中华微生物学和免疫学杂志
中華微生物學和免疫學雜誌
중화미생물학화면역학잡지
CHINESE JOURNAL OF MICROBIOLOGY AND IMMUNOLOGY
2010年
5期
431-437
,共7页
焦丽燕%鲍作义%李韩平%郭东星%王铮%庄道民%李林%耿庆茂%刘永健%刘思扬%李敬云
焦麗燕%鮑作義%李韓平%郭東星%王錚%莊道民%李林%耿慶茂%劉永健%劉思颺%李敬雲
초려연%포작의%리한평%곽동성%왕쟁%장도민%리림%경경무%류영건%류사양%리경운
原代HIV-1耐药毒株%耐药突变%复制动力学
原代HIV-1耐藥毒株%耐藥突變%複製動力學
원대HIV-1내약독주%내약돌변%복제동역학
Primary HIV-1 drug resistant isolate%Drug resistant mutation%Replication dynamics
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.
目的 分離培養體外穩定傳代的原代HIV-1耐藥毒株,觀察失去藥物壓力下,耐藥毒株的體外生長以及主要耐藥突變的縯化趨勢.方法 採集15例服用拉米伕定+司他伕定+萘韋拉平(3TC+D4T+NVP)的HIV-1感染者的外週血單箇覈細胞(PBMC),用體外共培養的方法從中分離原代HIV-1毒株;RT-PCR擴增耐藥毒株歷代培養上清的HIV-1 pol區基因併測序,在Stanford HIV Drug Resistance Database數據庫進行耐藥性分析.結果 15例患者中病毒載量>1000拷貝/ml的有8例,均成功分離齣穩定傳代的原代毒株,其中2株為耐藥毒株,所攜帶的主要耐藥突變分彆是K103N/K238T和M184V/K103N/Y181C/H221Y,分彆對NVP和3TC/NVP高度耐藥;無藥物壓力的體外培養過程中,M184V、K103N、Y181C和H221Y等耐藥突變可以穩定傳代,但是K238T髮生瞭迴複突變.結論 分離齣2株穩定傳代的HIV-1耐藥毒株,無藥物壓力情況下,攜帶K103N突變的毒株具有較好的複製適應性,可穩定傳代;攜帶M184V和K103N/Y181C/H221Y的毒株也能夠穩定複製;本研究中髮現K238T耐藥突變在失去藥物的條件下穩定性差,提示該位點易髮生迴複突變.
목적 분리배양체외은정전대적원대HIV-1내약독주,관찰실거약물압력하,내약독주적체외생장이급주요내약돌변적연화추세.방법 채집15례복용랍미부정+사타부정+내위랍평(3TC+D4T+NVP)적HIV-1감염자적외주혈단개핵세포(PBMC),용체외공배양적방법종중분리원대HIV-1독주;RT-PCR확증내약독주역대배양상청적HIV-1 pol구기인병측서,재Stanford HIV Drug Resistance Database수거고진행내약성분석.결과 15례환자중병독재량>1000고패/ml적유8례,균성공분리출은정전대적원대독주,기중2주위내약독주,소휴대적주요내약돌변분별시K103N/K238T화M184V/K103N/Y181C/H221Y,분별대NVP화3TC/NVP고도내약;무약물압력적체외배양과정중,M184V、K103N、Y181C화H221Y등내약돌변가이은정전대,단시K238T발생료회복돌변.결론 분리출2주은정전대적HIV-1내약독주,무약물압력정황하,휴대K103N돌변적독주구유교호적복제괄응성,가은정전대;휴대M184V화K103N/Y181C/H221Y적독주야능구은정복제;본연구중발현K238T내약돌변재실거약물적조건하은정성차,제시해위점역발생회복돌변.
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.
