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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 detect and analyze the expression of prokaryotic ubiquitin (Pup) -protein system genes in clinical isolates of drug-resistant Mycobacterium tuberculosis and in the original drug-resistant Mtb clinical isolates after different concentrations of anti-TB drugs were cultured To investigate whether the gene expression of Pup-proteasome in each group of drug-resistant Mtb strains is related to the drug resistance of the widely-available drug-resistant Mtb clinical isolates in Xinjiang after the antibiotic selection pressure of different concentrations of anti-tuberculosis drugs . Methods: The Mtb isolates (INH-Mtb), Mtb isolates (RFP-Mtb), Mtb isolates (SM-Mtb) , Mtb clinical isolates (EB-Mtb) and multidrug-resistant Mtb isolates (MDR), which were resistant to ethambutol, were cultured in the original drug-resistant, low-drug-containing and high drug- To the logarithmic growth phase, the total RNA was extracted from each group of resistant Mtb strains, SYBR Green I real-time quantitative PCR method was used to detect the antibiotic selection pressure of each group of resistant Mtb strains in different concentrations of anti-TB drug Pup - Proteasome system gene expression levels. Results: The expression of Pup gene in INHMtb, RFP-Mtb, SM-Mtb and MDR groups was significantly higher than that of Mtb strains resistant to virulent drug resistance Down-regulated 0.74,0.23,0.28,0.57 times; Dop gene expression were up-regulated 1.33,1.63,1.14,2.88 times; Paf A gene expression were up 1.69,1.30,1.58,1.32 times; Mpa gene expression were increased 3.05,1.79,1.31, 2.27-fold, the difference was statistically significant (P <0.05); compared with the original drug-resistant Mtb strains, the high-concentration anti-TB drug strains cultured Mtb strains, Pup gene in INH-Mtb, RFP-Mtb, SM-Mtb and MDR decreased by 0.58, 0.37, 0.43 and 0.78 times, Dop gene expression increased by 2.62, 2.49, 1.69 and 2.95 times respectively; Paf A gene expression increased by 2.16,1.48 and 2.02, 2.21-fold; Mpa gene expression were increased 1.63,3.22,1.13,3.94 times, the difference was statistically significant (P <0.05). Conclusion: The expression of Pup gene, Dop gene, Mpa gene and Paf A gene in Pup-proteasome system of Mtb strains in different groups after antibiotic selection pressure of different concentrations of anti-tuberculosis drugs are different, suggesting that the Mycobacterium tuberculosis Pup-proteasome system Of the gene expression and resistance in Xinjiang widely resistant Mtb clinical isolates.