferation of PBMCs from patients with AD (stimulation index =75 ± 2.12,P ＜ 0.05),as well as the release of tumor necrosis factor-α (TNF-α),interferon (IFN)-γ,interleukin (IL)-2,IL-12,IL-4,IL-5 and IL-13 by the PBMCs (all P ＜ 0.05).Compared with the blank control group,100 μmol/L lidocaine significantly inhibited the TSST-1-stimulated proliferation of PBMCs from patients with AD (stimulation index =58 ± 3.14,P＜ 0.05),as well as the release of IL-4,IL-5,IL-13,TNF-α and IFN-γ by the stimulated PBMCs (all P ＜ 0.05).Western blot showed that 100 μmol/L lidocaine significantly blocked the down-regulation of filaggrin expression in HaCaT cells (P ＜ 0.01).Conclusion Lidocaine has a significant inhibitory effect on the activation of TSST-1-stimulated PBMCs from patients with AD.
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<p><b>OBJECTIVE</b>To investigate the role of histone acetylation in regulating influenza virus replicative intermediate double-stranded RNA (dsRNA)-induced interleukin-6 (IL-6) expression in A549 cells.</p><p><b>METHODS</b>A549 cells were treated with influenza virus replicative intermediate dsRNA, histone deacetylase (HDAC) inhibitor trichostatin A (TSA), or HADC small interfering RNA (siRNA). The changes in the cellular IL-6 promoter activities were detected by dual-luciferase assay, and IL-6 mRNA and protein expressions in the cells were determined using real-time RT-PCR and ELISA, respectively.</p><p><b>RESULTS</b>Influenza virus replicative intermediate dsRNA obviously up-regulated IL-6 expression in the cells. HDAC inhibitor TSA significantly enhanced the activity of IL-6 promoter and increased IL-6 mRNA expression in A549 cells, and HDAC3 may play an important role in this process. HDAC inhibitor TSA and DNMT inhibitor DAC showed no synergic effect in regulating IL-6 expression.</p><p><b>CONCLUSIONS</b>Influenza virus replicative intermediate dsRNA-induced IL-6 expression in A549 cells is regulated by histone acetylation.</p>
Subject(s)
Humans , Acetylation , Cell Line, Tumor , Gene Expression Regulation , Histone Deacetylase Inhibitors , Pharmacology , Histones , Metabolism , Interleukin-6 , Metabolism , Orthomyxoviridae , Genetics , Metabolism , Promoter Regions, Genetic , RNA, Double-Stranded , RNA, Messenger , Genetics , RNA, ViralABSTRACT
The article discusses the curriculum truss of medical biosciences major and the curriculum setup with basic medical courses as the foundation,highlighting the life science scopes and emphasizing integrated curriculum and the training of molecular biology technology to culture practical cross-subject talents in the life sciences for society.
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<p><b>OBJECTIVE</b>To construct the specific stable expression and high efficiency small interfering RNA(siRNA) expression vector that can block DNMT1 gene function.</p><p><b>METHODS</b>Using vector-based RNA interference technique, the authors constructed a vector to transcribe functional short interfering RNA (RNAi). After transfection by lipofectmine (TM) reagent, the treated cells were selected by G418. The expression levels of RNA and protein of DNMT1 were analyzed by reverse transcription polymerase chain reaction(RT-PCR) and Western blotting. The status of methylation of E-cadherin was analyzed by methylation-specific PCR(MSP).</p><p><b>RESULTS</b>The expression level of endogenous DNMT1 mRNA in transfected SMMC-7721 cell lines with DNMT1 RNAi construct was 43% less than that in control cell 7721-pSU cell lines. The protein level in the former was about 10% less than that in the latter. The efficiency of the siRNA of DNMT1 was found to be higher than 90%. Demethylation of promoter of E-cadherin was obtained due to the inhibition of DNMT1.</p><p><b>CONCLUSION</b>DNMT1 siRNA stable expressing vector was obtained by gene-recombined technology. There was no complete sameness between the levels of protein and RNA in gene silenced cell lines. The efficiency of the siRNA should be confirmed by Western-blotting.</p>