Toll样受体8在人宫颈癌细胞株HeLa中的表达及其激动剂对HeLa细胞增殖和凋亡的影响

Expression of TLR8 in human cervical cancer HeLa cells and the effect of TLR8 agonist on the cell proliferation and apoptosis

目的观察Toll样受体8（TLR8）在人宫颈癌细胞株HeLa中的表达，探讨TLR8激动剂CL075对HeLa细胞增殖和凋亡的影响。方法采用实时荧光定量聚合酶链反应（PCR）法，检测13种肿瘤细胞系中TLR8mRNA和经CL075作用后HeLa细胞中环氧化酶2（COX-2）、Bcl-2和血管内皮生长因子（VEGF）mRNA的表达水平；采用免疫荧光技术对HeLa细胞中TLR8蛋白的表达进行定位观察；采用流式细胞术检测不同浓度CL075作用下HeLa细胞的细胞周期和凋亡的变化；采用四甲基偶氮唑蓝（MTT）法检测HeLa细胞的增殖状态。结果与其他肿瘤细胞株相比，HeLa细胞中TLR8mRNA的表达水平最高，达703．7±20．6。TLR8蛋白主要定位于HeLa细胞的胞浆中。0．1、0．5和1．0μg／ml的CL075分别作用于HeLa细胞48h后，G2／M＋s期细胞所占的比例逐渐增高，其中1．0μg／mlCL075作用组G2／M＋S期细胞所占的比例最高，达（57．67±1．73）％，明显高于空白对照组[（39．02±2．33）％，P〈0．01]。经不同浓度的CL075处理后，HeLa细胞的凋亡水平与空白对照组相比，无明显改变（P〉0．05）；但经顺铂处理后，凋亡细胞明显增多（P〈0．01）。MTT法检测结果显示，与空白对照组比较，CL075作用于HeLa细胞48和72h后，HeLa细胞的增殖能力明显增强（P〈0．01）。CIA）75作用于宫颈癌HeLa细胞24和48h后，COX-2、Bcl-2和VEGF mRNA的表达水平明显升高（P〈0．05）。结论宫颈癌HeLa细胞中TLR8的表达水平及其与配体相互作用的信号，可能是肿瘤发生和发展的重要因素之一。TLR8可能是宫颈癌治疗的一个潜在靶点。

Objective To observe the expression of Toll-like receptor 8 （TLRS） in human cervical cancer cell-line HeLa cells, and the effects of TLR8 agonist CL075 on the survival and proliferation of HeLa cells. Methods PCR and RT-PCR were used to detect the expression of TLR8 in 13 cancer cell lines, and the expression of COX-2, Bcl-2, VEGF mRNA in the HeLa cells stimulated by TLR8 agonist CID75 were also measured by RT-PCR. Immunofluorescence technique was used to determine the exact location of TLR8 in the cells. The percentage of viable cells was determined by trypan blue exclusion after the HeLa cells were stimulated with TLR8 agonist CL075 （0.1 μg/ml, 0. 5 μg/ml, 1.0 μg/ml, 2.5 μg/ml）, and cell cycle and apoptosis were analyzed by flow cytometry, and the proliferation was measured by M＇IT. Results Compared with the other cancer cell lines, the expression of TLR8 in HeLa cells was the highest （703.7 ± 20.6）. After stimulation by CL075, the cells had a remarkable increase of the percentage of cells in G2/M ＋ S phases. In the control group, the percentage of cells in GJM ＋ S phases was （ 39.02± 2.33 ） % , whereas after stimulated with 1.0 μg/ml CL075, the percentage of cells in G2/M ＋ S phases reached the highest ratio （57.67 ±1.73）% , and the percentage of cells in G2/M ＋ S phases had a less decrease after 2.5 wg/ml CL075 stimulation and the percentage was （ 56.14 ±3.73 ） %. After the CL075 treatment, there was no significant changes of apoptosis compared with that of the control cells （ P 〉 0.05 ）, but after DDP treatment the apoptosis had a significant change （ P 〈 0. 01 ）. After stimulation by 1.0 μg/ml CL075 for 24 h, no significant difference （ P 〉 0.05 ） was found by MTr test, but a significant difference was found at 48 h and 72 h （P 〈0.01 ）. An increased expression of COX-2, Bel-2 and VEGF mRNA was observed in HeLa cells after stimulation by TLR8 agonist CL075 for 24 h and 48 h （ P 〈 0.05 ）. Conclusions Expression of TLR8 is significantly increased in HeLa cells. The proportion of cells at different phases has a significant change after CI.075 stimulation, which may up-regulate the proliferation of HeLa cells. These data suggested that TLR8 agnnist may influence the tumor development and TLR8 may become a potential target in the treatment for cervical cancer.