外周血单个核细胞中肺炎衣原体的分离、培养及连续传代  

The Isolation,Culture,and Serial Passage of Chlamydia pneumoniae from PBMC

作　　者：金前[1] 孙书明[2] 王卫群[3] 周颖[1] 余竹元[4] 

机构地区：[1]浙江省立同德医院呼吸科,浙江杭州310012 [2]复旦大学附属金山医院呼吸科,上海200540 [3]复旦大学附属金山医院心内科,上海200540 [4]复旦大学附属中山医院免疫室,上海200032

出　　处：《微生物学通报》2010年第11期1650-1655,共6页Microbiology China

基　　金：上海市科委基金项目(No.034119811)

摘　　要：开发一种分离并增殖肺炎衣原体(Cpn)的简易方法极具意义。该方法先分离血液标本中的外周血单个核细胞(PBMC),用PEG使Cpn-Ag阳性的PBMC裂解释放出Cpn,然后与人喉表皮癌细胞(Hep-2)一起离心,继续培养Hep-2细胞,再将Hep-2细胞冻融破碎后,放入到新的Hep-2细胞中进行离心,以完成Cpn的1次传代,然后以同样的方法进行2-4次传代。分别用微量免疫荧光法(MIF)及PCR法检测Hep-2细胞中的Cpn-Ag和CpnDNA,并用FITC标记的属特异性衣原体脂多糖单克隆抗体检测实验分离以及进口菌株传代后的包涵体形成单位数量。结果显示MIF法检测Cpn感染后的Hep-2细胞,其胞内Cpn-Ag强阳性;MIF法检测1次传代及2次传代的Hep-2细胞,其胞内Cpn-Ag亦均强阳性,3次传代为阳性,而4次传代为阴性;PCR法检测2次传代后CpnDNA阳性。该简化方法可以实现PBMC中Cpn的分离,分离菌株传代4次后出现退化(优于进口菌株),但该方法仍可实现进一步的培养及传代。Developing a new method to isolate and proliferate Chlamydia pneumoniae is meaningful.This new method begins with isolating peripheral blood mononuclear cell from blood samples,and use polyethylene glycol to break peripheral blood mononuclear cells which are positive with Chlamydia pneumoniae antigen,then centrifugate these broken peripheral blood mononuclear cells together with Hep-2 cells.After 7-day＇s culture,these cells were broken by freeze-thaw cycle,and put into new Hep-2 cells,then centrifugated together to finish the first passage of Chlamydia pneumoniae.The second to the fourth passages were conducted in the same way.At the same time,we used Micro-immunofluorescence and PCR methods to detect Chlamydia pneumoniae in Hep-2 cells,also used a genus-specific,fluorescein isothiocyanate-conjugated monoclonal antibody to Chlamydia lipopolysaccharide,to stain inclusions,and got the IFUs counts of both imported and isolated strains after each passage.With the method of Micro-immunofluorescence,we found that Hep-2 cells centrifugated with peripheral blood mononuclear cells,Hep-2 cells of the first passage,Hep-2 cells of the second passage were all strong positive with Chlamydia pneumoniae antigen,Hep-2 cells of the third passage was positive,and the fourth passage negative.We also detected Chlamydia pneumoniae DNA existing in Hep-2 cells of the second passage with the method of PCR.So this simplified way can successfully achieve the isolation of Chlamydia pneumoniae from peripheral blood mononuclear cells.The phenomenon of degeneration appeared in the fourth passage of isolated strain,which was still superior to imported strain,but it will not largely affect the culture and passage of Chlamydia pneumoniae from peripheral blood mononuclear cells.

关 键 词：肺炎衣原体 单个核细胞 连续传代 

分 类 号：Q93-3[生物学—微生物学]