Characteristic comparison of mouse primary macrophages cultured in L929 cell conditioned medium / 生物工程学报

The purpose of this study is to provide a culture for mouse bone marrow-derived macrophages (BMDM) and peritoneal macrophages (PM) and to characterize their molecular and cellular biology. The cell number and purity from the primary culture were assessed by cell counter and flow cytometry, respectively. Morphological features were evaluated by inverted microscope. Phagocytosis by macrophages was detected by the neutral red dye uptake assay. Phenotypic markers were analyzed by real-time fluorescent quantitative PCR. Our results show that the cell number was much higher from culture of BMDM than PM, while there was no significant difference regarding the percentage of F4/80+CD11b+ cells (98.30%±0.53% vs. 94.83%±1.42%; P>0.05). The proliferation rate of BMDM was significantly higher than PM in the presence of L929 cell conditioned medium, by using CCK-8 assay. However, PM appeared to adhere to the flask wall and extend earlier than BMDM. The phagocytosis capability of un-stimulated BMDM was significantly higher than PM, as well as lipopolysaccharide (LPS)-stimulated BMDM, except the BMDM stimulated by low dose LPS (0.1 μg/mL). Furthermore, Tnfα expression was significantly higher in un-stimulated BMDM than PM, while Arg1 and Ym1 mRNA expression were significantly lower than PM. The expression difference was persistent if stimulated by LPS+IFN-γ or IL-4. Our data indicate that bone marrow can get larger amounts of macrophages than peritoneal cavity. However, it should be aware that the molecular and cellular characteristics were different between these two culture systems.

Effects of paeonol on lipopolysaccharide/adenosine 5′-triphosphate induced NLRP3 inflammasome activation in primary rat microglia / 中国药理学通报

Aim To investigate the effects of paeonol on lipopolysaccharide ( LPS) and adenosine 5′-triphos-phate ( ATP) induced NLRP3 inflammasome activation in primary rat microglia and the mechanisms responsi-ble for this anti-inflammatory effects. Methods Pri-mary rat microglia were identified immunohistochemi-cally using the cluster of differentiation 11 b ( CD11 b ) antibody. Proinflammatory cytokine IL-1β was deter-mined by ELISA. Western blot was performed to ob-serve the protein expression of NLRP3 , ASC and caspase-1 in cultured primary rat microglia. The level of intracellular reactive oxygen species ( ROS) was mo-nitored by using the fluorescent probe 2′, 7′-dichlo-rofluorescein diacetate ( DCFH-DA ) . Results LPS (0. 5 mg · L-1 )/ATP ( 5 mmol · L-1 ) significantly increased intracellular ROS level and IL-1β secretion and upregulated NLRP3 , ASC and caspase-1 protein expression in primary rat microglia. Paeonol signifi-cantly decreased intracellular ROS level and IL-1β se-cretion, and inhibited LPS/ATP induced overexpres-sion of NLRP3 , ASC and caspase-1 in cultured primary rat microglia. Conclusion Paeonol can inhibit LPS/ATP induced NLRP3 inflammasome activation in pri-mary rat microglia, and this inhibitory effect may be through the suppression of intracellular ROS.

Enhanced erythromycin production in Saccharopolyspora erythraea by tandem expres-sion of metK, vhbS and adpA / 军事医学

Objective To construct erythromycin-overproducing mutants by tandemly expressing S-adenosylmethionine synthetase gene metK, Vitreoscilla hemoglobin gene vhbS and pleiotropic regulatory gene adpA in Saccharopolyspora eryth-raea.Methods Through PEG-mediated protoplast transformation , the integrative plasmid carrying metK, vhbS and adpA was respectively introduced into erythromycin-producing wild-type strain S.erythraea A226 and industrial strain WB .The engineered strains were generated by apramycin resistance screening and PCR identification .The erythromycin production was compared in original strains and their mutants by the inhibition test of Bacillus subtilis and HPLC analysis .Results and Conclusion Four A226-derived mutants A226-P1-P4 and three WB-derived mutants WB-P1-P3 were independently obtained.Compared with wild-type strain A226, the relative erythromycin titer of the four engineered strains A 226-P1-P4 was increased from 8%to 25%by scoring the growth-inhibition zones .Further HPLC analysis showed that the four mutants had increased erythromycin A yield by 64%-94%.Likewise, the relative erythromycin titer and erythromycin A yield of the three engineered strains WB-P1-P3 were enhanced by 6%-10%and 31%-62%, respectively, in comparison with the original strain WB.The results show the universality of enhancing erythromycin productionvia tandem expression of metK, vhbS and adpA in S.erythraea.