Résumé
Objective To study the relationship between the expression of Mycobacterium tuberculosis small heat shock protein Hsp16.3 and the apoptosis of infected mouse alveolar macrophages.Methods The laboratory mice were infected with bacterial suspension of the international standard virulent strain of Mycobacterium tuberculosis H37Rv strains (H37Rv),Hsp16.3 gene deletion mutants of the international standard virulent of Mycobacterium tuberculosis H37Rv strains(△H37Rv),or sterile saline solution (normal control)by the tail vein. After successful replication of mouse infection model in each group,we cleaved the alveolus of each group of mice and collected lavage fluid to obtain alveolar macrophages of the infected mice at days 1 ,3 ,5 ,7 ,9 ,1 1 ,1 3 and 1 5 .Then the infection status of macrophages was observed with confocal laser scanning microscopy;flow cytometry was used to detect the apoptosis rate of alveolar macrophages of the infected mice;Caspase-3 and Bcl-2 expressions were examined by Western blot.Results The apoptosis rate of Hsp16.3 gene was higher in deletion strain (△H37Rv)group and H37Rv strains (H37Rv)group than in control group.The apoptosis rate of alveolar macrophages in △ H37Rv group gradually increased,peaked at day 7 ,and then gradually decreased.It was significantly higher in H3 7 Rv group than in H3 7 Rv strain group from day 1 to 7 and from day 1 3 to 1 5 (P<0 .0 5 ).Caspase-3 and Bcl-2 protein expressions in the macrophages of△H37Rv group and H37Rv group were higher than those of control group.Caspase-3 expression in the microphages of △H3 7 Rv group and H3 7 Rv group gradually increased from day 1 to 7 and peaked at day 7;it peaked again at day 13 in H37Rv group.However,Caspase-3 expression remained significantly higher in△H37Rv group than in H3 7 Rv group (P<0 .0 5 ).Bcl-2 expression in △H3 7 Rv group did not change much at the early stage of infection (P<0 .0 5 ),but gradually increased after day 9 .Bcl-2 expression in H3 7 Rv group did did not change much from day 1 to 7 (P<0.05),but gradually increased after day 7.However,it remained lower in△H37Rv group than in H37Rv group,especially after 7 days(P<0.05).Conclusion Mycobacterium tuberculosis small heat shock protein Hsp16.3 can inhibit the apoptosis of macrophages during the early and late stages of infection,and this inhibition may be achieved by inhibiting the expression of apoptotic protease Caspase-3 and promoting the expression of Bcl-2 protein.
Résumé
Objective To explore the correlation between Mycobacterium tuberculosis ( MTB ) PhoPR two-component system and drug resistance of MTB clinical isolates widespread in Xinjiang region by analyzing the expression of PhoP gene and PhoR gene among different isolates .Methods Total RNA of MTB was extracted from drug-susceptible strains , the strains only resistant to a single first-line anti-TB drugs (INH, RFP, SM and EB) and multidrug-resistant (MDR) strains, respectively.The purity of total RNA was checked by agarose gel electrophoresis .The expressions of PhoP gene and PhoR gene were quantified by using SYBR Green I qRT-PCR and the differences of their gene expression in different isolates were ana-lyzed.Results Compared with the drug-susceptible strains of MTB, the expression of PhoP gene was up-regulated for about 1.48 times in MTB strains resistant to RFP (RFP-MTB) and 2.74 times in MDR strain (P<0.05).Compared with MDR strain, the expressions of PhoP gene in the isolates resistant to INH (IN-HMTB), RFP (RFP-MTB), SM (SM-MTB) and EB (EB-MTB) were down-regulated for 0.70, 0.50, 0.25 and 0.21 times respectively.The expressions of PhoR gene were down-regulated for 0.36, 0.54, 0.35 and 0.19 times, respectively (P<0.05).The expressions of PhoR gene in the isolates of INH-MTB, RFP-MTB, SM-MTB and EB-MTB were up-regulated for 6.33, 4.56, 2.34, 1.85 and 9.06 times, respectively as compared with the drug-susceptible strains (P<0.05).Conclusion Significant differences of PhoR gene and PhoP gene expressions were observed among drug-susceptible strains , INH-MTB, RFP-MTB, SM-MTB, EB-MTB and MDR strains.Therefore, the Mycobacterium tuberculosis PhoPR two-component system is asso-ciated with the drug resistance of MTB strains prevalent in Xinjiang region .