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目的探讨10-23脱氧核酶(deoxyribozyme,DRZ)抑制结核分枝杆菌异柠檬酸裂合酶(isocitrate lyase,ICL)的表达及其在抗结核分枝杆菌感染中的作用。方法根据计算机模拟的结核分枝杆菌ICL mRNA的二级结构设计合成5条ICL特异的10-23 DRZ(DZ1～DZ5),在无细胞反应体系中鉴定其对结核分枝杆菌ICL mRNA的切割活性后,在不同条件下用DZ4对结核分枝杆菌进行处理,于处理后不同时间检测ICL酶活性变化,间接酶联免疫吸附测定法(ELISA)检测ICL的表达。用异烟肼单独处理及DZ4与异烟肼联合处理后的结核分枝杆菌感染人巨噬细胞系THP-1细胞,于感染后不同时间用Ziehl-Heelson染色法和结核分枝杆菌培养计数的方法,观察10-23DRZ对结核分枝杆菌感染巨噬细胞的影响,同时取经异烟肼单独处理及DZA与异烟肼联合处理后的结核分枝杆菌接种于Middle Brook 7H10(M7H10)培养基,观察10-23DRZ对在巨噬细胞外生长的结核分枝杆菌的影响。结果DZ1、DZ3、DZ4和DZ5在无细胞反应体系中可对ICL mRNA进行有效切割,且其活性具有高度特异性。5μmol／L DZ4与异烟肼联合应用时可显著抑制结核分枝杆菌ICL的表达,与单用相应浓度的异烟肼比较,处理1、2、3周后结核分枝杆菌ICL酶活性分别下降34．9％、46．6％、46．7％(异烟肼10μg／L)或21．9％、33．48、36．9％(异烟肼5μg/L)。DZ4与异烟肼联合处理后的结核分枝杆菌在巨噬细胞内存活的能力显著下降,在感染后4 d和7 d时,THP-1细胞内的荷菌量分别由单用相应浓度异烟肼处理对照组的126．5×104 CFU、307．5×104 CFU(异烟肼10μg／L)和133．0×104 CFU、325．4×104 CFU(异烟肼5μg／L)下降至54．6×104 CFU、114．3×104 CFU和71．7×104 CFU、174．4×104 CFU。10-23DRZ对结核分枝杆菌在M7H10培养基中生长无明显影响。结论异烟肼可有效促进10-23DRZ进入结核分枝杆菌;10-23DRZ与亚抑菌浓度的异烟肼联用可有效抑制结核分枝杆菌ICL的表达,降低其在巨噬细胞中的存活能力。
Objective To investigate the inhibitory effect of 10-23 deoxyribozyme (DRZ) on the expression of isocitrate lyase (ICL) in Mycobacterium tuberculosis and its role in anti-Mycobacterium tuberculosis infection. Methods Five ICL specific 10-23 DRZ (DZ1 ~ DZ5) were designed and synthesized according to the secondary structure of ICL mRNA of Mycobacterium tuberculosis. The cleavage activity of Mycobacterium tuberculosis ICL mRNA in cell-free reaction system Mycobacterium tuberculosis was treated with DZ4 under different conditions, ICL enzyme activity was detected at different times after treatment, and ICL expression was detected by indirect enzyme-linked immunosorbent assay (ELISA). Mycobacterium tuberculosis infected with isoniazid and DZ4 combined with isoniazid were used to infect human macrophage THP-1 cells, which were counted by Ziehl-Heelson staining and Mycobacterium tuberculosis culture at different time after infection Methods The effect of 10-23 DRZ on Mycobacterium tuberculosis infection was observed. At the same time, Mycobacterium tuberculosis treated with isoniazid alone and combined with isoniazid (DZA) was inoculated into Middle Brook 7H10 (M7H10) The effect of 10-23DRZ on Mycobacterium tuberculosis grown outside macrophages was observed. Results DZ1, DZ3, DZ4 and DZ5 efficiently cleave ICL mRNA in a cell-free reaction system, and its activity is highly specific. The combination of 5μmol / L DZ4 and isoniazid significantly inhibited the expression of Mycobacterium tuberculosis ICL, compared with the corresponding concentration of isoniazid alone, the activity of ICL of Mycobacterium tuberculosis decreased after 1, 2, and 3 weeks of treatment 34.9%, 46.6%, 46.7% (isoniazid 10 μg / L) or 21.9%, 33.48, 36.9% (Isoniazid 5 μg / L). The ability of Mycobacterium tuberculosis to survive in macrophages after treatment with DZ4 and isoniazid was significantly decreased. At 4 d and 7 d after infection, the amount of bacteria in THP-1 cells was increased from 126.5 × 104 CFU, 307.5 × 104 CFU (isoniazid 10 μg / L) and 133.0 × 104 CFU, 325.4 × 104 CFU (isoniazid 5 μg / L) of the control group were decreased To 54.6 × 104 CFU, 114.3 × 104 CFU and 71.7 × 104 CFU, 174.4 × 104 CFU. 10-23DRZ had no significant effect on the growth of M. tuberculosis in M7H10 medium. Conclusions Isoniazid can effectively promote the entry of 10-23DRZ into M. tuberculosis. In combination with isoniazid at a concentration of 10-23DRZ, the expression of ICL of Mycobacterium tuberculosis can be effectively inhibited and its survival in macrophages can be reduced ability.