ObjectiveTo identify the expression functions of human NF-κBp65 nuclear localization signals' deletion mutant plasmids(namely pcDNA3.1(+)-NF-κBp65ΔNLS, NF-κBp65ΔNLS, for short) and the changes of proliferation, migration and adhesion ability of A549 lung cancer cells with low expression of NF-κBp65 (namely A549/NF-κBp65 shRNA cells). MethodsHuman A549/NF-κBp65 shRNA cells were cultivated and divided into a control group, a transfection pcDNA3.1 (+) group, and a transfection NF-κBp65ΔNLS group. Indirect immunofluorescence, real-time fluorescent quantitative PCR and Western blot techniques were used to detect the NF-κBp65 intracellular localization and the change of NF-κBp65 mRNA and protein expression level. MTT, Transwell and cell adhesion experiments were used to analyze the changes of proliferation, migration and adhesion ability of A549/NF-κBp65 shRNA cells. ResultsThe human NF-κBp65ΔNLS eukaryotic expression plasmid was successfully constructed. Compared with the control group and the transfection pcDNA3.1(+) group, NF-κBp65 mRNA expression level in A549/NF-κBp65 shRNA cells was increased in the transfection NF-κBp65ΔNLS group(10.63±0.84 vs. 1.04±0.21 and 1.23±0.22, P < 0.01) and NF-κBp65 protein expression level was also increased (1.07±0.06 vs. 0.53±0.02 and 0.59±0.04, P < 0.01). NF-κBp65 protein mainly located in the cytoplasm, and did not significantly transferred into the nucleus after stimulated by TNF-α. At the same time, A549/NF-κBp65 shRNA cells' proliferation, migration and adhesion ability were enhanced compared with the control group and the transfection pcDNA3.1(+) group. ConclusionsThrough gene mutation technology to build the human NF-κBp65ΔNLS eukaryotic expression plasmid and transfect into A549/NF-κBp65 shRNA lung cancer cell lines, both mRNA and protein expression levels of NF-κBp65 were increased significantly, and NF-κBp65 protein mainly located in the cytoplasm. The overexpressed NF-κBp65 in cytoplasm can obviously enhance the A549/NF-κBp65 shRNA cell's proliferation, migration and adhesion ability. It suggests that NF-κBp65 stranded in the cytoplasm can still regulate biological behavior of lung cancer cells by influencing the NF-κB signaling pathway related proteins.
Objective To investigate the clinical characteristics and bacterial drug resistance of bloodstream infection of gram-negative bacteria, and provide guidance for clinical rational drug use and control of hospital infection. Methods A retrospective analysis was conducted in the patients diagnosed as severe pneumonia with blood culture of gram-negative bacteria from January 2015 to December 2017 in Beijing Anzhen Hospital. Results A total of 60 severe pneumonia patients suffered from bloodstream infection of gram-negative bacteria were recruited including 34 males and 26 females aging from 42 to 89 years and 73.4 years in average. In the 60 patients, 32 cases were infected with Klebsiella pneumonias, 20 cases were infected with Acinetobacter baumanni, and 8 cases were infected with Escherichia coli. The antimicrobial susceptibility testing result of Klebsiella pneumonias showed that the drug susceptibility rate was 100% to tigecycline, and 6.3% to amikacin. Escherichia coli was sensitive to Amikacin, imipenem, ceftazidime and meropenem while resistance to other drugs. The antimicrobial resistance of Acinetobacter baumanni was 28.6% for cefoperazone/sulbactam, and 14.3% for tigecycline. C-reactive protein, procalcitonin and SOFA scores were higher in the patients infected with Acinetobacter baumanni. Neutrophils and blood lactic acid were higher in the patients infected with Klebsiella pneumonias. There were no statistical differences in white blood cell, platelet or motality rate between the patients infected with Acinetobacter baumanni and the patients infected with Klebsiella pneumonias. SOFA scores and blood lactic acid had significantly statistical relevance with prognosis. Conclusion There is a high proportion of drug resistance of Klebsiella pneumoniae and Acinetobacter baumanni in the bloodstream infection of gram-negative bacteria.