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Objective To investigate the effect of microRNA-24 (miR-24) on cell proliferation and migration in osteosarcoma cell line U2OS and its possible mechanism.Methods U2OS cell line with miR-24 over-expression was established by transfecting miR-24 mimic, and then cell proliferation and migration in control group, negative control group and miR-24 over-expression group were detected with real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) assay, cell counting kit-8 (CCK-8) assay and Transwell assay, respectively.Epithelial-mesenchymal transition (EMT) progression and activation of nuclear factor-κB (NF-κB) pathway were detected by Western blotting.Results The expressions of miR-24 in the control group, negative control group and miR-24 over-expression group were 1.00±0.00, 1.03±0.08 and 2.46±0.29, with significant difference (F=11.026, P=0.012).Compared with the control group, the expression of miR-24 in human osteosarcoma U2OS cell line was significantly increased after transfecting miR-24 mimic (t=4.604, P=0.009).After over-expression of miR-24 for 24 h, 48 h and 72 h, U2OS cell viabilities were decreased significantly compared with the control group [(3.56±0.27)% vs.(8.63±0.79)%, t=3.896, P=0.016;(20.16±1.09)% vs.(54.77±5.42)%, t=4.813, P=0.008;(45.47±3.16)% vs.(95.52±8.56)%, t=7.173, P=0.002)].After over-expression of miR-24 for 24 h, the cell migration rates in control group, negative control group and miR-24 over-expression group were (100.00±0.00)%, (99.26±5.85)% and (31.37±2.09)%, respectively, and there was statistically significant difference among the three groups (F=12.175, P=0.009);and compared with control group, cell migration rate was decreased significantly after over-expression of miR-24 (t=3.843, P=0.004).Meanwhile, over-expression of miR-24 up-regulated the expression levels of epithelial cell markers E-cadherin (t=3.852, P=0.018) and β-catenin (t=3.512, P=0.024), while down-regulated the expression levels of mesenchymal cell markers N-cadherin (t=3.832, P=0.018) and vimentin (t=4.058, P=0.012), with a suppressed EMT progress.Other than that, miR-24 over-expression inhibited the expressions of NF-κB (p65) (t=4.813, P=0.008), phosphorylation inhibitor of nuclear factor kappa-B kinase α (p-IKK-α) (t=3.764, P=0.013) and phosphorylation inhibitor of nuclear factor kappa-B kinase complex α (p-IκB-α) (t=4.064, P=0.012), suppressing the activation of NF-κB pathway.Conclusion miR-24 can suppress cell proliferation and migration of osteosarcoma cell line U2OS in vitro, and its mechanism may be related to the suppression of NF-κB activation and EMT progression.It hints that miR-24 may be used as a potential new target for osteosarcoma therapy.
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Objective:To study the clinical effect and safety of ubenimex capsules and SOX chemotherapy on the advanced gastric cancer.Methods:90 patients with advanced gastric cancer who were treated in our hospital from September 2013 to September 2015 were selected and randomly divided into the observation group (n=45) and the control group (n=45).The patients in the control group were treated with SOX chemotherapy,while the patients in the observation group were treated with ubenimex capsules on the basis of control group.Then the serum levels of MMP-2 and MMP-9,the immune functions,the clinical efficacy,the adverse reactions and survival rate of two groups were observed and compared before and after the treatment.Results:After treatment,the CD4+,CD4+/CD8+ in the observation group were higher than those of the control group (P＜0.05);The levels of MMP2 and MMP-9 in the observation group were lower than those of the control group (P＜0.05);The total effective rate of the observation group was higher than that of the control group [68.89％(31/45) vs 48.89％(22/45)] (P＜0.05);The incidence of thrombocytopenia,leukopenia,nausea and vomiting and abnormal liver functions in the observation group was lower than that of the control group (P＜0.05);The survival rate of the observation group was higher than that of the control group at 6 months and 12 months [93.33％ (42/45) vs 77.78％ (35/45),82.22％ (37/45) vs 57.78％ (26/45)](P＜0.05).Conclusion:Compared with SOX chemotherapy alone,ubenimex capsules and SOX chemotherapy could effectively improve the immune function,enhance the long-term survival rate with high safety of patients with advanced gastric cancer.
