耐药结核杆菌原核类泛素蛋白(Pup)-蛋白酶体系统基因表达的研究

Expression of prokaryotic ubiquitin-like protein (Pup)-proteasome system in drug-resistant Mycobacterium tuberculosis

目的:检测分析经过不同浓度的抗结核药物培养后的各组耐药结核杆菌临床分离株和原始耐药状态的耐药Mtb临床分离株原核类泛素蛋白(Pup)-蛋白酶体系统基因的表达差异,探讨研究经过不同浓度抗结核药物的抗生素选择压力作用后,各组耐药Mtb菌株中的Pup-蛋白酶体系统的基因表达是否与新疆地区广泛流行的耐药Mtb临床分离株耐药性相关。方法:将单纯耐异烟肼的Mtb临床分离株(INH-Mtb)、单纯耐利福平的Mtb临床分离株(RFP-Mtb)、单纯耐链霉素的Mtb临床分离株(SM-Mtb)、单纯耐乙胺丁醇的Mtb临床分离株(EB-Mtb)、耐多药的Mtb临床分离株(MDR),分别在原始耐药状态、低浓度含药培养基和高浓度含药培养基上培养至对数生长期,分别提取各组耐药Mtb菌株的总RNA,利用SYBR Green I实时荧光定量PCR的方法检测各组耐药Mtb菌株在不同浓度抗结核药物的抗生素选择压力作用的Pup-蛋白酶体系统基因的表达水平。结果:与原始耐药状态下耐药Mtb菌株相比较,经低浓度抗结核药物压力作用培养后的各组Mtb菌株,Pup基因在INHMtb、RFP-Mtb、SM-Mtb、MDR组中的表达分别下调0.74、0.23、0.28、0.57倍;Dop基因表达分别上调1.33、1.63、1.14、2.88倍;Paf A基因表达分别上调1.69、1.30、1.58、1.32倍;Mpa基因表达分别上调3.05、1.79、1.31、2.27倍,差异有统计学意义(P<0.05);与原始耐药状态下耐药Mtb菌株相比较,经高浓度抗结核药物压力作用培养后的各组Mtb菌株,Pup基因在INH-Mtb、RFP-Mtb、SM-Mtb、MDR组中的表达分别下调0.58、0.37、0.43、0.78倍;Dop基因表达分别上调2.62、2.49、1.69、2.95倍;Paf A基因表达分别上调2.16、1.48、2.02、2.21倍;Mpa基因表达分别上调1.63、3.22、1.13、3.94倍,差异有统计学意义;(P<0.05)。结论:经过不同浓度抗结核药物的抗生素选择压力作用后各组Mtb菌株中Pup-蛋白酶体系统的Pup基因、Dop基因、Mpa基因和Paf A基因的表达有差异,提示结核杆菌Pup-蛋白酶体系统的基因表达与新疆地区广泛流行的耐药Mtb临床分离株耐药性相关。

Objective： To study the different gene expressions of Pup-proteasome system between the original drug resistance Mtb and the cultured Mtb（ INH-Mtb,RFP-Mtb,SM-Mtb,EB-Mtb,MDR） which were at the different concentrations of the Mtb drug,and to explore whether the different Pup-proteasome system gene expression was relevent to the clinical isolates of Mtb-resistant which was widely spreaded in Xinjiang region. Methods： Culturing INH-Mtb,RFP-Mtb,SM-Mtb,EB-Mtb,MDR strains at Original drug resistance Medium,low concentrations of drug Medium and high concentrations of drug Medium to the logarithmic phase,and extract total RNA from each group of Mtb. Using SYBR Green Ⅰ real-time PCR to detect the Pup-proteasome system expression level in different concentrations of drug Mtb in each group of Mtb. Results： Compared with the original state of drug-resistant strains,Pup gene in INH-Mtb,RFP-Mtb,SM-Mtb and MDR strains group were down-regulated 0. 74,0. 23,0. 28,0. 57 times; Dop gene were up-regulated 1. 33,1. 63,1. 14,2. 88 times; Paf A gene were up-regulated 1. 69,1. 30,1. 58,1. 32 times; Mpa gene were up-regulated 3. 05,1. 79,1. 31,2. 27 times in low concentrations of Anti-Mtb drugs condition,the difference was statistically significant（ P〈0. 05）. Compared with the original state of drug-resistant strains,Pup gene in INH-Mtb,RFP-Mtb,SM-Mtb,MDR strains group were down-regulate 0. 58,0. 37,0. 43,0. 78 times; Dop gene were up-regulated 2. 62,2. 49,1. 69,2. 95 times; Paf A gene were up-regulated 2. 16,1. 48,2. 02,2. 21 times; Mpa gene were up-regulated 1. 63,3. 22,1. 13,3. 94 times in the high concentrations of Anti-Mtb drugs condition,the difference was statistically significant（ P 0. 05）. Conclusion： Through the different concentrations of antibiotic selection pressure,these groups of Mtb strains expressions in the Pup-proteasome system of Pup gene,Dop gene,Mpa gene and Paf A gene were different,The results reveal that Pup-proteasome system is associated with the drug resistance in Mtb which was spreaded in Xinjiang region.