结核休眠菌模型构建及复苏探索

发布时间：2018-01-10 01:01

本文关键词：结核休眠菌模型构建及复苏探索　出处：《重庆医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的构建结核休眠菌缺钾模型,并探索结核菌培养上清液(supernatant,SN)、分枝杆菌噬菌体TM4、分枝杆菌噬菌体Guo1对模型菌的复苏作用。方法1.构建结核休眠菌缺钾模型采用缺钾Sauton培养基培养对数生长期结核菌,诱导其进入休眠状态。采用菌落计数、刃天青试验和最大或然数法(MPN)检测结核菌存活率及休眠菌比例;用透射电子显微镜(transmission electron microscope,TEM)观察结核菌细胞壁的厚度改变;采用金胺O-尼罗红双荧光染色法检测结核菌是否失去抗酸染色特性和发生脂质聚集;药敏测试检测结核菌对异烟肼及利福平的耐药情况。2.复苏结核休眠菌将对数期结核菌的培养上清液、分枝杆菌噬菌体TM4、分枝杆菌噬菌体Guo1分别与缺钾建模的结核休眠菌混合培养,采用菌落计数法检测复苏情况。结果1.构建结核休眠菌缺钾模型缺钾培养30天后,结核菌从杆状变为球形,细胞壁增厚,失去抗酸染色特性,发生脂质聚集,并对抗结核药物耐药。2.复苏结核休眠菌混合培养第1日时,空白对照组、上清组、TM4组、Guo1组菌落计数分别为(2.67±0.58)×102CFU/mL、(3.00±1.00)×102CFU/mL、(2.67±0.58)×102CFU/mL、(2.33±0.58)×102CFU/mL;培养第15日时,各组菌落计数分别为(7.33±1.53)×104CFU/mL、(6.67±2.08)×1010CFU/mL、(3.00±1.00)×1010CFU/mL、(3.33±1.53)×1010CFU/mL。结论缺钾培养可诱导对数期结核菌进入休眠状态。对数期结核菌培养上清液、分枝杆菌噬菌体TM4、分枝杆菌噬菌体Guo1均能够复苏结核休眠菌。其中以上清液复苏能力最强,分枝杆菌噬菌体TM4和Guo1复苏能力无明显差异。
[Abstract]:Objective to establish the potassium deficiency model of Mycobacterium tuberculosis dormant bacteria and to explore the supernatant TM4 of Mycobacterium tuberculosis culture supernatant. The resuscitation of Mycobacterium bacteriophage Guo1 on the model bacteria. 1. The model of Mycobacterium tuberculosis dormancy bacteria was established and cultured in the logarithmic growth phase using Sauton medium. 2. The survival rate of tuberculous bacteria and the proportion of dormant bacteria were detected by colony count, edge azurol test and maximum probability method (MPN). The thickness of the cell wall of tuberculous bacilli was observed by transmission electron microscope (TEM) and transmission electron microscopesimetry (TM). Whether the mycobacterium tuberculosis lost its acid fast staining characteristics and the accumulation of lipid was detected by the double fluorescence staining method of Amidine O- Nile red. Drug sensitivity test to detect the drug resistance of Mycobacterium tuberculosis to isoniazid and rifampicin. 2. Resuscitation tuberculosis dormancy bacteria culture supernatant of logarithmic phase tuberculous bacteria, Mycobacterium bacteriophage TM4. Mycobacterium bacteriophage Guo1 was mixed with Mycobacterium tuberculosis dormancy bacteria which was modeled by potassium deficiency, and the resuscitation was detected by colony counting method. Results 1. After 30 days of potassium deficiency culture, the model of Mycobacterium tuberculosis dormant bacteria was cultured. 2. Tuberculous bacteria from rod-shaped to spherical, cell wall thickening, loss of acid-fast staining characteristics, lipid accumulation, and anti-tuberculosis drug resistance .2. resuscitation tuberculosis dormancy bacteria mixed culture on 1st, blank control group. The colony count of Guo1 group was 2.67 卤0.58 脳 10 ~ 2 CFU 路m ~ (-1) 脳 10 ~ (2) CFU / mL in supernatant group (TM4) and 3.00 卤1.00 脳 10 ~ (2) CFU / mL respectively. 2.67 卤0.58) 脳 10 ~ (2) CFU / m ~ (-1) (2.33 卤0.58) 脳 10 ~ (2) CFU / m ~ (-1); On 15th, the colony count of each group was 7.33 卤1.53 脳 10 ~ 4 CFU / m ~ (-1) (6.67 卤2.08) 脳 10 ~ (10) CFU / mL. 3.00 卤1.00) 脳 10 ~ (10) CFU / mL. Conclusion potassium deficiency culture can induce mycobacterium tuberculosis into dormancy state in logarithmic phase. The supernatant of logarithmic culture of Mycobacterium tuberculosis, Mycobacterium phage TM4. Mycobacterium bacteriophage Guo1 was able to resuscitate the dormancy bacteria of tuberculosis, among which the supernatant was the strongest, but the TM4 and Guo1 of Mycobacterium bacteriophages had no significant difference.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R378

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