结核分枝杆菌持留状态体内外模型的建立与检测

目的建立体内外结核分枝杆菌持留状态模型，检测在不同条件和化疗阶段中结核分枝杆菌持留菌，探讨其与化学治疗的关系。方法应用低氧培养、定量逆转录聚合酶链反应测定结核分枝杆菌异柠檬酸裂解酶（ICL）、小分子热休克蛋白（Acr）及85B蛋白的mRNA表达变化的方法检测体内外模型中结核分枝杆菌持留菌。结果体外及动物模型中均存在结核分枝杆菌低氧培养阳性，体外模型中结核分枝杆菌ICLmRNA和Acr蛋白mRNA在低氧条件下表达逐渐增加，4d时显著增加，其对数值分别为（5．3±0．9）和（6．4±1．6）拷贝／ml，而85BmRNA在有氧条件下明显增加，10d时的对数值为（6．1±0．9）拷贝／ml，在低氧条件下无明显变化。在小鼠感染与治疗模型中，ICLmRNA在感染的2周和4周均有表达，在治疗4周后下降；AermRNA在感染4周及治疗4周不表达或表达很少，治疗8和10周表达量增加，10周的对数值为（6．2±1．7）拷贝／ml，治疗12周直至停药后4周仍有表达，停药4周的对数值为（3．0±1．6）拷贝／ml；而85B蛋白mRNA则在治疗前高表达，对数值为（6．4±1．1）拷贝／ml，随着治疗时间延长而逐渐降低，治疗12周时测定值为阴性。结论成功建立了结核分枝杆菌持留状态的体外及动物模型，ICLmRNA、Acr蛋白mRNA高表达可作为持留菌存在的标志，应用液体低氧培养并联合mRNA检测有可能检测到结核分枝杆菌持留菌。

A study on in vitro and in vivo models of Mycobacterium tuberculosis persistence

OBJECTIVE: To establish in vivo and in vitro models of persistent Mycobacterium tuberculosis infection, and therefore to study the persisters in different conditions and periods of chemotherapy, and to explore the relationship between the persisters and chemotherapy. METHODS: The persisters in the two models were examined by culturing Mycobacterium tuberculosis in oxgen-starved condition and determining the mRNA expression of isocitrate lyase (ICL), alpha-crystallin chaperone (Acr) and 85B through quantitative PCR. RESULTS: The bacteria which could be cultured in oxgen-starved condition were discovered in both models. The mRNA expression of ICL and Acr increased gradually and dramatically after culture for 4 days in the in vitro model, their values being (5.3 +/- 0.9) and (6.4 +/- 1.6) log copy/ml respectively. While the mRNA expression of 85B showed no significant change in oxgen-starved condition, it increased significantly in the standard condition, the values being (6.1 +/- 0.9) log copy/ml at 10th day. In the mice infected with Mycobacterium tuberculosis, the mRNA expression of ICL was detected in 2 and 4 weeks post-infection, and decreased 4 weeks after treatment. The Acr mRNA showed no or very low expression 4 weeks post-infection or 4 weeks after treatment, but it increased significantly 8 and 10 weeks after treatment, with a value of (6.2 +/- 1.7) log copy/ml at 10 weeks, and its expression was still detected 12 weeks after treatment and 4 weeks after the cessation of treatment [(3.0 +/- 1.6) log copy/ml]. The expression of 85B mRNA was high before the treatment [(6.4 +/- 1.1) log copy/ml], and decreased gradually during the therapy. CONCLUSION: The models of in vitro and in vivo persistence of Mycobacterium tuberculosis were established. ICL and Acr mRNAs were highly expressed, which may be the markers of persisters. Persisters can be detected by culture in oxgen-starved conditions and the measurement of mRNA expression.