中华妇产科杂志
中華婦產科雜誌
중화부산과잡지
CHINESE JOUNAL OF OBSTETRICS AND GYNECOLOGY
2015年
5期
367-373
,共7页
任虹%任国丽%孙丽敏%范秀华%王钰然%李晓茜
任虹%任國麗%孫麗敏%範秀華%王鈺然%李曉茜
임홍%임국려%손려민%범수화%왕옥연%리효천
宫颈肿瘤%趋化因子CCL5%环AMP%环AMP依赖性蛋白激酶类%地诺前列酮%环氧化酶2%肿瘤,实验性
宮頸腫瘤%趨化因子CCL5%環AMP%環AMP依賴性蛋白激酶類%地諾前列酮%環氧化酶2%腫瘤,實驗性
궁경종류%추화인자CCL5%배AMP%배AMP의뢰성단백격매류%지낙전렬동%배양화매2%종류,실험성
Uterine cervical neoplasms%Chemokine CCL5%Cyclic AMP%Cyclic AMP-dependent protein kinases%Dinoprostone%Cyclooxygenase 2%Neoplasms,experimental
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。
目的：探討感染期子宮頸癌U14細胞荷瘤小鼠巨噬細胞分泌CC型趨化因子配體5（CCL5）受到抑製的機製。方法取6~8週齡的雌性C57BL/6小鼠隨機（採用隨機數字錶法）分為4組，每組6隻。無瘤組：皮下註射DMEM培養基；荷瘤組：皮下註射子宮頸癌細胞繫U14細胞；無瘤+脂多糖（LPS；用于誘導小鼠感染）組：皮下註射DMEM培養基，腹腔內註射LPS；荷瘤+LPS組：皮下註射U14細胞，腹腔內註射LPS。（1）採用ELISA法及熒光定量PCR技術檢測各組小鼠血清及巨噬細胞中CCL5和前列腺素E2（PGE2）的錶達。（2）提取各組荷瘤小鼠血清製備腫瘤條件培養基（TCM），體外誘導巨噬細胞，檢測誘導前、後各組上清液和巨噬細胞中CCL5、PGE2和環燐痠腺苷（cAMP）的錶達。（3）荷瘤+LPS組選用環氧閤酶2（COX-2）抑製劑——NS398阻斷PGE2的產生（即荷瘤+LPS+NS398組），蛋白激酶A（PKA）阻滯劑——H89阻斷cAMP/PKA信號通路（即荷瘤+LPS+H89組），分彆製備TCM併檢測兩組上清液和巨噬細胞中CCL5、PGE2和cAMP的錶達。結果（1）荷瘤組小鼠血清中CCL5蛋白含量及巨噬細胞中CCL5 mRNA的錶達水平[分彆為（151±35）pg/ml、1.0]均明顯低于無瘤組[分彆為（691±85）pg/ml、4.5±0.8；P<0.05]；其血清中PGE2蛋白含量及巨噬細胞中PGE2 mRNA的錶達水平[分彆為（1198±83）pg/ml、5.8±0.8]均明顯高于無瘤組[分彆為（187±25）pg/ml、1.0；P<0.05]。無瘤+LPS組小鼠血清中CCL5蛋白含量及巨噬細胞中CCL5 mRNA的錶達水平[分彆為（4049±141）pg/ml、31.5±2.0]均高于明顯高于荷瘤+LPS組[分彆為（1951±71）pg/ml、12.1±2.8；P<0.05]；其血清中PGE2蛋白含量及巨噬細胞中PGE2 mRNA的錶達水平[分彆為（676±70）pg/ml、3.4±0.4]均低于荷瘤+LPS組[分彆為（2550±382）pg/ml、11.6±0.9；P<0.05]。（2）各組小鼠血清製備的TCM在體外培養巨噬細胞後，荷瘤組上清液中CCL5、PGE2、cAMP蛋白含量[分彆為（1626±177）、（790±156）、（164±30）pg/ml]及巨噬細胞中CCL5、PGE2、cAMP mRNA的錶達水平（分彆為28.6±1.2、1.7±0.3、1.6±0.3）均明顯高于無瘤組[其蛋白含量分彆為（27±3）、（448±115）、（118±25）pg/ml，其mRNA的錶達水平均為1.0；P<0.05]。無瘤+LPS組上清液中CCL5蛋白含量及巨噬細胞中CCL5 mRNA的錶達水平[分彆為（10475±742）pg/ml、212.0±5.7]均明顯高于荷瘤+LPS組[分彆為（6375±530）pg/ml、142.3±2.5；P<0.05]；無瘤+LPS組上清液中PGE2、cAMP蛋白含量[分彆為（2438±95）、（340±13）pg/ml]及巨噬細胞中PGE2、cAMP mRNA的錶達水平（分彆為4.3±0.7、4.1±0.4）均明顯低于荷瘤+LPS組[其蛋白含量分彆為（3441±163）、（542±42）pg/ml，其mRNA的錶達水分彆為5.9±0.3、5.4±0.5；P<0.05]。（3）與荷瘤+LPS組比較，荷瘤+LPS+NS398組上清液中CCL5蛋白含量及巨噬細胞中CCL5 mRNA的錶達水平[分彆為（7691±269）pg/ml、159.0±8.9]明顯升高（P<0.05），而上清液中PGE2、cAMP蛋白含量及巨噬細胞中PGE2、cAMP mRNA的錶達水平均明顯降低（P<0.05）；荷瘤+LPS+H89組上清液中CCL5蛋白含量及巨噬細胞中CCL5 mRNA的錶達水平[分彆為（8375±520）pg/ml、177.0±8.8]進一步升高（P<0.05），而上清液中PGE2、cAMP蛋白含量及巨噬細胞中PGE2、cAMP mRNA的錶達水平均進一步降低（P<0.05）。結論感染期子宮頸癌U14細胞荷瘤小鼠血清可在體外抑製巨噬細胞分泌CCL5，這種抑製效應可能是通過cAMP/PKA通路而受PGE2的調控。
