ABSTRACT
Objective To evaluate the regulatory role of azithromycin-induced persistent Chlamydia trachomatis (Ct) infection in the apoptosis of Hela229 cells.Methods Hela229 cells were firstly co-cultured with Ct for 22 hours,and then cultured with Dulbecco's modified Eagle's medium (DMEM) containing 0.08 mg/L azithromycin for 26 hours to establish a cell model of persistent Ct infection (persistent infection group).These infected Hela229 cells cultured with azithromycin-free DMEM served as a cell model of acute Ct infection (acute infection group).After 48-hour infection with Ct,azithromycin was removed,and infected Hela229 cells in the above 2 groups were successively cultured with DMEM for the resurgence of Ct.Immunofluorescence assay and electron microscopy were performed to verify the persistent Ct infection model.The Hela229 cells in the persistent infection group and acute infection group as well as uninfected Hela229 cells (control group) were treated with staurosporine (STS) for 4 hours to induce the apoptosis,and then cell apoptosis was detected by Hoechst 33258 staining,annexin V/propidium iodide staining and flow cytometry.Results After the treatment with azithromycin,atypical inclusions with aberrant reticulate bodies appeared in the Ct-infected cells.After removing azithromycin,cells were cultured until 96 hours after infection,and infectious elementary bodies reappeared in the Ct inclusions.After the treatment with STS,Hoechst staining showed that there was loose chromatin in the persistently infected cells,while chromatin condensation was observed in the uninfected cells.After 24-hour infection with Ct and 4-hour induction with STS,the apoptosis rate was significantly higher in the persistent infection group (45.567％ ± 2.631％) than in the acute infection group (38.567％ ± 1.701％,t =2.686,P =0.028),but significantly lower in the persistent infection group than in the uninfected group (69.800％ ± 2.835％,t =8.187,P ＜ 0.001).After 48-hour infection with Ct and 4-hour induction with STS,there was a significant difference in the apoptosis rate between the persistent infection group (46.700％ ± 5.257％) and acute infection group (61.767％ ± 1.815％,t =5.781,P ＜ 0.001),as well as between the persistent infection group and the uninfected group (68.667％ ± 3.156％,t =7.421,P ＜ 0.001).Conclusion This study showed that azithromycin-induced persistent Ct infection regulated the apoptosis of host cells,and this effect lasted 48 hours.
ABSTRACT
We expressed B cell epitopes of dengue virus envelope protein and NS1 protein in prokaryotic cells,and purified and evaluated for its serological activities.A recombinant multi-epitope chimeric gene named rE including eight B cell epitopes was connected by linker peptide (EAAAK)2 and cloned into prokaryotic expression vector pET-28a(+),and transformed into E.coli BL21(DE3) cells for expression under induction of IPTG.The expressed recombinant protein was purified with 6× His purification media,and identified by SDS-PAGE and Western blot,and its antigenicity was analyzed by using an indirect ELISA assay.The recombinant expression vector pET28a-rE was constructed and expressed in BL21 (DE3) successfully,but the recombinant proteins mainly appeared as inclusion bodies.The target protein was obtained with high purity through the purification of affinity chromatography.SDS-PAGE and Western blot analysis showed that the molecular weight of fusion protein was in the expected line.The established indirect ELISA has high accuracy.This recombinant peptide antigen expressed in E.coli has good potential for serum testing.
ABSTRACT
Objective To investigate the role of PPAR-γ in the gene expression of T-bet/GATA-3 in Jurkat T cells,and to explore the mechanisms underling this sensitizing effect of the change of TH cell subpopulation group.Methods Jurkat T cells were stimulated with PPAR-γ agonist pioglitazone.TH cell related cytokine IFN-γ and IL-10 was detected by ELISA,and the expression of transcription factors(T-bet and GATA-3)mRNA was detected by RT-PCR.To prove the PPAR-γ-dependent effect.the PPAR-γ-specific antagonist GW9662 was used.Results Stimulated with agonist PPAR-γpioglitazone.the concentration of IFN-γ and IL-10 and the expression of transcription factor T-bet and GATA-3 mRNA were both significantlY decreased in Jurkat T cells obviously,and these actions were dependent on the time and the concentrations of pioglitazone.Added with antagonist GW9662 at the same time,such inhibitory actions of IFN-γ and T-bet expression were recovered.but not IL-10 and GATA-3.Conclusion Pioglitazone can inhibite T cells proliferation and their secretion of cytokines.Pioglitazone can inhibit TH1 cells from secreting cytokines,and it is a PPAR-γ-dependent effect related to T-bet.The inhibition on TH2 is not a PPAR-γ-dependent effect and it is GATA-3 related.