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为探讨伤寒沙门菌质粒pRST98上毒力基因与小鼠巨噬细胞J774A.1凋亡的关系及可能机制,将鼠伤寒沙门菌标准毒株SR-11、低毒株RIA和pRST98经接合转移导入RIA的接合子pRST98/RIA,体外分别与小鼠巨噬细胞J774A.1共培养,Giemsa染色法确定细菌对细胞的感染复数(multiplicity,MOI)。于0、1、3、6、12和24h用Annexin V-FITC试剂盒检测细菌感染后细胞凋亡情况;用透射电镜观察细胞的变化;用JC-1染色法、激光扫描细胞仪及激光共聚焦显微镜检测线粒体膜电位(△Ψm)的改变。结果显示,细菌与J774A.1的最佳MOI为100∶1。从感染后3h起,J774A.1的凋亡率表现为SR-11组>pRST98/RIA组>RIA组(P<0.05);电镜观察的结果显示3h起各组细胞内可见染色质边聚等凋亡改变,12h见凋亡小体,以SR-11组最为明显,pRST98/RIA组多于RIA组,24h各组细胞多为坏死改变;感染后J774A.1线粒体△Ψm下降的百分率表现为SR-11组、pRST98/RIA组和RIA组依次降低。以上结果表明携带质粒pRST98的宿主菌毒力增强,感染后使巨噬细胞的凋亡率随时间延长而增加。
To explore the relationship between the virulence gene of Salmonella typhi plasmid pRST98 and the apoptosis of mouse macrophage J774A.1 and the possible mechanism, Salmonella typhimurium standard strain SR-11 and low strain RIA and pRST98 were transferred by conjugation The RIA conjugate, pRST98 / RIA, was co-cultured with mouse macrophage J774A.1 in vitro, and the multiplicity of infection (MOI) of the cells was determined by Giemsa staining. Annexin V-FITC kit was used to detect the apoptosis after bacterial infection at 0, 1, 3, 6, 12 and 24h. The changes of cell apoptosis were observed by transmission electron microscope. The cell apoptosis was detected by JC-1 staining, laser scanning cytometry and laser Changes of mitochondrial membrane potential (△ Ψm) were detected by focusing microscope. The results showed that the best MOI for bacteria and J774A.1 was 100: 1. From 3 h after infection, the apoptotic rate of J774A.1 was significantly higher in SR-11 group than in pRST98 / RIA group and RIA group (P <0.05). The results of electron microscopy showed that the apoptosis rate of J774A.1 The apoptotic bodies were observed at 12h, the most obvious was in SR-11 group, the more in pRST98 / RIA group than in RIA group, the most of them were necrotic cells in 24h group. The percentage of △ Ψm in J774A.1 decreased SR-11 group, pRST98 / RIA group and RIA group in turn decreased. The above results showed that host strain carrying plasmid pRST98 enhanced virulence, and the rate of apoptosis of macrophages increased with time after infection.