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目的比较结核分枝杆菌(Mycobacterium tuberculosis,MTB)原始耐药菌株与经过含不同浓度培养基培养后的菌株中PhoPR双组分系统基因表达水平的差异,研究MTB PhoPR双组分系统与MTB耐药的相关性。方法分别在原始状态下和用低浓度含药培养基及高浓度含药培养基培养单纯耐异烟肼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MTB临床分离株(MDR)养至对数期,提取各组MTB总RNA,并进行纯度鉴定;运用SYBR GreenⅠ实时荧光定量PCR,检测各组MTB的PhoP基因和PhoR基因的表达,比较不同耐药的MTB临床分离株PhoP基因和PhoR基因表达水平的差异。结果在低浓度抗结核药物条件下培养的INH-MTB、RFP-MTB、SM-MTB和MDR组MTB与原始状态下MTB相比,PhoP基因表达分别上调2.19、2.04、1.72、2.73倍,PhoR基因分别上调1.57、2.57、1.56和2.83倍,差异有统计学意义(P<0.05);在高浓度抗结核药物条件下培养,INH-MTB、RFP-MTB、SMMTB、EB-MTB和MDR组MTB中的PhoP基因表达分别上调1.79、1.44、2.10、1.27和1.97倍,PhoR基因在INHMTB、RFP-MTB、SM-MTB和MDR组MTB分别上调1.05、1.91、1.76和2.13倍,差异有统计学意义(P<0.05)。结论 PhoP和PhoR基因的差异表达与MTB的耐药性相关,提示PhoPR双组分系统与MTB耐药具有相关性。
OBJECTIVE: To compare the difference of gene expression levels of two-component system of PhoPR in Mycobacterium tuberculosis (MTB) -resistant strains with those cultured in different concentrations of media, and to investigate the relationship between MTB PhoPR two-component system and MTB resistance Relevance. Methods The INH-MTB isolates of Isoniazid-resistant isolates (INH-MTB) and the pure MTB-resistant clinical isolates of RFB (Rifampicin) were respectively cultured in low concentration drug- MTB, SM-MTB, EB-MTB and MDR-resistant MDR to logarithmic phase The total RNA of MTB in each group was extracted and identified by purity. The expression of PhoP gene and PhoR gene in MTB of each group were detected by SYBR Green Ⅰ real-time fluorescent quantitative PCR. The PhoP gene and PhoR gene expression levels of different resistant MTB clinical isolates were compared The difference. Results The MTT of INH-MTB, RFP-MTB, SM-MTB and MDR group cultured in low concentration of anti-TB drug were up-regulated by 2.19,2.04,1.72 and 2.73 times as much as that of MTB under MTB. The PhoR gene MTB, RFP-MTB, SMMTB, EB-MTB and MTT in MDR group were significantly higher than those in control group (P <0.05) , Respectively. The PhoP gene levels were up-regulated by 1.79,1.44,2.10,1.27 and 1.97 times, respectively. The MTT of PhoR gene in INHMTB, RFP-MTB, SM-MTB and MDR group were increased by 1.05, 1.91, 1.76 and 2.13 times, P <0.05). Conclusion The differential expressions of PhoP and PhoR genes are related to the resistance of MTB, suggesting that PhoPR two-component system is related to MTB resistance.