沙眼衣原体pORF5质粒蛋白对HeLa细胞自噬的影响
发布时间：2018-11-14 08:56
【摘要】：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;P0.05);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;P0.01)和0.85倍(t=16.56;P0.05),较PLenO-DCE-HeLa细胞分别增加1.95倍(t=79.12;P0.01)和1.57倍(t=23.95;P0.05);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;P0.05)和4.34倍(t=112.23;P0.01)。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。
[Abstract]:Objective to investigate the effect of Chlamydia trachomatis (Chlamydia trachomatis,Ct) plasmid pORF5 on autophagy of HeLa cells, and to provide experimental evidence for further elucidation of the pathogenesis of chlamydia trachomatis (Chlamydia trachomatis,Ct). Methods Ct pORF5 plasmid protein gene was amplified by PCR and cloned into PLenO-DCE lentivirus plasmid. The recombinant expression vector of lentivirus was identified by double enzyme digestion and sequencing. The recombinant vector was co-transfected into 293T cells to prepare lentivirus. The lentivirus was collected and reinfected with HeLa cells. The stable transfection cell line (PLenO-DCE/pORF5-HeLa) of pORF5 gene was obtained by flow cytometry. The control cell line (PLenO-DCE-HeLa) was also established. Real-time PCR and Western blot were used to detect the protein and mRNA expression of microtubule-associated protein 1 (LC3), Becin1 and LC3-II/LC3I, and indirect immunofluorescence was used to detect the fluorescent spots of autophagy. Results the number of LC3 red fluorescent spots in PLenO-DCE/pORF5-HeLa and PLenO-DCE-HeLa cells was (97.6 卤12.1) / cell and (34.0 卤2.6) / cell, respectively. The difference was statistically significant (t = 45.36). P0.05); The expression of LC3,Beclin1 mRNA in PLenO-DCE-HeLa and PLenO-DCE/pORF5-HeLa cells after starvation was significantly higher than that in the untreated group, and the expression of LC3,Beclin1mRNA in PLenO-DCE/pORF5-HeLa cells was 3.10-fold higher than that in the untreated group (tb95.25). P0.01) and 0.85 times (tampon 16.56%), 1.95 times than PLenO-DCE-HeLa cells (79.12% P0.01) and 1.57 times (tF23.95% P0.01); The ratio of LC3- 鈪，
本文编号：2330711
[Abstract]:Objective to investigate the effect of Chlamydia trachomatis (Chlamydia trachomatis,Ct) plasmid pORF5 on autophagy of HeLa cells, and to provide experimental evidence for further elucidation of the pathogenesis of chlamydia trachomatis (Chlamydia trachomatis,Ct). Methods Ct pORF5 plasmid protein gene was amplified by PCR and cloned into PLenO-DCE lentivirus plasmid. The recombinant expression vector of lentivirus was identified by double enzyme digestion and sequencing. The recombinant vector was co-transfected into 293T cells to prepare lentivirus. The lentivirus was collected and reinfected with HeLa cells. The stable transfection cell line (PLenO-DCE/pORF5-HeLa) of pORF5 gene was obtained by flow cytometry. The control cell line (PLenO-DCE-HeLa) was also established. Real-time PCR and Western blot were used to detect the protein and mRNA expression of microtubule-associated protein 1 (LC3), Becin1 and LC3-II/LC3I, and indirect immunofluorescence was used to detect the fluorescent spots of autophagy. Results the number of LC3 red fluorescent spots in PLenO-DCE/pORF5-HeLa and PLenO-DCE-HeLa cells was (97.6 卤12.1) / cell and (34.0 卤2.6) / cell, respectively. The difference was statistically significant (t = 45.36). P0.05); The expression of LC3,Beclin1 mRNA in PLenO-DCE-HeLa and PLenO-DCE/pORF5-HeLa cells after starvation was significantly higher than that in the untreated group, and the expression of LC3,Beclin1mRNA in PLenO-DCE/pORF5-HeLa cells was 3.10-fold higher than that in the untreated group (tb95.25). P0.01) and 0.85 times (tampon 16.56%), 1.95 times than PLenO-DCE-HeLa cells (79.12% P0.01) and 1.57 times (tF23.95% P0.01); The ratio of LC3- 鈪，
本文编号：2330711
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