体外无药物选择压力条件下HIV-1耐药基因突变的演变  

Evolvement of HIV-1 drug resistant mutations in vitro without drug pressure

作　　者：焦丽燕[1] 鲍作义[1] 李韩平[1] 郭东星[1] 王铮[1] 庄道民[1] 李林[1] 耿庆茂[1] 刘永健[1] 刘思扬[1] 李敬云[1] 

机构地区：[1]军事医学科学院微生物流行病研究所病原微生物生物安全国家重点实验室,北京100071

出　　处：《中华微生物学和免疫学杂志》2010年第5期431-437,共7页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金（30800938,30830088）

摘　　要：目的 分离培养体外稳定传代的原代HIV-1耐药毒株,观察失去药物压力下,耐药毒株的体外生长以及主要耐药突变的演化趋势.方法 采集15例服用拉米夫定＋司他夫定＋萘韦拉平（3TC＋D4T＋NVP）的HIV-1感染者的外周血单个核细胞（PBMC）,用体外共培养的方法从中分离原代HIV-1毒株;RT-PCR扩增耐药毒株历代培养上清的HIV-1 pol区基因并测序,在Stanford HIV Drug Resistance Database数据库进行耐药性分析.结果 15例患者中病毒载量〉1000拷贝/ml的有8例,均成功分离出稳定传代的原代毒株,其中2株为耐药毒株,所携带的主要耐药突变分别是K103N/K238T和M184V/K103N/Y181C/H221Y,分别对NVP和3TC/NVP高度耐药;无药物压力的体外培养过程中,M184V、K103N、Y181C和H221Y等耐药突变可以稳定传代,但是K238T发生了回复突变.结论 分离出2株稳定传代的HIV-1耐药毒株,无药物压力情况下,携带K103N突变的毒株具有较好的复制适应性,可稳定传代;携带M184V和K103N/Y181C/H221Y的毒株也能够稳定复制;本研究中发现K238T耐药突变在失去药物的条件下稳定性差,提示该位点易发生回复突变.Objective To isolate stable passage primary HIV-1 drug resistant strains and observe replication dynamics of the drug resistant isolates and evolvement tendency of the drug resistant mutations in vitro.Methods Peripheral blood mononuclear cells（PBMCs）from 15 AIDS patients receiving highly active antiretroviral therapy（HAART）were collected,and the primary HIV-1 stains were separated utilizing co-cultivated with PBMCs from normal people.HIV-1 pol genes from those strains were obtained by RT-PCR and sequenced.The drug resistant mutations were analyzed in the Stanford HIV Drug Resistance Database.Results Eight strong positive strains were isolated from 15 AIDS patients with viral loads higher than 1000 copies/ml,and two of them were drug resistant.Drug resistant mutations of the two strains were respectively K103N/K238T and M184V/K103N/Y181C/H221Y which show high-level resistance to NVP and 3TC/NVP,respectively.K103N,M184V,Y181C and H221Y exist stably in the environment without drug pressure,however,RT K238T reverted to K238.Conclusion Two drug resistant strains were successfully isolated in vitro without drug pressure.Strains with K103N shows superior fitness and can exist steadily.Strains with M184V and K103N/Y181C/H221Y can also replicate stably in vitro without drug pressure.NNRTI mutation K238T reproduces astatically,which suggests that RT 238 codon might revert gradually to wild genotype.

关 键 词：原代HIV-1耐药毒株 耐药突变 复制动力学 

分 类 号：R512.91[医药卫生—内科学]