利用噬菌体杀灭巨噬细胞内耻垢分枝杆菌的实验研究

目的探讨噬菌体能否杀灭巨噬细胞内耻垢分枝杆菌,并成为减少细胞内活菌量的手段。方法将体外培养的已感染耻垢分枝杆菌的小鼠腹腔巨噬细胞随机分为4组生理盐水组,噬菌体高剂量组、中剂量组和低剂量组。分别加入生理盐水0.1ml、10#噬菌体2.1×107噬菌斑形成单位(PFU)、2.1×106PFU和2.1×105PFU。通过洗涤去除细胞外的噬菌体与耻垢分枝杆菌,观察加入噬菌体24h后巨噬细胞内耻垢分枝杆菌活菌数量变化情况。并在电子显微镜下观察巨噬细胞吞噬噬菌体前后胞内改变情况。结果24h后生理盐水组巨噬细胞内活菌数的对数值为5.74±0.18,噬菌体高、中、低剂量组的活菌数对数值分别为4.77±0.08、4.97±0.17、5.33±0.13。生理盐水组的活菌数高于噬菌体高、中、低剂量组,其中生理盐水组与噬菌体高、中剂量组之间比较差异有统计学意义(P<0.01)。高、中剂量组间差异无统计学意义(P>0.05),高、低剂量组间差异有统计学意义(P<0.01)。电子显微镜显示噬菌体能感染细胞内的耻垢分枝杆菌,合成子代噬菌体。结论感染了分枝杆菌的巨噬细胞可以同时吞噬10#噬菌体,进入细胞内的噬菌体能感染裂解胞内分枝杆菌使活菌量减少,使噬菌体治疗分枝杆菌感染成为可能。

The in vitro killing of intracellular Mycobacterium smegmatis by Mycobacteriophage

OBJECTIVE: To study the effect of Mycobacteriophage on the lysis of intracellular Mycobacterium smegmatis. METHODS: Peritoneal macrophages from BALB/C mice were incubated with Mycobacterium smegmatis for 4 h, and the extracellular bacteria were removed. Then the infected macrophages were treated for 2 h with normal saline, or different doses of Mycobacteriophages (2.1 x 10(7) PFU, 2.1 x 10(6) PFU, and 2.1 x 10(5) PFU, respectively), all in a volume of 0.1 ml, and then the extracellular phages and Mycobacterium smegmatis were removed by washing. After incubation for 24 h, the number of viable intracellular bacteria was determined. The intracellular changes after infection of host bacteria by bacteriophages in the macrophages were observed by electron microscopy. RESULTS: The logarithm 10 of viable intracellular bacteria unit was 5.74 +/- 0.18 in the saline group, 4.77 +/- 0.08 in the high dose phage group (P < 0.01), 4.97 +/- 0.17 in the moderate dose phage group (P < 0.01), and 5.33 +/- 0.13 in the low dose phage group (P > 0.05). Electron microscopy confirmed the infection of intracellular bacteria by the bacteriophages and the production of filial bacteriophages. CONCLUSIONS: Mycobacteriophages phagocytosed by macrophages are capable of killing the infected mycobacteria. The result suggests that the use of Mycobacteriophages is a potentially novel strategy in the treatment of intracellular bacterial infection.