雷帕霉素对伤寒沙门菌质粒pR_(ST98)与巨噬细胞相互作用的影响

Effect of rapamycin on the interaction between Salmonella typhi plasmid pR_(ST98) and macrophages

研究自噬诱导剂雷帕霉素(RAPA)对伤寒沙门菌质粒pRST98与巨噬细胞相互作用的影响。用加入RAPA和未加药的RPMI1640培养巨噬细胞J774A.1后,分别与携带pRST98的伤寒沙门菌ST8,无质粒的伤寒沙门菌ST10和鼠伤寒沙门菌标准毒株SR-11共作用,观察细胞自噬和凋亡情况。结果显示,MDC染色后2h和4h细胞自噬泡的数量SR-11组<ST8组<ST10组,RAPA作用后各组均有所增加;自噬蛋白Beclin-1表达量在2hSR-11组<ST8组<ST10组,用药后0点即表现上述规律;sub-G1凋亡峰SR-11组>ST8组>ST10组,RAPA干预后ST8组凋亡率下降,ST10组凋亡率升高(P<0.05);电镜下2～4h可见部分细菌被双层膜包裹,8h起可见凋亡改变。RAPA作用后,ST8组细胞破坏减轻,ST10组加重。提示伤寒沙门菌质粒pRST98与细胞自噬有关,在早期可抑制自噬,其诱导的巨噬细胞凋亡可被RAPA减轻,而RAPA能使ST10诱导的巨噬细胞凋亡增强。

To study the effect of autophagy inducer rapamycin （RAPA） on the interaction between Salmonella typhi plasmid pRST98 and macrophage,strain J774A.1 of macrophages was cultivated in RPMI 1640 medium with RAPA or without overnight,and then Salmonella typhi carrying pRST98,ST8and Salmonella typhi without pRST98,ST10 and Salmonella typhimurium strain SR-11 carrying a 100 kb virulence plasmid were added. The situation of autophagy and apoptosis of cells was observed. It was showed that the number of autophagy vacuoles at 2 and 4 hrs after staining with MDC was SR-11 groupST8groupST10 group,and the number increased after the treatment with RAPA. The expression of Beclin-1 at 2 hr was SR-11 groupST8 groupST10 group,and the tendency appeared at 0 h time point after RAPA treatment;the apoptosis rate displayed a tendency of SR-11 groupST8 groupST10 group. After interference with RAPA the apoptosis rate of ST8decreased,meanwhile ST10 increased （P0. 05）. The observation by transmission electron microscope showed that bacteria were surrounded by a bilayer membrane at 2～4 hrs,and apoptosis was observed from 8 hrs. while the infection of ST8 lessened,the infection of ST10 was aggratated with RAPA treatment. All these results suggest that pRST98 can inhibit autophagy at an early stage and RAPA can reduce the apoptosis induced by Salmonella typhi carrying pRST98,but enhance the apoptosis caused by Salmonella typhi without pRST98.