摘要目的 探讨结核分枝杆菌(MTB)ATP结合区外排泵基因Rv1217c-Rv1218c的表达与耐药表型的关系.方法 选择对利福平、异烟肼、链霉素、乙胺丁醇联合或单一耐药,对二线抗结核药物至少有一种以上耐药的MTB菌株24株和对上述4种一线药物和二线药物均敏感的菌株10株(全敏感组).提取菌株RNA,反转录,实时RT-PCR方法检测ATP结合区外排泵基因Rv1217c、Rv1218c的表达量,采用t检验和Logistic回归分析法分析外排泵基因表达量在不同菌株耐药表型中的差异.结果 与全敏感组(0.42±0.31)比较,Rv1217c基因表达量在耐利福平组(2.13±1.89,t=3.44,P＜0.01)、耐异烟肼组(1.84±1.86,t=3.16,P＜0.01)、耐链霉素组(1.86±1.96,t=2.78,P＜0.05)和耐乙胺丁醇组(3.36±2.35,t=3.04,P＜0.05)均升高,差异有统计学意义.与全敏感组(0.65±0.42)比较,Rv1218c基因表达量在耐利福平组(2.54±1.84,t=3.82,P＜0.01)、耐异烟肼组(2.34±1.84,t=3.72,P＜0.01)、耐链霉素组(2.15±1.86,t=3.01,P ＜0.01)和耐乙胺丁醇组(3.78±1.78,t=4.22,P＜0.01)均升高,差异有统计学意义.耐多药组Rv1217c、Rv1218c基因表达量为2.74±2.07和3.33±1.77,高于多耐药组的0.79±0.47和1.03±0.79,差异有统计学意义(t=2.91,P＜0.05；t=3.84,P＜0.01).Logistic回归分析显示,Rv1217c基因高表达与利福平耐药呈正相关,但与Rv1218c基因高表达呈负相关(P＜0.01)；Rv1217c基因高表达与异烟肼耐药呈负相关,但与Rv1218c基因高表达呈正相关(P＜0.01)；两基因高表达与乙胺丁醇耐药均呈正相关(P＜0.01)；两基因高表达与耐多药呈正相关(P＜0.01).链霉素耐药与两基因的表达无关(P＞0.05).结论 ATP结合区外排泵基因Rv1217c-Rv1218c表达量与MTB对多种药物的耐药有关,表达增高可能是耐多药发生的机制之一.

abstractsObjective To explore the association of ATP-binding cassette (ABC) efflux pump gene Rv1217c-Rv1218c and the drug resistance of Mycobacterium tuberculosis.Methods A total of 34 Mycobacterium tuberculosis clinical isolates including 24 drug-resistance isolates which were resistant to riffampicin,isoniazid,streptomycin or ethambutol and resistant to at least one second-line antituberculosis drug,and 10 drug-sensitive isolates were involved in this study.The RNA of isolated strains was extracted and then reverse transcribed. Gene expression was performed by real-time polymerase chain reaction (PCR) and data was analyzed by t test and Logistic regression analysis.Results The expressions of Rv1217c in rifampicin-resistant group (2.13 ± 1.89,t =3.44,P＜0.01),isoniazid-resistant group ( 1.84 ± 1.86,t =3.16,P＜ 0.05),streptomycin-resistant group ( 1.86 ±1.96,t=2.78,P＜0.05) and ethambutol-resistant groups (3.36±2.35,t=3.04,P＜0.05) were all higher than sensitive isolates (0.42 ± 0.31).The expressions of Rv1218c in rifampicin-resistant group (2.54±1.84,t=3.82,P＜0.01),isoniazid-resistant group (2.34± 1.84,t=3.72,P＜0.01),streptomycin-resistant group (2.15±1.86,t=3.01,P＜0.01) and ethambutol-resistant groups (3.78± 1.78,t=4.22,P＜0.01 ) were all higher than sensitive isolates (0.65 ± 0.42).The expressions of Rv1217c and Rv1218c in multidrug resistant group were 2.74±2.07 and 3.33± 1.77,respectively,which were both higher than polydrug resistant group (0.79 ± 0.47 and 1.03 ± 0.79,respectively; t =2.91,P＜0.05 ; t =3.84,P＜0.01,respectively).Logistic regression analysis found that high Rv1217c expression was positively correlated with rifampicin resistance and negatively correlated with isoniazid resistance (both P＜ 0.01 ),while high Rv1218c expression was negatively correlated with rifampicin resistance and positively correlated with isoniazid resistance (both P＜0.01 ).High expressions of two genes were both positively correlated with ethambutol resistance and multidrug resistance (both P＜0.01 ) and not statistically correlated with streptomycin resistance (P＞0.05).Conclusions The expressions of ABC efflux pump gene,Rv1217c-Rv1218c,are correlated with multiple drug resistance.The overexpression may contribute to the multidrug resistance of Mycobacterium tuberculosis.