沙眼衣原体pORF5蛋白通过激活PI3K/Akt信号通路抑制细胞凋亡  

Chlamydia trachomatis pORF5 protein inhibits apoptosis through activating PI3K-Akt pathway

作　　者：卜继常 邹燕[1] 聂倩 雷文波[1] 周洲[1] 陆春雪[1] 陈超群[1] 李忠玉[1] BU Ji-Chang;ZOU Yan;NIE Qian;LEI Wen-Bo;ZHOU Zhou;LU Chun-Xue;CHEN Chao-Qun;LI Zhong-Yu

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

出　　处：《中国免疫学杂志》2018年第3期321-324,330,共5页Chinese Journal of Immunology

基　　金：国家自然科学基金(No.81772210;No.31470277);2017年地方高校国家级大学生创新创业训练计划项目(教高司函[2017]40号);2017年度湖南省大学生研究性学习和创新性实验计划项目(湘教通[2017]205号);特殊病原体防控湖南省重点实验室(2014-5);湖南省高等学校"分子靶标新药研究"协同创新中心(2014-405)资助项目

摘　　要：目的:探讨沙眼衣原体(Chlamydia trachomatis,Ct)pORF5蛋白对细胞凋亡的影响,并进一步分析其分子机制,为阐明Ct致病机制提供实验依据。方法:p GEX-6p/pORF5重组质粒转化XL1-blue大肠杆菌,IPTG诱导表达GST-pORF5融合蛋白,融合蛋白经谷胱甘肽琼脂糖凝胶4B纯化及蛋白酶切除GST标签后得到pORF5蛋白。用不同浓度的pORF5蛋白刺激HeLa细胞,Western blot检测不同时间Bax和Bcl-2的表达水平以及PI3K/Akt磷酸化水平,Hoechst 33342及流式细胞技术分析细胞凋亡情况;HeLa细胞经PI3K/Akt特异性抑制剂LY294002预处理1 h后,再用pORF5蛋白刺激24 h,测定细胞凋亡率,并进一步分析凋亡相关蛋白Bax和Bcl-2的表达水平及PI3K/Akt磷酸化水平。结果:凋亡相关蛋白Bax和Bcl-2的表达变化与pORF5蛋白浓度呈现一定的浓度和时间依赖性,当pORF5蛋白浓度达10μg/ml时,Bax表达下调,Bcl-2的表达上调,当升高至15μg/ml时,Bax和Bcl-2的表达量变化最明显;15μg/ml的pORF5蛋白刺激HeLa细胞24 h,Bax和Bcl-2表达变化最明显;流式细胞检测结果显示:pORF5蛋白刺激组较TNF-α处理组和未处理组细胞凋亡率分别降低了27.3%(P<0.01)和8.4%(P<0.05);Akt在pORF5蛋白刺激15 min后发生磷酸化,30 min后磷酸化水平达到峰值,用PI3K抑制剂LY294002预处理Hela细胞后,发现Akt的磷酸化显著减少,Bax蛋白表达明显上调,Bcl-2表达明显下调;LY294002处理组细胞凋亡率相比于对照组增加了13.0%(P<0.01)。结论:pORF5蛋白通过激活PI3K/Akt信号通路调节Bcl-2和Bax蛋白的表达抑制细胞凋亡。Objective:To study the relationship between apoptosis and the pORF5 protein of Chlamydia trachomatis,and further to explore its molecular mechanisms,which could lay a foundation for chlamydial pathogenic mechanisms.Methods:pGEX-6p/pORF5 recombinant expression vector was transformed to XL1-blue E.Coli to express GST-pORF5 fusion protein,and GST-pORF5 fusion protein was purified with Glutathione Sepharose 4B Beads,and cleaved to get pORF5 protein without GST tag by PreScission protease.The pORF5 protein was used to stimulate HeLa cells at different concentrations,then Western blot was used to evaluate the expression of Bax,Bcl-2 and phosphorylation of PI3K/Akt at different time points,Hoechst staining and Flow cytometry were applied to measure the apoptosis of HeLa cells.Before treated with pORF5 protein for 24 h,HeLa cells were pretreated with PI3K inhibitor LY294002 for 1 h,the expression of Bax,Bcl-2 and the phosphorylation of Akt were evaluated by Western blot,apoptosis rates were also determined.Results:The pORF5 protein changed the expression of Bax and Bcl-2 in dose-and time-dependent manners,pORF5 increased the expression of Bcl-2 and decreased the expression of Bax at the concentration of 10μg/ml,and there was obvious change at concentration of 15μg/ml for 24 h.The apoptosis rates of pORF5 treated group were reduced by 27.3%and 8.4%respectively when compared with TNF-αtreated group(P<0.01)and untreated group(P<0.05).Akt was phosphorylated after stimulated with pORF5 protein for 15 min,and reached its peak at 30 min.PI3K/Akt inhibitor led to the decrease of the expression of Bcl-2 and phosphorylation of Akt and increase of the expression of Bax,furthermore,PI3K/Akt inhibitor reversed pORF5-mediated anti-apoptosis,the apoptosis rate in LY294002 treated group was increased by 13.0%,when compared with the control group(P<0.01).Conclusion:pORF5 protein could inhibit apoptosis through activating PI3K/Akt signal pathway by induction of Bcl-2 and suppression of Bax.

关 键 词：沙眼衣原体 pORF5蛋白 细胞凋亡 PI3K/AKT信号通路 

分 类 号：R374.1[医药卫生—病原生物学]