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目的:构建颗粒溶素(GNLY)真核表达质粒,并将重组质粒肌肉注射结核分枝杆菌感染小鼠观察其保护效应。方法:将人颗粒溶素cDNA插入pcDNA3.1(-)构建真核表达质粒。18只感染结核分枝杆菌H37Rv株的BALB/c小鼠随机分为生理盐水组、pcDNA3.1(-)质粒组和GNLY质粒组。感染第1天和第6天各组小鼠分别从后腿股四头肌注射生理盐水、pcDNA3.1(-)质粒、GNLY质粒,免疫2次。感染4周后,处死各组小鼠,检测器官活菌数[lgCFU/(g.ml)],脾重指数(WI),并观察肺、脾组织病理情况及抗酸染色情况。结果:GNLY质粒组肺组织活菌数为6.688±0.003,显著低于生理盐水组(7.821±0.090,P<0.01)和pcDNA3.1(-)质粒组(7.848±0.017,P<0.01);GNLY质粒组脾组织活菌数为7.140±0.034,显著低于两对照组(7.805±0.080,7.858±0.043,P<0.01)。GNLY质粒组脾重指数(2.575±0.649)也显著低于生理盐水组(4.580±0.967,P<0.01)和pcDNA3.1(-)质粒组(3.885±0.228,P<0.01)。GNLY质粒组肺脏病理学损害明显轻于两对照组,组织抗酸染色细菌荷载数量明显较两对照组低。结论:GNLY对结核分枝杆菌感染小鼠具有一定的保护作用。
OBJECTIVE: To construct the eukaryotic expression plasmid of granulocyte lysozyme (GNLY) and infect mice with M. tuberculosis intramuscular injection to observe its protective effect. Methods: Human granulysin cDNA was inserted into pcDNA3.1 (-) to construct eukaryotic expression plasmid. Eighteen BALB / c mice infected with Mycobacterium tuberculosis H37Rv were randomly divided into saline group, pcDNA3.1 (-) plasmid group and GNLY plasmid group. On day 1 and day 6 of infection, mice in each group were injected with normal saline, pcDNA3.1 (-) plasmid and GNLY plasmid respectively from the quadriceps femoris and immunized twice. After 4 weeks of infection, the mice in each group were sacrificed and viable cells of the organ [lgCFU / (g.ml)] and spleen weight index (WI) were detected. Pathological changes and acid-fast staining of lung and spleen were observed. Results: The number of viable cells in lungs of GNLY plasmid group was 6.688 ± 0.003, which was significantly lower than that of the control group (7.821 ± 0.090, P <0.01) and pcDNA3.1 (-) plasmid group (7.848 ± 0.017, P <0.01) The number of viable cells in the plasmid group was 7.140 ± 0.034, which was significantly lower than that in the two control groups (7.805 ± 0.080, 7.858 ± 0.043, P <0.01). The spleen mass index of GNLY plasmid group (2.575 ± 0.649) was also significantly lower than that of the normal saline group (4.580 ± 0.967, P <0.01) and pcDNA3.1 (-) plasmid group (3.885 ± 0.228, P <0.01). The pathological lung damage of GNLY plasmid group was lighter than that of the two control groups, and the number of bacterial load of acid-fast staining was significantly lower than that of the two control groups. Conclusion: GNLY can protect Mycobacterium tuberculosis mice.