目的 探讨鼠伤寒沙门菌感染早期巨噬细胞内过氧化物酶体及诱导型一氧化氮合酶(iNOS)的作用.方法用pTassC-GFP质粒转染鼠巨噬细胞RAW 264.7,以观察标记了绿色荧光蛋白的靶向沙门菌分泌的SpiC蛋白(TassC)在胞内的存在形式.用pDsRed2-Perxi和pTassC-GFP质粒共转染RAW 264.7细胞,将阳性细胞命名为RAW-DT,用pDsRed2-Perxi质粒转染RAW 264.7细胞,阳性细胞命名为RAW-D.采用Alexa Fluor 350对结合单抗的鼠伤寒沙门菌显色,用Alexa Fluor 488对iNOS进行显色,并在荧光显微镜下观察.用鼠伤寒沙门菌感染RAW-DT细胞,以观察鼠伤寒沙门菌与TassC和过氧化物酶体的关系；用鼠伤寒沙门菌感染RAW-D细胞,以观察iNOS与过氧化物酶体和鼠伤寒沙门菌的关系.结果 免疫荧光观察显示,呈现绿色荧光的TassC-GFP有膜性结构,可与标记了红色荧光的过氧化物酶体共定位；感染1h的RAW-DT胞内鼠伤寒沙门菌吞噬泡可招募TassCGFP和过氧化物酶体；感染1h的RAW-D胞内鼠伤寒沙门菌吞噬泡可与iNOS和过氧化物酶体共定位；在1h的观察期间,一旦鼠伤寒沙门菌进入巨噬细胞,膜性小泡(SCV)周围即可募集较多过氧化物酶体.结论 野生型鼠伤寒沙门菌可诱导巨噬细胞产生iNOS,iNOS和TassC均可与过氧化物酶体结合,可能具有一定的杀菌作用.%Objective To investigation on the early carrying inducible nitric oxide synthase for peroxisomes to Salmonella typhimurium during the bacteria infection mouse macrophages. Methods RAW 264. 7 macrophages were transfected with pTassC-GFP plasmids to analysis the existence form of green fluorescent protein labeled target for Salmonella secreted protein SpiC( TassC ) protein in the celL The interaction between the fusion protein TassC-GFP and peroxisomes were analyzed by co-transfection of pTassC-GFP and pDsRed2-Perxi (labels peroxisomes red) plasmids to RAW264. 7 macrophages, the positive transfected cells named RAW-DT. RAW-D cells were named by transfecting RAW 264. 7 with pDsRed2-Perxi plasmids. S typhimurium was detected with mono-antibody and visualized with Alexa Fluor 350 conjugated donkey anti-mouse antibodies. Inducible nitric oxide synthase (iNOS or NOS2) was detected with iNOS-antibody and visualized with Alexa Fluor 488 conjugated goat anti-rabbit antibodies. S. Typhimurium were used to infect the RAW-DT cells to analyze the interaction among bacteria, TassC-GFPs and peroxisomes. The RAW-D cells were infected with S. Typhimurium 1h to analyze the interaction among bacteria, iNOS and peroxisomes. Results TassC vesicles co-localized with peroxisomes when RAW264.7 macrophages were co-transfected with pTassC-GFP and pDsRed2-Perxi plasmids. It was determined by a three-dimensional (xyz) fluorescence microscopy that the recruitment or overlapping of TassC-GFP and pemxiomes to the Salmonella-containing vacuoles (SCV) after infection of RAW-DT macrophages with S typhimurium for 1h. The SCVs also could co-localized with peroxisomes and iNOS after infection of RAW-D cells with S typhimurium for 1h. Upon entry of Salmonella, peroxisomes were recruited to the Salmonella-containing vesicles and remain aggregated around the SCV for the duration of the 60 minutes observation time. Conclusion These findings indicated that, wild type S typhimuriwn could induce iNOS production in RAW264. 7 macrophages, both of iNOS and TassC could bind with peroxisomes. Host cell TassC has been identified as the host cell target for S typhimuriwn secreted SpiC The findings suggest that if SpiC was still attached with bacteria, TassC would mediated peroxisomes binding with iNOS involved in the early germicidal phase of wild type S. Typhimurium infection. If SpiC was secreted to the cytosol, the bactericidal effection of iNOS would be decrease.
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