缺氧模型和多因素模型构建结核休眠菌模型比较研究  

Comparative study of two in vitro dormant models for Mycobacterium tuberculosis

作　　者：江莉莎[1,2] 姚义勇[1] 张莉[1] 柳岩[1] 郭述良[1] 

机构地区：[1]重庆医科大学附属第一医院呼吸与危重症医学科,重庆400016 [2]重庆医科大学附属第一医院第一分院,重庆400016

出　　处：《中国人兽共患病学报》2016年第4期327-331,共5页Chinese Journal of Zoonoses

基　　金：国家"十二五"重大专项课题(No.2012ZX10003009);国家临床重点专科专项经费(No.2012-649)联合资助~~

摘　　要：目的比较缺氧模型及多因素模型两种结核休眠菌建模方法的优劣。方法利用缺氧因素构建缺氧模型;利用低氧(5%O2)、高CO2(10%CO2)、弱酸(pH 5.0)及营养缺乏(10%7H9液体培养基)多种因素构建多因素模型。分别在第20d、30d时取样,用金胺O-尼罗红染色法染色,在共聚焦显微镜下观察结核菌脂质聚集及失去抗酸性的情况;用电镜观察结核菌细胞壁增厚情况;并检测其对利福平的耐药率。在建模30d时,将等量的两种模型分别转入新鲜7H9液体培养基中,观察并比较其复苏情况。结果建模20d时,两模型组细菌均出现脂质聚集及失去抗酸性,无明显差异。两模型组细菌均未出现明显细胞壁增厚。建模30d时,多因素模型组细菌几乎都表现出脂质聚集及失去抗酸性,而缺氧模型组仍有部分细菌未表现出脂质聚集及失去抗酸性。同时,多因素模型组在建模30d还出现了细胞壁增厚的细菌,缺氧模型组则无明显改变。多因素模型组细菌对利福平的耐药率明显高于缺氧模型组,且多因素模型组细菌复苏的菌量远小于缺氧模型组。结论多因素模型较缺氧模型能更快诱导结核菌进入休眠状态,且诱导出的结核休眠菌状态更稳定。To compare the oxygen-deficit model with the multiple-stress model,and understand their pros and cons,the oxygen-deficit model was constructed by applying gradually low oxygen.The multiple-stress model was developed by applying combined stress of low oxygen（5% O2）,high carbon dioxide（10% CO2）,faintly acid（pH 5.0）and lack of nutrient（10%7H9broth）.After both the models were constructed for 20 days and 30 days,respectively,the sample was collected.Loss of acid fastness and accumulation of lipid bodies was observed through confocal microscopy after dual staining of Mycobacterium tuberculosis（Mtb）with the combination Auramine-O and Nile Red.Cell wall thickness of Mtb was observed by electron microscope.And the drug-resistance rate to rifampicin（Rif）was also detected.At 30 th day,equivalent Mtb of two models was subcultured into fresh 7H9 broth to observe the auto-resuscitation.We discovered that Mtbof two models showed loss of acid fastness and accumulation of lipid bodies but no transformation of cell wall at 20 th day.Mtb of multiple-stress model almost showed loss of acid fastness and accumulation of lipid bodies at 30 th day.However,portions of Mtb did not show loss of acid fastness and accumulation of lipid bodies in oxygen-deficit model.At 30 th day,cell wall thickness of Mtb was observed in multiple-stress model but did not in oxygen-deficit model.The drug-resistance rate to rifampicin of multiple-stress model was obviously higher than that of oxygen-deficit model,and the amount of Mtbin multiple-stress model was smaller than that of oxygendeficit model after auto-resuscitation in fresh 7H9 broth.In Conclusion,Mtbcould develope dormant state more rapid in multiple-stress model,and its dormant state was more stable.

关 键 词：结核休眠菌 缺氧模型 多因素模型 比较 

分 类 号：R378[医药卫生—病原生物学]