E型沙眼衣原体持续性感染模型的建立与鉴定

Establishment and identification of persistent infection model of Chlamydia trachomatis serovar E

目的构建E型沙眼衣原体(Ct)持续性感染细胞模型,为深入研究Ct致病机制提供实验基础。方法用不同剂量人重组干扰素-γ(IFN-γ)处理Ct感染的HeLa细胞,体外诱导Ct持续性感染,间接免疫荧光法检测不同剂量IFN-γ下Ct包涵体形态的变化;移除处理因素,继续培养16 h后观察Ct包涵体形态,并计算Ct包涵体形成单位(IFUs)。实时定量PCR检测Ct DNA修复和重组(dnaA和dnaK)、细菌分裂(ftsK)、蛋白水解和肽转运(oppA.4和htrA)、膜结构(ompA)、发育调节(htcA)和分子伴侣(groEL)等相关基因转录水平。流式细胞术和Hoechst染色检测持续性感染12 h、24 h、40 h时肿瘤坏死因子-α(TNF-α)诱导后的HeLa细胞凋亡率。结果75 U/mL IFN-γ可显著降低Ct感染能力,包涵体形态亦发生明显改变;移除IFN-γ后Ct IFUs为(12.60±0.52)×10^(9)/L,而未移除IFN-γ组IFUs为(3.60±0.52)×10^(9)/L,Ct感染力得到显著提高(P<0.05)。持续性感染组dnaA、dnaK、groEL、ompA、ftsK和htcA基因转录水平在不同的感染时间发生不同程度的改变;而oppA.4和htrA在急性和持续性感染状态下转录水平差异无显著性(P>0.05)。凋亡诱导后,与HeLa细胞凋亡率33.63%比较,流式细胞检测Ct持续性感染12 h、24 h、40 h时细胞凋亡率分别为14.77%(P<0.05)、18.71%(P<0.05)、26.32%(P>0.05);Ct在感染12 h和24 h时呈现抗凋亡效应,随着感染时间延长,衣原体抗凋亡能力下降。结论成功构建IFN-γ诱导的E型Ct持续性感染体外模型,持续性感染状态的Ct能抑制细胞凋亡。

Aim To construct a cell model of persistent infection of Chlaydia trachomatis(Ct)serovar E which provides an experimental basis for further study of Ct pathogenesis.Methods HeLa cells were treated with human recombinant interferon-gamma(IFN-γ)at different concentrations to induce Ct persistent infection model,indirect immunofluorescence assay was used to detect morphological changes of Chlamydial inclusion bodies.Ct inclusion bodies were observed after removal of IFN-γfor 16 h,and Ct inclusion forming units(IFUs)were also calculated.Quantitative real-time polymerase chain reaction was used to detect the relative transcript levels of genes related to DNA repair and recombination(dnaA and dnaK),bacterial division(ftsK),protein hydrolysis and peptide transport(oppA.4 and htrA),membrane structure(ompA),developmental regulation(htcA)and molecular chaperone(groEL).Flow cytometry and Hoechst staining were used to detect apoptotic rates of HeLa cells after treated with tumor necrosis factor-α(TNF-α)at 12 h,24 h,and 40 h during Ct persistent infection.Results IFN-γsignificantly reduced the Ct infectivity at the concentration of 75 U/mL,the morphologies of inclusion bodies were also changed remarkably.The IFUs of IFN-γremoval group and non-removal group were(12.60±0.52)×10^(9)/L and(3.60±0.52)×10^(9)/L respectively,the Chlamydial infectivity was significantly increased after IFN-γremoval(P<0.05).In the persistent infection group,the transcriptional levels of dnaA,dnaK,groEL,ompA,ftsK and htcA genes were changed with different degrees at different infection time,while the transcriptional levels of oppA.4 and htrA had no significant difference between the acute and persistent infection groups(P>0.05).After inducing apoptosis,compared with HeLa cells(33.63%),the apoptotic rates of persistent infection were 14.77%(P<0.05),18.71%(P<0.05)and 26.32%(P>0.05)at 12 h,24 h and 40 h respectively.Ct showed an anti-apoptotic effect at 12 h and 24 h after infection,and the anti-apoptotic ability decreased with the infected time.Conclusion The in vitro model of Ct serovar E persistent infection induced by IFN-γwas successfully constructed,and persistent Ct infection could inhibit cell apoptosis.