鹿源结核菌中药抗菌增效剂的筛选及其对PhoPR双组份系统基因转录水平的影响

发布时间：2018-05-19 23:13

  本文选题：鹿源 + 结核菌　； 参考：《吉林农业大学》2017年硕士论文

【摘要】：结核病(Tuberculosis,TB)是由结核分枝杆菌(Mycobacterium tuberculosis)引起的慢性消耗性、人畜共患的重大传染性疾病。抗结核化学药物用于临床治疗结核病已有数十年,加之用药不合理、AIDS的流行等一系列原因,导致了耐药结核菌的产生。耐药结核病的治疗困难,治愈率较低,治疗过程中对机体伤害极大。寻找新型治疗药物和治疗方案迫在眉睫。中药被用于治疗疾病历史悠久,其作用细菌机制复杂,不易使细菌产生耐药性,且毒副作用较小。所以,本试验旨在丰富的中药中寻找对耐药结核菌的抑制剂或抗菌增效剂,并对其机理进行初步研究和探讨。本实验对多株结核菌的耐药性进行检测,结果表明MT-7、MB-1两株结核菌为耐多药结核菌。因此选择MT-7、MB-1为本实验的实验菌株,以敏感菌株H37Rv作为对照。对17种中药分别进行水提和醇提,得到17种中药的水提物和醇提物,以微量稀释法作用于三株结核菌,结果表明,白屈菜、黄芩、蒲公英三种中药具有较好的体外抗结核菌活性,其中白屈菜提物效果最好。将三种中药的提取物与抗结核药物联合作用于三株结核菌,结果表明,白屈菜醇提物与利福平联合作用于H37Rv、MB-1、MT-7时,两者间具有一定的协同作用。提取白屈菜中总生物碱并纯化。测定白屈菜水提物与醇提物中总生物碱含量,通过微量稀释法比较白屈菜总生物碱与水提物、醇提物的体外抑结核菌活性。结果表明,白屈菜总生物碱抑菌活性最好,白屈菜醇提物中生物碱含量比水提物中生物碱含量高,体外抗结核效果也比水提物效果好。将白屈菜总生物碱与抗结核药物联合作用于三株结核菌,结果表明两者间具有协同作用,FIC≤0.5,说明白屈菜总生物碱为利福平的抗结核菌增效剂。此外,对白屈菜总生物碱对细胞毒性进行了检测,白屈菜总生物碱的MIC值远远低于白屈菜总碱对细胞有毒性作用的浓度值。用白屈菜总生物碱、白屈菜总生物碱和利福平分别作用于三株结核菌,提取结核菌的RNA,以荧光定量PCR方法检测结核菌PhoP、PhoR基因的表达量。检测结果表明,在高浓度白屈菜总生物碱作用下,两株耐药结核菌的PhoP、PhoR基因表达量均有下降,说明白屈菜总生物碱可以降低耐药结核菌的耐药性。在白屈菜总生物碱和利福平联合作用下的两株耐药结核菌的PhoP、PhoR基因表达量也均有有明显下降,说明白屈菜生物碱和利福平间存在协同作用,也证实了白屈菜生物碱是利福平的抗结核菌增效剂,其抗结核菌增效机制有待进一步研究。
[Abstract]:Tuberculosis (TB) is a serious infectious disease caused by Mycobacterium tuberculosism. Antituberculous chemicals have been used in clinical treatment of tuberculosis for decades, and a series of reasons, such as irrational use of drugs and the prevalence of AIDS, have led to the emergence of drug-resistant TB bacteria. Drug-resistant tuberculosis treatment is difficult, the cure rate is low, the treatment process is very harmful to the body. It is urgent to look for new therapeutic drugs and therapeutic schemes. Chinese medicine has been used to treat diseases for a long time, its mechanism is complex, it is not easy to make bacteria drug resistance, and the toxicity and side effects are relatively small. Therefore, the purpose of this study was to search for inhibitors or synergists against drug-resistant tuberculous bacilli in Chinese medicine, and to explore its mechanism. The results showed that two strains of MT-7 MB-1 were multidrug resistant. Therefore, MT-7 MB-1 was selected as the experimental strain and the sensitive strain H37Rv as control. The water and alcohol extracts of 17 kinds of traditional Chinese medicine were extracted by water and alcohol, respectively, and three strains of tuberculous bacilli were treated with microdilution method. The results showed that Scutellaria baicalensis. Dandelion has good anti-tuberculous activity in vitro. Three strains of tuberculous bacteria were treated with the extracts of three kinds of traditional Chinese medicine and antituberculous drugs. The results showed that there was a certain synergism between the alcohol extract and rifampicin in H37Rvv1 MB-1 MT-7. Total alkaloids were extracted from Dioscorea chinensis and purified. The contents of total alkaloids in water extract and alcohol extract were determined and the antibacterial activity of alcohol extract against tuberculous bacteria in vitro was compared by microdilution method. The results showed that the total alkaloids of Dioscorea albicans had the best antibacterial activity. The alkaloid content in the alcohol extract was higher than that in the water extract, and the antituberculous effect in vitro was better than that in the water extract. Three strains of tuberculous bacteria were treated with total alkaloids and antituberculous drugs. The results showed that there was a synergistic effect of FIC 鈮，

本文编号：1912113