体外诱导沙眼衣原体持续性感染对宿主细胞凋亡的调控  

Regulation of apoptosis of host cells by in vitro azithromycin- induced persistent Chlamydia trachomatis infection

作　　者：陈文韬 薛耀华 黄进梅 杨结仪 赵云虎 蓝银苑 方铭恒 郑碧英 郑和平 Chen Wentao;Xue Yaohua;Huang Jinmei;Yang Jieyi;Zhao Yunhu;Lan Yinyuan;Fang Mingheng;Zheng Biying;Zheng Heping(Clinical Laboratory, Dermatology Hospital of Southern Medical University, Guangdong Provincial Dermatology Hospital, Guangzhou 510000, China;Graduate College, Guangdong Medical University, Dongguan 523808, Guangdong, China)

机构地区：[1]南方医科大学皮肤病医院广东省皮肤病医院检验科,广州510000 [2]广东医科大学研究生学院

出　　处：《中华皮肤科杂志》2018年第5期347-351,共5页Chinese Journal of Dermatology

基　　金：广东省自然科学基金（2015A030313895）;广东省医学科学技术研究基金（A2016563）

摘　　要：目的探讨阿奇霉素诱导持续性感染下沙眼衣原体对Hela229细胞凋亡的调控作用。方法沙眼衣原体感染Hela229细胞22h后，加入含0．08mg／L阿奇霉素的DMEM培养液继续培养至感染48h，构建持续性感染细胞模型，以不含阿奇霉素的DMEM培养液培养构建急性感染细胞模型。感染48h后，去除阿奇霉素继续培养进行沙眼衣原体复活试验。免疫荧光和电镜检测技术验证持续性感染模型。将沙眼衣原体急性感染和持续性感染的Hela229细胞用星状孢子素处理4h诱导凋亡后，用Hoechst33258染色和膜联蛋白V-碘化丙锭流式细胞仪检测宿主细胞凋亡，以未感染Hela229细胞为对照。结果加入阿奇霉素后，沙眼衣原体感染的细胞内形成异形包涵体，其内为畸形网状体。去除阿奇霉素继续培养至96h，沙眼衣原体包涵体内见具有感染性的原体。Hoechst染色显示，经星状孢子素诱导细胞凋亡，持续性感染的细胞染色质疏松，而无沙眼衣原体感染的细胞染色质浓缩。在感染24h时诱导凋亡，持续性感染组和急性感染组细胞凋亡率分别为45．567％±2．631％和38．567％±1．701％，两者差异有统计学意义（t=2．686，P=0．028），持续性感染组与未感染组（69．800％±2．835％）差异有统计学意义（t=8．187，P〈0．001）。在感染48h时诱导凋亡，持续性感染和急性感染组细胞凋亡率分别为46．700％±5．257％和61．767％±1．815％，两者差异有统计学意义（t=5．781，P〈0．001）；持续性感染组与未感染组（68．667％±3．156％）差异有统计学意义（t=7．421，P〈0．001）。结论阿奇霉素诱导沙眼衣原体持续性感染可调控宿主细胞凋亡，且作用持久至48h。Objective To evaluate the regulatory role of azithromycin- induced persistent Chlamydia trachomatis （Ct） infection in the apoptosis of Hela229 cells. Methods Hela229 cells were firstly co-cultured with Ct for 22 hours, and then cultured with Dulbecco＇ s modified Eagle＇s medium （DMEM） containing 0.08 mg/L azithromycin for 26 hours to establish a cell model of persistent Ct infection （persistent infection group）. These infected Hela229 cells cultured with azithromycin-free DMEM served as a cell model of acute Ct infection （acute infection group）. After 48-hour infection with Ct, azithromycin was removed, and infected Hela229 cells in the above 2 groups were successively cultured with DMEM for the resurgence of Ct. Immunofluorescence assay and electron microscopy were performed to verify the persistent Ct infection model. The Hela229 cells in the persistent infection group and acute infection group as well as uninfected Hela229 cells （control group） were treated with staurosporine （STS） for 4 hours to induce the apoptosis, and then cell apoptosis was detected by Hoechst 33258 staining, annexin V/propidium iodide staining and flow cytometry. Results After the treatment with azithromycin, atypical inclusions with aberrant reticulate bodies appeared in the Ct- infected cells. After removing azithromycin, cells were cultured until 96 hours after infection, and infectious elementary bodies reappeared in the Ct inclusions. After the treatment with STS, Hoechst staining showed that there was loose chromatin in the persistently infected cells, while chromatin condensation was observed in the uninfected cells. After 24-hour infection with Ct and 4-hour induction with STS, the apoptosis rate was significantly higher in the persistent infection group （45.567% ± 2.631%） than in the acute infection group （38.567% ± 1.701%, t = 2.686, P = 0.028）, but significantly lower in the persistent infection group than in the uninfected group （69.800% ± 2.835%, t = 8.187, P 〈 0.001）. After 48-hou

关 键 词：沙眼衣原体 叠氮红霉素 治疗失败 细胞凋亡 持续性感染 

分 类 号：R518[医药卫生—内科学]