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Objective To construct human yippee-like 5(YPEL5) gene eukaryotic expression recombinant plasmid and to express in esophageal carcinoma EC9706 cells .Methods The cDNA from human normal tissue was taken as a template and amplified to YPEL5 gene coding sequence with 366 bp in length .Then this se-quence was inserted into the multiple cloning site areas of eukaryotic expression vector pCDH-CD513B for ob-taining the eukaryotic expression vector pCDH-CD513B-Flag-YPEL5 .After the bacterial colony PCR identifi-cation ,it was sent to the corporation for testing the sequence .The successfully constructed recombinant plas-mid was transfected into human esophageal carcinoma EC9706 cells .The expression of PEL5 gene in EC9706 cells was detected by QRT-PCR and Western Blot .Results The YPEL5 gene segment with 366 bp in length was successfully amplified .pCDH-CD513B-Flag-YPEL5 recombinant plasmid was obtained by double enzyme digestion ,connection ,conversion and screening .The gene sequencing identification showed that the inserted gene sequence in recombinant plasmid was consistent with that in the GenBank .After 2 d of transfecting into EC9706 cells ,the QRT-PCR and Western Blot revealed that YPEL5 gene expression was significantly up-reg-ulated .Conclusion The pCDH-CD513B-Flag-YPEL5 eukaryotic expression vector is successfully constructed and is expressed in esophageal squamous cancer cell line EC9706 ,thus which lays a foundation for studying its function in the progression of esophageal cancer .
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AIM To investigate the protective effects and mechanism of propofol on cultured rat hippocampal neurons subjected to anoxia injury METHODS Hippocampal neurons of neonatal rats, which had been cultured in vitro for 10 days, were allocated to control groups and propofol treating groups In propofol treated groups,the culture medium were loaded with propofol at the concentrations of 3, 6, 12, 24, 48 mg?L -1 respectively, and then the neurons were exposed to oxygen glucose deprivation for 24 h, to 40 ?mol?L -1 H 2O 2 for 24 h or to 100 ?mol?L -1 glutamate for 50 min The cell survival rate in each group was evaluated by MTT colorimetry. Using a laser scanning confocal microscope (LSCM), the effects of propofol on neuronal calcium overload and on the reduction of mitochondrial membrane potential (△?m) evoked by anoxia were observed with fluo 3 and rhodamine 123 for real time changes of [Ca 2+ ] i and △?m. The electron spin resonance (ESR) was used to measure the scavenging effects of propofol on hydroxyl radical and superoxide anion RESULTS Propofol at the concentrations of 6～48 mg?L -1 attenuated the anoxic injury ( P
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Aim To investigate the protective effects of etomidate on cultured rat hippocampal neurons subjected to anoxia injury and its mechanism.Methods Hippocampal neurons of neonatal rat,which had been cultured in vitro for 10 days,were allocated to control groups and etomidate-treating groups.The neurons were exposed to oxygen-glucose deprivation for 24 h.The cell survival rate in each group was evaluated using MTT colorimetry.To explore the effect of etomidate on neuronal calcium overload evoked by anoxia or 50 mmol?L~(-1) KCl or 1 mmol?L~(-1) glutamate,fluo-3,a fluorescent probe,was used for imaging of intracellular calcium in laser scanning confocal microscope(LSCM)to measure real-time changes of [Ca~(2+)]_i in cultured rat hippocampal neurons.Results The hippocampal neurons developed acute neuronal swelling and widespread neuronal degeneration following anoxia for 24 h.Etomidate at concentrations of 1.2～4.8 mg?L~(-1) attenuated the neuronal injury in a dose-dependent manner(P
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Aim To evaluate the analgesic effect of agmatine on inflammatory pain and its influence on the analgesic effect of morphine. To investigate whether the mechanism of analgesic effect of agmatine is related to activation of imidazoline receptor or to affect the release of endogenous glutamate and gamma-aminobutyric acid (GABA) from rat spinal cord slices. Methods The formalin test in rats was used as a long-lasting inflammatory pain model. Effects of agmatine on basal and K+ evoked release of endogenous glutamate and GABA from rat spinal cord slices were determined by high performance liquid chromatography (HPLC). Results Pretreatment with agmatine (ip or sc) inhibited the second phase of the nociceptive response of rats and potentiated the analgesic effect of morphine in phase 2, but not in phase 1. Idazoxan did not attenuate the analgesic effect of agmatine. Agmatine (1, 10, 100, 1000 ?mol?L -1 ) had no effect on the basal release of glutamate and GABA from spinal cord slices, nor did it affect the K+ (50 mmol?L -1 ) evoked release of glutamate and GABA contents. Conclusions Agmatine has an analgesic effect and enhances morphine analgesia in the second but not the first phase of formalin-induced nociception. Its analgesic effect does not likely involve imidazoline receptor. The mechanism of the analgesic effect of agmatine may not be associated with inhibiting glutamate release nor increasing the GABA content.