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Objective: To prepare and characterize the polyclonal antibody against KIAA0649 and identify the localization and the functional motif of KIAA0649. Methods: Three polypeptides were synthesized based on the bioinformatics analysis of KIAA0649 protein. New Zealand rabbits were immunized with the mixture of the three KIAA0649 peptides coupled with keyhole limpet hemocyanin ( KLH). The titer of the antisera was detected with ELISA. The antisera were purified with immuno-affinity chromatography when the titer reached 1:10~5. Western blot was performed with the purified antisera on the cell lysates of U2OS cells transfected with either Flag-KIAA0649 or KIAA0649-targeting siRNA. Immunofluorescence was performed with the purified antisera and anti-Flag antibody on the cells transfected with FlagKIAA0649. A series of Flag-K1AA0649 deletion mutants was constructed by PCR cloning. The cellular compartmentation of full-length Flag-KIAA0649 and its deletion mutants were analyzed with immunofluorescence. Results: The results of Western blot and immunofluorescence demonstrated that the antisera from the KIAA0649 polypeptides-immunized rabbits specifically recognized endogenous and exogenous KIAA0649. The full-length Flag-K1AA0649 displayed specific nuclear foci. The Flag-KIAA0649 deletion mutant containing PENF motif showed the same nuclear foci as the full length of Flag-KJAA0649, suggesting that the PENF motif could be the minimum functional motif of KIAA0649. Conclusion: We have obtained anti-KIAA0649 polyclonal antibody which will be useful for further investigation. The PENF motifcould be the minimum functional domain of KIAA0649.
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Objective Analysis the risk factors which maybe caused the cerebral hemorrhage after the liver transplantation, and then make out the counter nursing measures. Methods Selected 12 patients with cerebral hemorrhage after the liver transplantation as the experimental group, and then matching 48 patients without cerebral hemorrhage after the liver transplantation as the control group. Analysis the factor which related the cerebral hemorrhage. Results The factors which related the cerebral hemorrhage after the liver transplantation included in the Intensive Care Unite, heme level, Child level, APTT, albumin, blooding volume during the operation, operation time and the postoperative blood platelet level. The factors of Child level and the blooding volume during the operation were the independence risk factors of cerebral hemorrhage. Conclusion The factors of Child level and the blooding volume during the operation were the independence risk factors of cerebral hemorrhage, we should carry out counter nursing measures to prevent them.
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Objective: To investigate the effect of intracellular free Ca 2+ concentration ([Ca 2+ ] i ) on injury following transient Mg 2+ free treatment in vitro in developing cortical neurons. Methods: Embryo cortical neurons of rats cultured for 6 d and 17 d were directly exposed to Mg 2+ free media, or pretreated with NMDA receptor antagonists or calcium channel antagonist before being exposed to Mg 2+ free media. MTT assay was used to study the injury of neurons. [Ca 2+ ] i were measured using fluo 3, a fluorescent calcium sensitive dye and laser scanning confocal microscope, and calculated by the fluorescent intensity. Results:Compared with control, MTT conversion rates decreased after transient (3 h) Mg 2+ free treatment in neurons cultured for 6 d and 17 d in vitro, (59.1?6.87)% and (51.2?5.90)%, respectively. In neurons pre and co treated with 10 ?mol?L -1 MK 801, 50 ?mol?L -1 AP 5 and 10 ?mol?L -1 nifedipine, MTT conversion rates were higher than those of neurons with only Mg 2+ free treatment. Peak values of [Ca 2+ ] i in neurons cultured for 6 d and 17 d were 2.4?0.23 and 3.2?0.32, respectively Peak value of neurons 17 d in vitro was significantly higher than that of neurons 6 d in vitro (P
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AIM As one of the reactive oxygen species, toxic dose of H2O2 leads to the necrosis or apoptosis of many kinds of cells. It is nuclear whether these processes are related with the changes of nuclear Ca2+ elicited by H2O2. This study on the effect of taurine on changes of nuclear and cytosolic (Ca2+) elicited by H2O2 in rat vascular smooth muscle cells. METHODS The techniques of Fluo-3 and laser scanning confocal microscopy were used. RESULTS Rapid and sustaining rise of nuclear ［Ca2+］ (［Ca2+］n) and cytosolic ［Ca2+］(［Ca2+］c) were found in cultured rat vascular smooth muscle cells upon adding of 0.5% H2O2. Pretreatment of the cells with 20 mmol*L-1 taurine, a regulator of cellular Ca2+ homestasis, significantly decreased the rise amplitude of ［Ca2+］n (P<0.05), but failed to affect that of ［Ca2+］c (P>0.05) elicited by H2O2. It was suggested that the changes of ［Ca2+］n and ［Ca2+］c have different respond to taurine. CONCLUSION It was concluded that there exist the independent regulating mechanisms of Ca2+ in the nucleus from differences between the nucleus and cytosol Ca2+ in sensitivity to action of H2O2 and taurine.
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To identify the expression of the molecule recognized by Pf18-3 mAb (Pf18-3 molecule) on various cells. Meth-ods: The expression of pr18-3 molecule was assayed by flow cytometry and confocal laser scanning microscope . Result: The molecule recog-nized by Pf18-3 mAb expressed on TSC and other stromal cells. Whereas, fresh thymocytes were Pf18-3 negative. Interestingly, the expressionof Pf18-3 molecule was gradually up-regulated on thymocytes after activation by ConA. This molecule mainly expressed on CD4+ CD8+ andCD4+ CD8- cells. Under confocal laser scanning microscope, the staining of fluorescence showed as ring around the cell, it changed grsduallystronger and thicker with activation. Conclusion: This study indicated that the Pf18-3 molecule was co-expressive molecule of MTSC and acti-vated tlymocytes,it was concemed closely about the activation of CD4+ C D8+ and CD4+ CD8- cells.
