沙眼衣原体pORF5质粒蛋白抑制肿瘤坏死因子α诱导HeLa细胞凋亡  

Inhibitory effect of Chlamydia trachomatis plasmid-encoded protein pORF5 on HeLa cell apoptosis induced by tumor necrosis factor-alpha

作　　者：杨晓玉[1] 邹燕[1] 龚思露 卜继常 周洲[1] 刘良专[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医学微生物学教研室特殊病原体防控湖南省重点实验室,湖南衡阳421001

出　　处：《中华皮肤科杂志》2017年第5期341-345,共5页Chinese Journal of Dermatology

基　　金：国家自然科学基金（31470277、81102230、31300156）

摘　　要：目的 探讨沙眼衣原体质粒蛋白pORF5对肿瘤坏死因子α（TNF？α）诱导HeLa细胞凋亡的影响。方法 将含pORF5基因的慢病毒重组表达载体与辅助质粒共转染293T细胞制备慢病毒，慢病毒收集浓缩后再感染HeLa细胞，流式细胞仪分选获得pORF5基因稳定转染细胞株（pORF5？HeLa），同时建立空载体转染对照细胞株（对照HeLa）。将两种细胞株分别分为两组，一组用20 μg/L TNF？α处理（处理组），一组仅用新鲜培养基培养（未处理组），作用6 h，Hoechst33258染色观察凋亡细胞形态，流式细胞仪检测细胞凋亡率，实时 PCR检测凋亡相关蛋白Caspase3、Bcl？2和Bax mRNA表达水平，Western印迹检测Bax、Bcl？2蛋白表达水平。结果 TNF？α处理细胞6 h后，Hoechst33258染色发现pORF5？HeLa和对照HeLa细胞中均可见不同程度的核固缩、碎裂，高亮蓝色凋亡小体；pORF5？HeLa细胞的凋亡率为（35.5 ± 4.5）%，对照HeLa细胞凋亡率为（63.6 ± 5.8）%，均显著高于相应未处理组［（9.5 ± 1.5）%和（7.9 ± 0.9）%，t值分别为13.53、32.36，均P 〈 0.01］。处理组pORF5？HeLa细胞中Bax和Caspase3 mRNA表达水平较处理组对照HeLa细胞分别降低72.8%和84.5%（t值分别为35.29，42.25，均P 〈 0.01），但Bcl？2 mRNA的表达水平显著高于处理组对照HeLa细胞（t = 87.12，P 〈 0.01）。处理组pORF5？HeLa细胞中Bax蛋白表达水平亦显著低于处理组对照HeLa细胞（t = 17.58，P 〈 0.01），而Bcl？2蛋白表达水平较对照HeLa细胞增加6.8倍，差异有统计学意义（t = 18.93，P 〈 0.01）。结论 pORF5可能通过增强抗凋亡蛋白Bcl？2降低促凋亡蛋白Caspase3和Bax的表达，抑制TNF？α诱导的HeLa细胞凋亡。Objective To evaluate inhibitory effect of Chlamydia trachomatis plasmid-encoded protein pORF5 on HeLa cell apoptosis induced by tumor necrosis factor-alpha （TNF-α）. Methods The recombinant lentiviral vector containing pORF5 gene and helper plasmids were co-transfected into 293T cells to prepare the recombinant lentivirus. Then, the lentivirus particles were collected and concentrated, and used to infect HeLa cells. Flow cytometric screening identified stable pORF5-expressing HeLa （pORF5-HeLa） cells. Meanwhile, the empty plasmid was transfected into HeLa cells to prepare control HeLa cells. The two cell lines were both divided into two subgroups to be treated with 20 μg/L TNF-α and fresh culture medium respectively for 6 hours. Then, Hoechst 33258 staining was performed to observe morphological changes of apoptotic cells, flow cytometry to detect cell apoptosis, real-time PCR to measure the mRNA of Caspase3, Bax and Bcl-2, and Western blot analysis to determine the protein of Bax and Bcl-2. Results After 6-hour treatment with TNF-α, Hoechst 33258 staining showed variable degrees of karyopyknosis and karyorrhexis, and highly-refractive blue apoptotic bodies in the pORF5-HeLa cells and control HeLa cells. The pORF5-HeLa cells and control HeLa cells both showed significantly higher apoptosis rate in the treated subgroup than in the untreated subgroup （pORF5-HeLa cells： 35.5% ± 4.5% vs. 9.5% ± 1.5%, t = 13.53, P 〈 0.01; control HeLa cells： 63.6% ± 5.8% vs. 7.9% ± 0.9%, t = 32.36, P 〈 0.01）. Compared with treated control HeLa cells, treated pORF5-HeLa cells showed significant decreases in mRNA of Bax （72.8%） and Caspase 3 （84.5%） （t = 35.29, 42.25,respectively, both P 〈 0.01）, as well as in Bax protein （t = 17.58, P 〈 0.01）, but significant increases in Bcl-2 mRNA and protein （6.8 times） （t = 87.12, 18.93, respectively, both P 〈 0.01）. Conclusion pORF5 plasmid protein can inhibit TNF-α-induced HeLa cell apoptosis likely by increasing the of anti-apoptotic prot

关 键 词：沙眼衣原体 细胞凋亡 肿瘤坏死因子Α 半胱氨酸天冬氨酸蛋白酶3 BCL-2相关X蛋白质 B细胞淋巴瘤/白血病-2蛋白 pORF5质粒蛋白 

分 类 号：R374[医药卫生—病原生物学]