采用结晶紫染色检测结核分枝杆菌生物被膜的形成能力,实时荧光定量PCR(qRT-PCR)检测生物被膜态的不同结核分枝杆菌Rv3519表达水平,分析Rv3519基因表达与结核分枝杆菌生物被膜形成能力的关系.结果表明:临床分离株产膜能力较H37Rv明显偏低,差异显著(P＜0.01);临床分离株之间生物被膜产量差异不显著(P＞0.05);生物被膜态的分离株较标准株Rv3519基因表达量偏低,差异显著(P＜0.01).说明结核分枝杆菌生物被膜形成能力与Rv3519基因表达水平相关,Rv3519基因可以作为结核分枝杆菌生物被膜形成能力的潜在分子诊断标志.

To explore a reliable gene knockout method in Mycobacteria,and research the effect of MSMEG_6935deletion mutant on biofilm production in Mycobacteriumsmegmatis.The related gene knockout is important for Mycobacteria tuberculosis study.The upstream,downstream and kan flanking DNA fragments of target gene were fused by PCR,and then cloned into the shuttle vectorpPR27,the recombinant plasmid was introduced into mc2155 strain.In addition,we research the effect of MSMEG_6935deletion mutant on growth and biofilm production in Mycobacterium smegatis.The MSMEG_6935gene deletion strains were successfully constructed and the MSMEG_6935gene knockout has no effect on the growth curve of mc2155,but it has a great effect on biofilm production.

为了研究产单核细胞增生性李斯特菌(Lm)能否引起巨噬细胞产生胞外陷阱,并初步探讨巨噬细胞胞外陷阱(METs)对其杀伤作用.本文通过荧光显微镜、菌落计数和免疫印记等技术分析单增李斯特菌侵袭的2种巨噬细胞胞外陷阱形态、产量及抑菌效果.结果显示,小鼠巨噬细胞系RAW 264.7以及人巨噬细胞系Thp 1均可在单增李斯特菌的诱导下产生胞外陷阱;Lm在侵袭巨噬细胞15 min后已有METs产生,1～3 h产量明显增大,然后随时间的推移逐渐减弱;METs可以有效抑制Lm的生长.另外,本试验还发现Lm诱导胞外陷阱的产生与细胞外信号调节激酶相关.

To study the inhibitory activity of sodium houttuyfonate on biofilm of Staphylococcus aureus( S. aureus),a microdilution method was used measure the minimum inhibitory concentration( MIC) and the minimum bactericidal concentration( MBC) against S. aureus ATCC 25923and clinical isolates UAMS-1 in suspension. An agar plate method was used to determine the minimum biofilm inhibitory concentration( MBIC) and minimum biofilm bactericidal concentration( MBBC). The ability of sodium houttuyfonate which eliminated the biofilm was determined using confocal laser scanning microscopy,and the effect of sodium houttuyfonate on the secretion of S. aureus virulence factors was also determined using Western blot analysis. The results showed that sodium houttuyfonate had inhibitory activity against S. aureus in suspension with MIC and MBC values ranging from 16 to 64 μg /mL,and it had no obvious inhibitory effect on the mature biofilms with MBIC and MBBC values larger than 1 024 μg /mL. The sub-inhibitory concentration of sodium houttuyfonate had an inhibitory effect on the early stages of biofilm formation,which could significantly inhibit the secretion of α-haemolysin,enterotoxin A and B in dose-dependent manner. The results indicate that sodium houttuyfonate has good inhibitory activity against S. aureus in suspension and the early stages of biofilm formation.

为了研究柴胡皂甙D能否诱导HeLa细胞发生自噬,并初步探讨其机制,试验采用激光共聚焦显微镜、免疫印记等技术评价经柴胡皂甙D处理后的HeLa细胞的自噬水平.结果表明:经柴胡皂甙D处理后,激光共聚焦显微镜观察到GFP-LC3斑点显著增加;柴胡皂甙D能显著增加LC3-Ⅱ的表达;但自噬关键蛋白mTOR没有变化.说明柴胡皂甙D能诱导HeLa细胞发生自噬,并通过非mTOR依赖途径.

Phenol-soluble modulin(PSM) is a newly identified secreted virulence factor of Staphylococcus aureus.To study the effect of the subunits of PSM-α,the genes of PSM-α1,PSM-α2,PSM-α3 and PSM-α4 were cloned and the recombinant plasmid pGEX-PSM-α was constructed for expression in E.coli.The highly expressed soluble protein were purified and digested with thrombin to remove the GST-tag.Compared with negative control,the expression of human neutrophils IL-8 was increased significantly after co-culture with the purified PSM-α detected by ELISA kit.The current study demonstrated that PSM-α was able to cause disease by stimulating human neutrophils to release inflammatory factor.