摘要：
Objective To investigate the production and mechanism of chemokine (C-C motif) ligand 5 (CCL5) by macrophages in U14 cervical cancer-bearing mice during infection. Methods The U14 cervical cancer cells were injected in C57BL/6 mice to induce tumor-bearing condition. Lipopolysaccharide (LPS) was injected into C57BL/6 mice to induce infection. The protein expression of CCL5 in the serum and the CCL5 mRNA expression in inflammatory cells were measured by ELISA and fluorescence quantitative-PCR in four groups. Macrophages were induced in the tumor conditioned medium (TCM) which extracted from mice serum. The protein expression levels of CCL5, prostaglandin E2 (PGE2) and cyclic adenosine monophosphate (cAMP) in the medium and CCL5, PGE2 and cAMP mRNA expression in the macrophages were detected in different groups. In order to determine whether the inhibition was related to PGE2, selective cyclooxygenase 2(COX-2) inhibitor NS398 was used to reverse this phenomenon and protein kinase A (PKA) inhibitor H89 demonstrated the mechanism through blocking cAMP/PKA signaling pathway. Results (1) The protein and mRNA level of CCL5 in tumor-bearing mice were respectively (151±35) pg/ml and 1.0, which were lower than those in the tumor-free mice (691 ± 85) pg/ml and 4.5 ± 0.8, there were significant difference between them (all P<0.05). The protein and mRNA level of PGE2 in tumor-bearing mice were (1 198±83) pg/ml and 5.8±0.8, which were higher than those in the tumor-free mice (187±25) pg/ml and 1.0, the difference were significant (all P<0.05). The protein and mRNA level of CCL5 in tumor-free+LPS mice were (4 049±141) pg/ml and 31.5±2.0, which were higher than those in the tumor-bearing+LPS mice (1 951±71) pg/ml and 12.1±2.8, the difference were also significant (P<0.05). The protein and mRNA level of PGE2 in tumor-free+LPS mice were (676±70) pg/ml and 3.4±0.4, which were lower than those in tumor-bearing+LPS mice (2 550±382) pg/ml and 11.6±0.9, the difference were also significant (all P<0.05). (2) Macrophages were cultured in vitro using TCM derived from mice. The protein and mRNA level of CCL5 in tumor-bearing mice TCM were respectively (1 626 ± 177) pg/ml and 28.6 ± 1.2, which were higher than those in the tumor-free mice TCM [(27 ± 3) pg/ml and 1.0], there were significant difference (P<0.05). The protein and mRNA level of PGE2 in tumor-bearing mice TCM were (790 ± 156) pg/ml and 1.7 ± 0.3, which were higher than those in the tumor-free mice TCM [(448 ± 115) pg/ml, 1.0], the difference were significant (all P<0.05). The protein and mRNA level of cAMP in tumor-bearing mice TCM were (164 ± 30) pg/ml and 1.6 ± 0.3, which weres higher than those in the tumor-free mice TCM [(118 ± 25) pg/ml,1.0], the difference were significant (all P<0.05). The protein and mRNA level of CCL5 in tumor-free + LPS mice TCM were (10 475 ± 742) pg/ml and 212.0 ± 5.7, which were higher than those in the tumor-bearing+LPS mice TCM [(6 375±530) pg/ml, 142.3±2.5], the difference were significant (all P<0.05). The