婴幼与成年小鼠腹腔巨噬细胞吞噬体成熟化的差异研究

Study on the Difference Between Adult and Infant Mouse in Maturation of Pertinent Macrophage

目的：通过动物实验，研究婴幼与成年小鼠腹腔巨噬细胞吞噬体成熟化异同。方法1.采用腹腔灌洗法分离和纯化野生型成年鼠（10周龄）及野生型和TLR2（Toll样受体2）-/-型婴幼小鼠（2周龄）腹腔巨噬细胞，按1：1（细胞数：细菌数）比例分别与双标记有carboxyfluorescein-SE（pH-敏感性荧光探针）和carboxytetramethylrhodamine-SE（pH-非敏感性荧光探针）的革兰氏阳性金葡球菌共同孵育，细胞流式仪测定孵育5、10、15、30 min巨噬细胞吞噬体内pH（酸化）值的变化；2.分离野生型成年鼠（10周龄）及野生型和TLR2-/-型婴幼小鼠（2周龄）腹腔巨噬细胞，巨噬细胞与革兰氏阳性金葡球菌在加入（胞外细菌杀灭）或不加入（总的细菌杀灭）Cytochalasin后共同孵育不同时间，溶解细胞计数细菌菌落生长数，胞内细菌杀灭=总的细菌杀灭－胞外细菌杀灭，重复3次。结果1.与野生型的成年小鼠相比较，婴幼小鼠的巨噬细胞吞噬体的酸化延迟且不足；TLR2-/-型婴幼小鼠吞噬体的酸化进一步受损。2.与野生型的成年小鼠相比较，婴幼小鼠的巨噬细胞胞内杀菌力明显下降；TLR2-/-型婴幼小鼠的巨噬细胞胞内杀菌力严重受损。结论1.与野生型的成年小鼠相比较，婴幼小鼠吞噬体成熟存在缺陷。2.TLR2在调节吞噬体成熟抵御革兰氏阳性细菌感染中发挥重要作用。

Objective To investigate the difference between adult and infant mouse in maturation of pertinent macrophage and clearance of bacteria.Methods1.Isolation of adult and infant macrophage or adult and infant TLR2-/-macrophage by Celiac lavage and inoculation with carboxyfluorescein-SE labeled or carboxytetramethylrhodamine-SE labeled Staphylococcus aureus at the ratio of 1：10, then FACS evaluate the pH value of endosome in macrophage at the time of 5, 10, 15 and 30 minutes. 2.Isolation of adult and infant macrophage or adult and infant TLR2-/- macrophage by Celiac lavage and inoculation with Cytochalasin, then the cell was lysed and the bacteria clones were counted. Clearance of bacteria in macrophage = total bacteria clearance- bacteria clearance out of macrophage.Results 1.The time of endosome acidifi cation in macrophage of infant mice was longer than macrophage of adult mice.The ability of endosome acidifi cation in macrophage of TLR2-/- infant mice was weaker than macrophage of wild infant mice. 2.The ability of infant mice macrophage in clearance of bacteria decreased compared with adult mice, the ability of macrophage in clearance of bacteria was more impaired in TLR2-/- infant mice.Conclusion1.The maturation of endosome in infant mice macrophage is defected compared with adult mice. 2.The role of TLR2 in promoting the maturation of endosome and resisting gram＋ bacteria infection is very important.