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AIM: The purpose of the present study was to detect intracellular Ca 2+ changes in living brain slices during focal cerebral ischemia/reperfusion (I/R) and reveal the role of intracellular Ca 2+ in the cerebral I/R injury. METHODS: The model of focal cerebral I/R was established in rats by reversible inserting a nylon thread, and dynamic change of intracellular Ca 2+ in brain slices was determined using laser confocal imaging system. RESULTS: ① Ca 2+ gradually enhanced with increase in ischemic time in cortex and striatum. ②At 1 h ischemia/ 10 min reperfusion, Ca 2+ increased significantly in striatum, but Ca 2+ decreased at 3 h reperfusion compared with 10 min reperfusion. ③ Ca 2+ markedly enhanced at 6 h ischemia compared with 1 h ischemia, and after 3 h reperfusion Ca 2+ decreased, but was still higher than that in sham-operation group. ④The striatum is more sensitive than cortex to ischemia/reperfusion. CONCLUSION: Ca 2+ overload in the area of cortex and striatum may play an important role in cerebral ischemia/reperfusion injury in rats.
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To study the significance of calcium in the apoptosis of HL-60 cells induced by VP-16, the technology of flow cytometry, confocal laser scanning microscopy and Western blot were used. The results showed that VP-16 could induced the apoptosis of HL-60 cells and transient increase of intracellular calcium concentration; EGTA [ethylene glycol-bis(2-aminoethyl)-N,N,N',N'-tetraacetic acid], that could combine the extracellular calcium, did not prevent the apoptosis of HL-60 cells. BAPTA-AM [1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxy-methyl) ester], however, a chelating agent of intracellular calcium ions, could prevent apoptosis and the release of cytochrome C from HL-60 cells. It was concluded that the calcium plays an important role in apoptosis and the release of cytochrome C.
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AIM and METHODS: To observe the effects of glucose-free and Mg 2+ -free in the extracellular fluid on the changes of [Ca 2+ ] i in the cerebro-cortical neurons damaged by 1 mmol/L glutamate using laser confocal scanning microscope. RESULTS: Both frequency and amplitude of neuronal calcium oscillation induced by glutamate were lowered in glucose-free and Mg 2+ -free buffers. The basic [Ca 2+ ] i concentration was lowered in the former case , but it was elevated in the latter case. CONCLUSION: Mg 2+ -free aggravates [Ca 2+ ] i overload induced by 1 mmol/L glutamate ,under certain conditions the glucose-free might resist damage role of glutamate and Mg 2+ -free.
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AIM As one of the reactive oxygen species, toxic dose of H 2O 2 leads to the necrosis or apoptosis of many kinds of cells. It is nuclear whether these processes are related with the changes of nuclear Ca 2+ elicited by H 2O 2. This study on the effect of taurine on changes of nuclear and cytosolic (Ca 2+ ) elicited by H 2O 2 in rat vascular smooth muscle cells. METHODS The techniques of Fluo 3 and laser scanning confocal microscopy were used. RESULTS Rapid and sustaining rise of nuclear [Ca 2+ ] ([Ca 2+ ] n) and cytosolic [Ca 2+ ]([Ca 2+ ] c) were found in cultured rat vascular smooth muscle cells upon adding of 0 5% H 2O 2. Pretreatment of the cells with 20 mmol?L -1 taurine, a regulator of cellular Ca 2+ homestasis, significantly decreased the rise amplitude of [Ca 2+ ] n ( P 0 05) elicited by H 2O 2. It was suggested that the changes of [Ca 2+ ] n and [Ca 2+ ] c have different respond to taurine. CONCLUSION It was concluded that there exist the independent regulating mechanisms of Ca 2+ in the nucleus from differences between the nucleus and cytosol Ca 2+ in sensitivity to action of H 2O 2 and taurine.
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AIM: This research emphasizes the effect of ganglioside GM1 on generation of the intracellular amyloid ?-protein(A?) and investigates where and how GM1 promoted the production of intracellular A?, particularly the more highly amyloidogenic A? 42 which is basis of senile plaque.METHODS: Human neuroblastoma cells transfected with human amyloid precursor protein (APP) cDNA were used to analyze the effect of the various concentrations of GM1 on the level of intracellular total A? by IP-Western blot. Subcellular compartment localized and colocalized with intracellular A? 42 was determined by double or triple immunofluorescence labeling.RESULTS: The intracellular total A? was promoted by GM1, and the levels of intracellular A? were correlated to the concentrations of GM1 in a dose-dependent fashion ( P