protein and mRNA level of PGE2 in tumor-free+LPS mice TCM were (2 438±95) pg/ml and 4.3±0.7, which weres lower than those in the tumor-bearing + LPS mice TCM [(3 441 ± 163) pg/ml, 5.9 ± 0.3], the difference were significant (all P<0.05). The protein and mRNA level of cAMP in tumor-free+LPS mice TCM were (340 ± 13) pg/ml and 4.1 ± 0.4, which were lower than those in the tumor-bearing + LPS mice TCM [(542 ± 42) pg/ml, 5.4 ± 0.5], the difference were significant (all P<0.05). (3) Using COX-2 inhibitor NS398 in the tumor-bearing+LPS mice, the protein and mRNA level of CCL5, PGE2 and cAMP were (7 691±269) pg/ml and 159.0±8.9, (2 820±152) pg/ml and 4.9 ± 0.3, (465 ± 8) pg/ml and 4.3 ± 0.4, respectively, and there were significant difference (all P<0.05), compared to before treatment. Using PKA inhibitor H89 in the tumor-bearing+LPS mice, the protein and mRNA level of CCL5, PGE2 and cAMP were (8 375±520) pg/ml and 177.0±8.8, (2 650±35) pg/ml and 4.7 ± 0.4, (368 ± 13) pg/ml and 3.1 ± 0.7, respectively, and there were significant difference (all P<0.05), compared to before treatment. Conclusion TCM of U14 cells activated macrophages to release PGE2 could inhibit the expression of CCL5 levels by cAMP/PKA signaling pathway.%目的：探讨感染期子宫颈癌U14细胞荷瘤小鼠巨噬细胞分泌CC型趋化因子配体5（CCL5）受到抑制的机制。方法取6~8周龄的雌性C57BL/6小鼠随机（采用随机数字表法）分为4组，每组6只。无瘤组：皮下注射DMEM培养基；荷瘤组：皮下注射子宫颈癌细胞系U14细胞；无瘤+脂多糖（LPS；用于诱导小鼠感染）组：皮下注射DMEM培养基，腹腔内注射LPS；荷瘤+LPS组：皮下注射U14细胞，腹腔内注射LPS。（1）采用ELISA法及荧光定量PCR技术检测各组小鼠血清及巨噬细胞中CCL5和前列腺素E2（PGE2）的表达。（2）提取各组荷瘤小鼠血清制备肿瘤条件培养基（TCM），体外诱导巨噬细胞，检测诱导前、后各组上清液和巨噬细胞中CCL5、PGE2和环磷酸腺苷（cAMP）的表达。（3）荷瘤+LPS组选用环氧合酶2（COX-2）抑制剂——NS398阻断PGE2的产生（即荷瘤+LPS+NS398组），蛋白激酶A（PKA）阻滞剂——H89阻断cAMP/PKA信号通路（即荷瘤+LPS+H89组），分别制备TCM并检测两组上清液和巨噬细胞中CCL5、PGE2和cAMP的表达。结果（1）荷瘤组小鼠血清中CCL5蛋白含量及巨噬细胞中CCL5 mRNA的表达水平[分别为（151±35）pg/ml、1.0]均明显低于无瘤组[分别为（691±85）pg/ml、4.5±0.8；P<0.05]；其血清中PGE2蛋白含量及巨噬细胞中PGE2 mRNA的表达水平[分别为（1198±83）pg/ml、5.8±0.8]均明显高于无瘤组[分别为（187±25）pg/ml、1.0；P<0.05]。无瘤+LPS组小鼠血清中CCL5蛋白含量及巨噬细胞中CCL5 mRNA的表达水平[分别为（4049±141）pg/ml、31.5±2.0]均高于明显高于荷瘤+LPS组[分别为（1951±71）pg/ml、12.1±2.8；P<0.05]；其血清中PGE2蛋白含量及巨噬细胞中PGE2 mRNA的表达水平[分别为（676±70）pg/ml、3.4±0.4]均低于荷瘤+LPS组[分别为（2550±382）pg/ml、11.6±0.9；P<0.05]。（2）各组小鼠血清制备的TCM在体外培养巨噬细胞后，荷瘤组上清液中CCL5、PGE2、cAMP蛋白含量[分别为（1626±177）、（790±156）、（164±30）pg/ml]及巨噬细胞中CCL5、PGE2、cAMP mRNA的表达水平（分别为28.6±1.2、1.7±0.3、1.6±0.3）均明显高于无瘤组[其蛋白含量分别为（27±3）、（448±115）、（118±25）pg/ml，其mRNA的表达水平均为1.0；P<0.05]。无瘤+LPS组上清液中CCL5蛋白含量及巨噬细胞中CCL5 mRNA的表达水平[分别为（10475±742）pg/ml、212.0±5.7]均明显高于荷瘤+LPS组[分别为（6375±530）pg/ml、142.3±2.5；P<0.05]；无瘤+LPS组上清液中PGE2、cAMP蛋白含量[分别为（2438±95）、（340±13）pg/ml]及巨噬细胞中PGE2、cAMP mRNA的表达水平（分别为4.3±0.7、4.1±0.4）均明显低于荷瘤+LPS组[其蛋白含量分别为（3441±163）、（542±42）pg/ml，其mRNA的表达水分别为5.9±0.3、5.4±0.5；P<0.05]。（3）与荷瘤+LPS组比较，荷瘤+LPS+NS398组上清液中CCL5蛋白含量及巨噬细胞中CCL5 mRNA的表达水平[分别为（7691±269）pg/ml、159.0±8.9]明显升高（P<0.05），而上清液中PGE2、cAMP蛋白含量及巨噬细胞中PGE2、cAMP mRNA的表达水平均明显降低（P<0.05）；荷瘤+LPS+H89组上清液中CCL5蛋白含量及巨噬细胞中CCL5 mRNA的表达水平[分别为（8375±520）pg/ml、177.0±8.8]进一步升高（P<0.05），而上清液中PGE2、cAMP蛋白含量及巨噬细胞中PGE2、cAMP mRNA的表达水平均进一步降低（P<0.05）。结论感染期子宫颈癌U14细胞荷瘤小鼠血清可在体外抑制巨噬细胞分泌CCL5，这种抑制效应可能是通过cAMP/PKA通路而受PGE2的调控。
