沙眼衣原体pORF5质粒蛋白抑制肿瘤坏死因子α诱导HeLa细胞凋亡北大核心CSCD

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。

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

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 expression 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 20μg/L TNF-αand 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 expression of Caspase3, Bax and Bcl-2, and Western blot analysis to determine the protein expression 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 expression 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 expression(t = 17.58,P < 0.01), but significant increases in Bcl-2 mRNA and protein(6.8 times)expression(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 expression of anti-apoptotic protein bcl-2 and decreasing the expression of pro-apoptotic proteins Caspase-3 and Bax.