中文摘要：

目的明确结核分枝杆菌（MTB）异柠檬酸裂合酶（ICL）对耻垢分枝杆菌（MS）在巨噬细胞内存活的影响，并初步探讨其可能的机制。方法构建MTB—icl基因的穿梭表达质粒pUV15-icl，电转化法导入MS中后检测MTB—ICL在MS中的表达。分别用rMS—pUV15-icl和rMS-pUV15感染鼠巨噬细胞系RAW264．7细胞，于感染一定时间后测定细胞内存活的细菌数，评价MTB．ICL对MS在巨噬细胞内存活的影响。分别留取上述两组巨噬细胞的培养上清液，检测干扰素γ（IFN-γ）和一氧化氮（NO）的水平，同时采用原位TUNEL技术检测两组巨噬细胞的凋亡水平，探讨MTB-ICL对MS在巨噬细胞内存活影响的可能机制。结果RT—PCR、Western印迹和荧光显微镜检测结果证实MTB-ICL可在MS中有效表达。与rMS-pUV15比较，rMS—pUV15-icl在巨噬细胞内的存活率明显高（P〈0．01），感染后24h和48h，其菌落数分别为（32．78±2．90）×10^3和（23．33±2．34）×10^3，而rMS—pUV15菌落数分别为（14．67±2．45）×10^3和（2．28±0．25）×10^3。MTB-ICL对巨噬细胞产生IFN-γ和NO没有明显影响。原位凋亡检测结果显示，与rMS—pUV15感染组比较，rMS-pUV15-icl感染组巨噬细胞的凋亡水平明显下降。结论MTB-ICL可促进MS在巨噬细胞内的存活，其机制之一可能是抑制宿主巨噬细胞的凋亡。

英文摘要：

Objective To investigate the effects of isocitrate lyase （ICL） from Mycobacterium tuberculosis （MTB-icl） on the survival of Mycobacterium smegmatis （MS） in macrophage and illuminate the possible mechanisms. Methods MTB-icl gene was amplified by PCR and cloned into Ecoli-Mycobacterium shuttle plasmid pUV15 to obtain recombinant shuttle plasmid pUV15-icl expressing ICL-GFP. The recombinant shuttle plasmid pUV15-icl and blank plasmid pUV15 were induced into MS of the line 1-2c so as to obtain rMS-pUV15-icl and rMS-pUV15. Shuttle plasmid rMS-pUV15-IG expressing ICL-green fluorescent protein （GFP） was constructed, rMS-pUV15-IG and MS 1-2c were used to infect the murine macrophages of the line RAW264.7, fluorescence microscopy was used to observe the expression of ICL- GFP. The expression of ICL in the MS swallowed by the macrophages was verified by RT-PCR and Western blotting. Another macrophages RAW264.7 were cultured and infected with rMS-pUV15-icl and rMS-pUV15 respectively. 0, 24, and 48 hours later macrophages were collected and the number of MS colonies was calculated. The interferon （IFN）-γ and nitrogen oxide （NO） concentrations in the culture supernatants of macrophages infected by rMS-pUV15-icl and rMS-pUV15 were measured by ELISA and Griess assay respectively. The apoptotic rate of the macrophages was assayed by in situ TUNEL technique. Results Western blotting showed that the MTB ICL protein expression of the rMS-pUV15-icl was significantly higher than that of rMS-pUV15. Fluorescence microscopy showed green fluorescence in the RAW264. 7 cells infected with rMS-pUV15-IG, but not ion the RAW264.7 cells infected with MS 1-2c. 0 h after the infection of the macrophages there was not significant difference in the MS amount in the macrophages between the rMS-pUV15-isl and rMS-pUV15 groups, and 24 h and 48 h later the MS amounts of the rMS-pUV15-icl group were （32.78 ±2.90） ×10^3 and （23.33 ±2.34）×10^3 respectively, both significantly higher than those of the rMS-pUV15 group [