沙眼衣原体pORF5质粒蛋白对HeLa细胞自噬的影响  

Effects of the pORF5 plasmid protein of Chlamydia Trachomatis on the autophagy of HeLa cells

作　　者：杨晓玉[1] 雷文波[1] 粟盛梅[1] 陈超群[1] 李忠玉[1] 

机构地区：[1]南华大学医学院病原生物学研究所湖南省特殊病原体防控重点实验室,湖南衡阳421001

出　　处：《中国病原生物学杂志》2017年第4期311-316,共6页Journal of Pathogen Biology

基　　金：国家自然科学基金项目(No.81102230,31470277);特殊病原体防控湖南省重点实验室(No.2014-5);湖南省高等学校"分子靶标新药研究"协同创新中心项目(No.2014-405)

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;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 examine the effects of the pORF5 plasmid protein of Chlarnydia trachomatis on the autoph- agy on HeLa cells in order to provide further insight into the pathogenesis of chlamydia. Methods The pORF5 gene was amplified with PCR and ligated into the lentiviral plasmid PLenO-DCE to construct a recombinant lentiviral expression vector. After identification by restriction enzyme digestion and DNA sequencing, the recombinant lentiviral vector and helper plasmids were co-transfected into 293T cells to produce a recombinant lentivirus. The lentivirus was then used to infect HeLa cells to establish stably transfected HeLa cell lines including the PLenO-DCE/pORFS-HeLa cell line and the control cell line PLenO-DCE-HeLa. After serum starvation for 24 h, autophagy-related proteins such as LC3 and Becinl were detected with real time PCR and Western blotting in PLenO-DCE/pORF5-HeLa and PLenO-DCE-HeLa cells, and the ratio of LC3-II to LC3q was calculated to evaluate the maturation of autophagy. Punctate spots indicative of autoph- agy were measured with an indirect immunofluorescence assay. Results LC3 red fluorescent spots appeared in both PLenO-DCE/pORF5-HeLa and PLenO-DCE HeLa cells after serum starvation for 24 h. Quantification was performed and the results were 97.6 ±12.1 puncta/cell and 34.0± 2.6 puncta/eell, respectively. The number of LC3 puncta in PLe- nO-DCE/pORF5-HeLa cells increased significantly compared to that in PLenO-DCE-HeLa cells （t= 45.36; P〈0.05）. Levels of LC3 and Beclinl mRNA in PLenO-DCE/pORF5-HeLa cells increased 3.10-fold （t--95.25; P〈0.01） and 0.85- fold （t--16. 56; P〈0.05） compared to levels in untreated cells. Levels of LC3 and Beelinl mRNA in PLenO-DCE/pORF5-HeLa cells increased 1.95 fold （t=79.12; P〈0.01） and 1.57-fold （t=-23.95; P〈0.05） compared to levels in PLenO-DCE-HeLa cells. After serum starvation of PLenO-DCE/pORF5-HeLa cells, the ratio of LC3-Ⅱto LC3- I in- creased 52.17 % and Beclinl protein increased 76.00 % compared to corresponding values i

关 键 词：沙眼衣原体 pORF5质粒蛋白 细胞自噬 微管相关蛋白1轻链3 BECLIN1 

分 类 号：R374[医药卫生—病原生物学]