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目的探讨沙眼衣原体(Chlamydia trachomatis,Ct)质粒蛋白pORF5对HeLa细胞自噬的影响,为进一步阐明Ct致病机制提供实验依据。方法 PCR扩增Ct pORF5质粒蛋白基因,克隆入PLenO-DCE慢病毒质粒,构建慢病毒重组表达载体。慢病毒重组表达载体经双酶切及测序鉴定后与辅助质粒共转染293T细胞,制备慢病毒。收集慢病毒,再感染HeLa细胞,流式细胞仪分选获得pORF5基因稳定转染细胞株(PLenO-DCE/pORF5-HeLa)。实验同时建立对照细胞株(PLenO-DCE-HeLa)。血清饥饿处理两组细胞24h,Real-time PCR和Western blot检测微管相关蛋白1轻链3(LC3)、Becin1的蛋白和mRNA表达水平,计算LC3-II/LC3I比率;采用间接免疫荧光检测自噬荧光斑点。结果 PLenO-DCE/pORF5-HeLa和PLenO-DCE-HeLa细胞饥饿处理后均出现LC3红色荧光斑点,斑点数分别为(97.6±12.1)个/细胞和(34.0±2.6)个/细胞,差异有统计学意义(t=45.36;P<0.05);饥饿处理后PLenO-DCE-HeLa和PLenO-DCE/pORF5-HeLa细胞中LC3、Beclin1的mRNA表达水平均显著高于未处理组,其中PLenO-DCE/pORF5-HeLa细胞中LC3、Beclin1mRNA的表达水平较未处理组增加3.10倍(t=95.25;P<0.01)和0.85倍(t=16.56;P<0.05),较PLenO-DCE-HeLa细胞分别增加1.95倍(t=79.12;P<0.01)和1.57倍(t=23.95;P<0.05);PLenO-DCE/pORF5-HeLa经饥饿处理24h后LC3-Ⅱ/LC3-Ⅰ比率和Beclin1蛋白较未处理组分别增加52.17%和76.00%(t值分别是15.13,57.24;P均<0.05),较PLenO-DCE-HeLa细胞组分别增加1.05倍(t=35.21;P<0.05)和4.34倍(t=112.23;P<0.01)。结论 pORF5质粒蛋白可诱导HeLa细胞自噬,可能在Ct致病过程中发挥重要作用。
Objective To investigate the effect of plasmid pORF5 of Chlamydia trachomatis (Ct) on autophagy in HeLa cells and provide experimental evidence for further elucidating the pathogenesis of Ct. Methods The Ct pORF5 plasmid DNA was amplified by PCR and cloned into PLenO-DCE lentiviral plasmid to construct lentivirus recombinant expression vector. The recombinant lentiviral vector was co-transfected into 293T cells by double enzyme digestion and sequencing, then the lentivirus was prepared. The lentivirus was collected and then infected into HeLa cells. The pORF5 gene stably transfected cell line (PLenO-DCE / pORF5-HeLa) was obtained by flow cytometry. At the same time, a control cell line (PLenO-DCE-HeLa) was established. Serum starvation treatment of the two groups of cells 24h, Real-time PCR and Western blot detection of microtubule-associated protein 1 light chain 3 (LC3), Becin1 protein and mRNA expression levels calculated LC3-II / LC3I ratio; using indirect immunofluorescence detection Bite fluorescent spots. Results LC3 red fluorescent spots were observed after starvation in PLenO-DCE / pORF5-HeLa and PLenO-DCE-HeLa cells, with spots of (97.6 ± 12.1) cells / (34.0 ± 2.6) cells / The expression of LC3 and Beclin1 mRNA in PLenO-DCE-HeLa and PLenO-DCE / pORF5-HeLa cells after starvation treatment were significantly higher than those in untreated group (P = 0.05) The expression levels of LC3 and Beclin1 mRNA in pORF5-HeLa cells increased by 3.10 fold (t = 95.25; P <0.01) The ratio of LC3-Ⅱ / LC3-Ⅰ and Beclin1 protein in PLenO-DCE / pORF5-HeLa treated with starvation for 24 h were significantly higher than those in untreated group (t = 79.12; P <0.01) (T = 35.21; P <0.05) and 4.34 times (t = 112.23, P <0.05), respectively, compared with PLenO-DCE-HeLa cells ; P <0.01). Conclusion pORF5 plasmid protein can induce autophagy in HeLa cells, which may play an important role in the pathogenesis of Ct.