Effect Of Lentiviral Vector-mediated Omp25 Overexpression On Microglial Cells And Its Molecular Mechanism

Objective Through Omp25 lentivirus-mediated gene transfection of mouse microglia(BV2) cells, Omp25 protein expression was observed.Explore Brucella virulence factors Omp25 overexpressing inflammatory effects on BV2 cells,for further study Omp25 genes provide a theoretical basis for the central nervous system in Brucella molecular pathogenesis of infection.Methods Selected in the logarithmic growth phase BV2 cells,and divided into three groups:infection Omp25 recombinant overexpression lentivirus vectors as the experimental group(LV-Omp25-GFP group), group infectedwith lentivirus negative vector(LV-GFPgroup) blank in the control group. After infection 72h: fluorescence was observed underfluorescent microscope expression determines lentivirus transfection efficiency,fluorescence flowcytomet-ry sorting of BV2 cells stably expressing green fluorescent protein used in the following experiments:(1)transmission electron microscopy: observation of slow viral infection and cell structure of the control group, the cells were observed before and after the change in the ultrastructure of infection.(2) each group were extracted RNA, DNA, semi-quantitative reverse transcription- polymerase chain reaction(RT-PCR) and Western blot to detect the expression of cells,Omp25 gene m RNA and protein expression of Omp25 verification transcript and protein levels.(3) Experiment were collected LV-Omp25-GFP group, LV-GFP group and control group cell supernatants by ELISA kit to detect cell inflammatory cytokine TNF-?, IL-6, NO.(4)secretion with 5um NF-?B signaling pathway inhibitor BAY11-7082 pretreatment BV2 cells 60 min, added LV-Omp25-GFP 50ul(MOI=50) infected BV2 cells,while a control group, the experimental group and the control group were collected after 72 h and expression of RT-PCR and ELISA detection of inflammatory factor m RNA and their protein levels.Several groups were compared using ANOVA, P <0.05 was considered statistically significant.Results(1) after lentivirus infection BV2 cells for 72 hours to observe the expression of LV-GFP group and LV-Omp25-GFP under the fluorescence microscopic,group of GFP fluorescence inverted microscope, some more than 85% of the cells fluoresce green, because the control group no virus, no GFP express under the fluorescence microscope.(2)ELISA results showed that after infection 72 h, compare with LV-Omp25-GFP group and control group and LV-GFP group were statistically significant(P <0.05), LV-GFP and the control group was not statistically significant(P> 0.05).(3)Quantitative PCR results showed that: 1 blank control group were compared, lentivirus group Omp25 gene m RNA expression levels were significantly up-regulated expression level than the control group and GFP group was significantly increased, the differences were statistically significant(P < 0.01), while GFP group compared with the control group, the difference was not statistically significant(P> 0.05). Show BV2 cells successfully overexpressed Omp25 gene..(4)Western blot results: compare with the control group, the Omp25 lentivirus group,s gray level is higher, Omp25 lentivirus infected BV2 cells Omp25 protein overexpression successful.(5) transmission electron microscopy showed that: compared with the control group, LV-Omp25-GFP group BV2 cells can be seen a long and rich filopodia, a large number of lipid vacuoles, myeloid bodies, mitochondria are a lot of damage, cell endoplasmic some sizes of phagocytic vacuoles, which varying amounts of viral particles containing the slow aggregation, increased lysosomes.(6) after pretreatment with BAY11-7082 to BV2 cells 60 min, transfected with LV-Omp25-GFP, and cell culture supernatants were collected after 72 h, ELISA and fluorescence quantitative detection of TNF-?, IL-6 and NO's expression. The results showed that after adding the signal pathway inhibitors can inhibit the induction of TNF-?, IL-6, NO production, proved NF-?B signaling pathway may be involved in the induction of proinflammatory cytokines TNF-?, IL-6, NO production.Conclusions Establish Omp25 lentivirus overexpressing cells transfected BV2 cell model, presumably that BV2 cells may engulfed the omp25 overexpressed lentivirus engulfed into the cellthrough endocytosis, changes cell ultrastructure, thereby affecting the intracellular environment, leading it to apoptosis; Omp25 overexpression can activate NF-?B signaling pathway leading to BV2 cells to secrete pro-inflammatory cytokines TNF-?, IL-6, NO increase, which may be one of the molecular mechanisms of neuronal Brucellosis.