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Objective To determine if propofol can protect cultured rat hippocampal neurons from anoxia-induced injury and elucidate the underlying mechanism.Methods Neonatal Wistar rats were decapitated. Hippocampus was isolated, minced and digested with 0.125 % trypsin at 371 for 25 min, then centrifuged at 1000 r/min for 5 min. The supernatant was discartled and the precipitate was resuspended in growth medium. The cell suspension was incubated at 37 ℃ for 10 days. The cultured hippocampal neurons were randomly divided into 3 groups: control group(group C) ,anoxia group(group A), propofol + anoxia group (group PA) . Group PA was further divided into 3 subgroups of different end-propofol concentrations:3, 12,48 mg?L-1 . The cultured neurons were transferred to low glucose medium and incubated at 37 ℃ in closed incubator filled with anoxic atmosphere (95% N2-5% CO2) for 24 h in group A and group PA (following addition of propofol) . The cell survival rate in each group was measured by MIT colorimetry. The real-time changes in [Ca2+ ]i in cultured hippocampal neurons induced by anoxia or glutamate or KCL were measured by fluorescence and laser scan confocal microscopy ( LSCM) after staining with fluo-3/AM.Results The hippocampal neurons developed acute swelling and widespread degeneration following anoxia. Propofol attenuated the neuronal injury at 12 and 48 mg?L-1 in a dose-dependent manner and significantly increased the cell survival rate following anoxia (P
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Objective To investigate the effect of propofol on N-methyl-D-aspartate (NMDA) receptor-mediated currents. Methods Hippocampal neurons were prepared from newborn Wistar rats and cultured for 8-12 days. Whole cell currents were recorded using patch-clamp technique and cells were voltaged-clamped at - 80 mV. 100 ?mol?L-1 NMDA and 3 or 48 ?g?ml-1 propofol were applied with a multi-pipe ejection system. GABAA receptor was then blocked with 100 ?mol?L-1 bicuculline to investigate the effect of propofol on NMDA receptor without the influence of GABAA receptor.Results Run-down of INMDA induced by 100 ?mol?L-1 NMDA applied to neurons which were cultured for 8-12 days was 15%?8%. Propofol 3 or 48 ?g?ml-1 significantly inhibited spontaneous excitatory postsynaptic currents and elicited a Cl- -mediated response by direct activation of GABAA receptor in the absence of GABA. therefore produced a reversible inhibition of whole cell currents activated by NMDA. After the GABAA receptor was blocked by 100 ?mol?L-1 bicuculine propofol still inhibited NMDA currents slightly. Conclusion Propofol inhibits the NMDA subtype of glutamate receptor mainly through activation of GABAA receptor. It can also directly suppress the NMDA receptor slightly.
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Aim To investigate the changes in the expression of protein kinase C gamma (PKC?), phosphorylated cAMP response element binding protein(pCREB)and immediate-early gene(c-fos and c-jun) in the spinal cord in formalin-induced inflammatory pain and study the effect of agmatine on the changes of PKC? activation, phosphorylation of CREB and expression of c-fos and c-jun.Methods Rats were decapitated at 10, 20 min or 2 h after intraplantar injection of 50 ?l 5% formalin and L_4, 5 spinal cords were dissected. Immunohistochemistry and western blotting analyses were used to observe the expression of PKC?, pCREB, c-fos and c-jun in the spinal dorsal horn and the effect of agmatine on the changes of their expression. Results Unilateral peripheral inflammation induced PKC? activation and CREB phosphorylation bilaterally while c-fos and c-jun expression ipsilaterally in rat spinal cord. PKC activity increased in membrane fractions with unchanged levels in the cytosolic fractions. Pretreatment intraperitoneally with 160 mg?kg-1 agmatine 15 min before inflammation significantly inhibited the activation of PKC? in the membrane fraction, suppressed the phosphorylation of CREB and the expression of c-fos and c-jun. Conclusion The mechanism of the analgesic effect of agmatine may be associated with inhibiting PKC? activation in the plasma membrane, CREB phosphorylation, c-fos and c-jun up-regulation which play roles in the hyperalgesia with peripheral inflammation.