人源肺孢子虫纯培养株的建立及基因鉴定  

Axenic cultivation and gene identification of Pneumocystis jiroveci isolated from Pneumocystis pneumonia patients

作　　者：黄敏君[1] 李淑珍[1] 安亦军[1] 卢思奇[2] 郭增柱[1] 

机构地区：[1]首都医科大学附属北京友谊医院北京热带医学研究所,100050 [2]首都医科大学寄生虫学教研室

出　　处：《中华微生物学和免疫学杂志》2007年第8期771-774,共4页Chinese Journal of Microbiology and Immunology

基　　金：北京市自然科学基金项目(No.7052009)

摘　　要：目的建立人源肺孢子虫(Pneumocystis jiroveci,Pj)纯培养株。方法从肺孢子虫肺炎(PCP)患者的支气管肺泡灌洗液(BALF)中分离Pj,用改良IMDM培养基做原代、传代和冻存复苏培养;以四胺银染色计数法观察虫体增殖情况;以线粒体rRNA大亚基特异引物扩增培养物中的目的基因,与基因库中的鼠源和人源肺孢子虫基因做序列比较。结果用添加S-腺苷甲硫氨酸(SAM)等辅助剂的IMDM培养基从2例PCP患者的BAIF中分离出2个巧纯培养株。分离培养的Pj可进行冷冻保存和复苏培养。培养120 h的Pj包囊可增殖3.2倍。基因序列分析证实分离出的Pj株与鼠源性肺孢子虫的线粒体rRNA大亚基同源性为67.8%,与GenBank中Pj的同源性为93.2%。结论用本法建立了2个Pj纯培养株。Objective To establish axenic cultivation of Pneumocystis jiroveci （Pj） from Pnetumocystis pneumonia （PCP） patients. Methods Pj were isolated from bronchoalveolar lavage fluid （BALF） of 2 cases with PCP and cultured in the growth in IMDM （GIBCO） supplemented with S-adenesyl-L-methionine, putrescine, N- acetyl glucosamine, putroscine, L-cysteine and L-glutamine, and newbem calf serum. The organisms cultured in the system were identified by the morphology of cysts in smears stained with Gomori＇ methenamine silver. The sequences of mitoehondriallarge-subunit＇s rRNA were compared with the 2 isolates of Pj, P. carinii f. sp. ratti variant （GenBank U20173） and P. cariniif, sp. hominis （GenBank M58605）. Results Pj isolated freshly from BALF of the PCP patients could be cultured axenieally and continuously passaged in IMDM culture system. Cultured organisms could be frozen and reinitiate inneed. The organisms may proliferate 3.2 times in 120 h. The gene sequencing of the mitoehondrial large-subunit＇s rRNA showed that Pneumocystis organisms from human and rat had significantly different DNA sequences. Conclusion Continuous axenie culture of 2 isolates of Pj has been achieved.

关 键 词：人源肺孢子虫 培养 基因 

分 类 号：R383[医药卫生—医学寄生虫学]