结核分枝杆菌利福平依赖株的遗传稳定性和蛋白质组学初步研究

发布时间：2018-04-27 19:24

本文选题：结核分枝杆菌 + 抗结核药物依赖性　；参考：《中国人民解放军军事医学科学院》2007年硕士论文

【摘要】： 结核菌药物依赖现象的发现，给结核病的防治又提出新的挑战。结核菌药物依赖，则意味着临床继续使用该药不但治疗无效，而且在一定条件下可能促进病原菌生长繁殖，使病情加重。因此对结核菌药物依赖现象进行研究已刻不容缓。目前药物依赖现象研究的瓶颈在于这种依赖性在体外不能稳定传代，因此本研究挑选依赖利福平结核菌进行体外连续传代实验，结果表明其药物依赖性可稳定遗传约30代。将经传代依赖性完全消失的结核菌悬液尾静脉注射感染小鼠，以不同频率给予利福平灌胃，观察到部分结核菌恢复依赖性。依赖菌所致肺结核以病情重、耐多药、化疗效果差和预后不良为主要临床特征，远比一般耐药病例危害大。已知结核分枝杆菌利福平依赖株和耐受株的rpoB基因突变高发位点基本相同，DNA指纹类型及分布亦无明显特殊性，表明前者的变异主要为表型变异。因此，依赖现象的独特表现可能仅源于基因表达差异。本研究从蛋白水平着手，优化了结核分枝杆菌的蛋白质组分析中的二维凝胶电泳技术，并用于分析利福平依赖株和耐受株总蛋白质的差异，获得了利福平依赖株和耐受株总蛋白质表达谱，鉴定8个显著差异表达的蛋白质点，获得7种蛋白质：3-氧酰基[酰基载体蛋白]合酶(3-oxoacyl-ACP synthase)、抗原85B A链(chain A，Ag85B)、烯醇基辅酶A水解酶(ECH)、跨膜丝/苏氨酸蛋白激酶D(STPK D)、假想转移酶(putative transferase)、3-酮酸-辅酶A转移酶β亚单位(βsubunit，，SCOB)、热休克蛋白HSPX，初步讨论了药物依赖现象的分子机制。运用比较蛋白质组学方法有助于研究药物依赖结核菌的蛋白质表达谱及差异表达的蛋白质分子，为进一步探寻结核菌药物依赖性的发生发展奠定基础。
[Abstract]:The discovery of drug dependence of tuberculous bacteria poses a new challenge to the prevention and treatment of tuberculosis. The drug dependence of tuberculous bacteria means that clinical use of the drug is not only ineffective, but also may promote the growth and reproduction of pathogenic bacteria under certain conditions, and make the disease worse. Therefore, it is urgent to study the drug dependence of tuberculous bacteria. At present, the bottleneck of drug dependence research is that the drug dependence can not be subcultured stably in vitro. Therefore, this study selected rifampicin-dependent tuberculous bacilli for continuous passage in vitro. The results showed that drug dependence could be inherited steadily for about 30 generations. Mice infected with Mycobacterium tuberculosis suspension completely disappeared after passage dependence were injected with rifampicin at different frequencies and some of them were observed to recover their dependence. The main clinical characteristics of pulmonary tuberculosis caused by dependent bacteria are serious condition, multidrug resistance, poor chemotherapeutic effect and poor prognosis, which are far more harmful than those of common drug resistant cases. It is known that the high frequency of rpoB gene mutation in rifampicin dependent strain and resistant strain of Mycobacterium tuberculosis has no obvious particularity, indicating that the variation of the former is mainly phenotypic variation. Therefore, the unique manifestation of dependence may be due only to differences in gene expression. In this study, we optimized two-dimensional gel electrophoresis for proteome analysis of Mycobacterium tuberculosis at protein level, and used to analyze the differences of total proteins between rifampicin dependent and tolerant strains. The total protein expression profiles of rifampicin dependent and tolerant strains were obtained and eight differentially expressed protein spots were identified. Seven kinds of protein synthase, 3-oxoacyl-ACP synthase, antigen85B A chain, enol coenzyme A hydrolase, transmembrane silk / threonine protein kinase D(STPK, hypothetical transferase putative transfer enzyme 3-ketoacyl-ACP synthase, antigen 85B A chain, enol coenzyme A hydrolase, transmembrane silk / threonine protein kinase D(STPK, were obtained. The molecular mechanism of drug dependence was preliminarily discussed by 尾 subunit SCOBX, heat shock protein HSPX. The application of comparative proteomics is helpful to study the protein expression profile and differentially expressed protein molecules of drug-dependent tuberculous bacteria and lay a foundation for further exploration of the occurrence and development of drug dependence of tuberculous bacteria.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R378

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