목적：탐토감염기자궁경암U14세포하류소서거서세포분비CC형추화인자배체5（CCL5）수도억제적궤제。방법취6~8주령적자성C57BL/6소서수궤（채용수궤수자표법）분위4조，매조6지。무류조：피하주사DMEM배양기；하류조：피하주사자궁경암세포계U14세포；무류+지다당（LPS；용우유도소서감염）조：피하주사DMEM배양기，복강내주사LPS；하류+LPS조：피하주사U14세포，복강내주사LPS。（1）채용ELISA법급형광정량PCR기술검측각조소서혈청급거서세포중CCL5화전렬선소E2（PGE2）적표체。（2）제취각조하류소서혈청제비종류조건배양기（TCM），체외유도거서세포，검측유도전、후각조상청액화거서세포중CCL5、PGE2화배린산선감（cAMP）적표체。（3）하류+LPS조선용배양합매2（COX-2）억제제——NS398조단PGE2적산생（즉하류+LPS+NS398조），단백격매A（PKA）조체제——H89조단cAMP/PKA신호통로（즉하류+LPS+H89조），분별제비TCM병검측량조상청액화거서세포중CCL5、PGE2화cAMP적표체。결과（1）하류조소서혈청중CCL5단백함량급거서세포중CCL5 mRNA적표체수평[분별위（151±35）pg/ml、1.0]균명현저우무류조[분별위（691±85）pg/ml、4.5±0.8；P<0.05]；기혈청중PGE2단백함량급거서세포중PGE2 mRNA적표체수평[분별위（1198±83）pg/ml、5.8±0.8]균명현고우무류조[분별위（187±25）pg/ml、1.0；P<0.05]。무류+LPS조소서혈청중CCL5단백함량급거서세포중CCL5 mRNA적표체수평[분별위（4049±141）pg/ml、31.5±2.0]균고우명현고우하류+LPS조[분별위（1951±71）pg/ml、12.1±2.8；P<0.05]；기혈청중PGE2단백함량급거서세포중PGE2 mRNA적표체수평[분별위（676±70）pg/ml、3.4±0.4]균저우하류+LPS조[분별위（2550±382）pg/ml、11.6±0.9；P<0.05]。（2）각조소서혈청제비적TCM재체외배양거서세포후，하류조상청액중CCL5、PGE2、cAMP단백함량[분별위（1626±177）、（790±156）、（164±30）pg/ml]급거서세포중CCL5、PGE2、cAMP mRNA적표체수평（분별위28.6±1.2、1.7±0.3、1.6±0.3）균명현고우무류조[기단백함량분별위（27±3）、（448±115）、（118±25）pg/ml，기mRNA적표체수평균위1.0；P<0.05]。무류+LPS조상청액중CCL5단백함량급거서세포중CCL5 mRNA적표체수평[분별위（10475±742）pg/ml、212.0±5.7]균명현고우하류+LPS조[분별위（6375±530）pg/ml、142.3±2.5；P<0.05]；무류+LPS조상청액중PGE2、cAMP단백함량[분별위（2438±95）、（340±13）pg/ml]급거서세포중PGE2、cAMP mRNA적표체수평（분별위4.3±0.7、4.1±0.4）균명현저우하류+LPS조[기단백함량분별위（3441±163）、（542±42）pg/ml，기mRNA적표체수분별위5.9±0.3、5.4±0.5；P<0.05]。（3）여하류+LPS조비교，하류+LPS+NS398조상청액중CCL5단백함량급거서세포중CCL5 mRNA적표체수평[분별위（7691±269）pg/ml、159.0±8.9]명현승고（P<0.05），이상청액중PGE2、cAMP단백함량급거서세포중PGE2、cAMP mRNA적표체수평균명현강저（P<0.05）；하류+LPS+H89조상청액중CCL5단백함량급거서세포중CCL5 mRNA적표체수평[분별위（8375±520）pg/ml、177.0±8.8]진일보승고（P<0.05），이상청액중PGE2、cAMP단백함량급거서세포중PGE2、cAMP mRNA적표체수평균진일보강저（P<0.05）。결론감염기자궁경암U14세포하류소서혈청가재체외억제거서세포분비CCL5，저충억제효응가능시통과cAMP/PKA통로이수PGE2적조공。
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.
