【作者】 赵勇；

【导师】 韩文瑜；

【作者基本信息】 中国人民解放军军需大学 ， 预防兽医学， 2003， 硕士

【摘要】 结核分枝杆菌(Mycobacterium tuberculosis，MTB)是结核病的病原菌。全世界每年约有200余万人死于结核病。近年，由于耐药结核菌的出现，特别是多重耐药结核菌的出现以及人类免疫缺陷病毒／艾滋病的流行，使这一全球性的疾病卷土重来，其感染率、发病率和死亡率均居高不下。目前临床常用的一线抗结核药物主要为INH、RFP、SM、PZA和EMB，但均已出现耐药菌，使得药物治疗效果不断下降，而且耐药菌比例仍在不断上升。耐药结核菌使得临床治疗工作变得十分困难，更增加了结核病的危险性，引起世界各国对结核菌耐药问题的极度重视。本实验在对结核菌耐药性基因检测的基础上，筛选对耐药结核菌的耐药性有抑制作用或可增强一线抗结核药物作用效果的中草药，为耐药结核菌的防治提供实验依据。本实验通过BACTEC-TB460检测系统和MTT微量板法从临床分离出38株结核菌，并确定分离菌株的耐药谱，从中筛选到17株耐利福平结核菌，9株耐链霉素结核菌。根据结核菌耐利福平基因rpoB和耐链霉素基因rpsL的核酸序列设计合成引物，对耐药基因进行PCR扩增、克隆及核酸序列测定，分析其耐药基因突变，检测到3种耐利福平基因rpoB的突变，2种耐链霉素基因rpsL的突变，从分子水平确定耐药产生的原因。根据药性选择部分中草药通过煎煮、浸渍、蒸馏等方法制备成中草药粗提物。采用MTT微量板法检测所制备中草药粗提物的敏感性对耐利福平、耐链霉素的结核菌的最小抑菌浓度(MIC)，结果大部分中草药粗提物高浓度可抑菌或杀菌，低浓度无作用。将上述耐药菌在含药培养基上培养及传代，用MTT法检测其对RFP、SM的耐药性，通过分析菌株耐药性的变化，确定中草药对菌株耐药性的影响，获得2种中草药(黄连、百部)对细菌SM耐药性有抑制作用，同时还发现有5种中草药可以不同程度增强链霉素的作用效果；但未发现中草药对耐RFP菌耐药性有抑制作用。根据细菌耐药机制，对中草药抑制耐药性的作用机制进行分析，相对应地检测了中草药作用作用前后上述耐药菌的菌体形态，菌体蛋白图谱和SM耐药基因rpsL的耐药突变变化情况。结果表明，细菌菌体形态和菌体蛋白图谱无明显变化。SM耐药基因rpsL序列发生突变，突变点有4处。更多还原

【Abstract】 Mycobacterium tuberculosis (MTB) is the pathogenic bacterium of tuberculosis (TB). One-third of the global population is sure to be infected with bacteria of the Mycobacterium tuberculosis complex. More than 8 million new cases of tuberculosis occur annually leading to 2 million deaths. Nowadays, because of occurrence of drug resistance tuberculosis, especially multiple-drug resistance tuberculosis (MDR-TB) and outbreak in association with human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS), tuberculosis have became more dangerous. Therapeutic efficacy of the anti-tuberculosis drugs commonly used in clinical, such as INH RFP SM PZA and EMB, is on descent. There have been ample warnings that MDR-TB will continue to emerge if countries do not strengthen their control of TB. The aim of this study was to detect drug-resistance genes and screen herbs that could inhibit drug resistance of MTB or enhance the anti-tuberculosis drugs’ effect in order to prevent and cure MTB.In this study, we obtained 38 MTB isolations from clinical by BACTEC-TB 460 system. Using of 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and microtiter plate, we assayed the minimal inhibitory concentration (MIC) of rifampin and streptomycin to the isolations. There are 17 rifampin-resistance isolations and 9 streptomycin-resistance isolations. According to the drug-resistance gene sequences of rpoB and rpsL, we designed 2 pairs of primers for PCR. By PCR and DNA sequencing, we analyzed the rifampin- and streptomycin-resistance isolations. The results were including 2 mutations identified in the rpsL gene and 3 mutations in the rpoB gene.we made some crude preparation of Chinese medicine herb selected on the basis of nature of a drug, we detected crude preparation susceptibility to the rifampin- and streptomycin-resistance isolations by MTT and microtiter plate, resulting in most of non-sensitivity. Analyzing the changes of MIC of those resistance strains after cultivation and transferring of culture, we found 2 herbs could decrease the streptomycin-resistance of isolations and 5 herbs could enhance the drug’s effect. But none could take effect on rifampin-resistance isolations. Based on mechanisms of drug resistance of MTB, we analyzed the changes of the morphology, protein electrophoresis map and drug-resistance genes occurred in those isolations. The results showed that no changes occurred in the two formers and the later changed for 4 sites by contrasted with the primordial drug-resistant isolatons.更多还原