CRH对腹腔巨噬细胞分泌IL-10的影响及其信号机制  

Effects of corticotrophin-releasing hormone on IL-10 secretion from peritoneal macrophages and its signal mechanism

作　　者：王伍超[1] 陈金萍[1] 张岫竹[1] 代维[1] 刘大维[1] 周继红[1] 

机构地区：[1]第三军医大学大坪医院野战外科研究所第四研究室,全军/重庆市交通医学研究所,创伤,烧伤与复合伤国家重点实验室,重庆400042

出　　处：《第三军医大学学报》2009年第22期2228-2231,共4页Journal of Third Military Medical University

基　　金：国家重点基础研究发展计划(973计划)(2005CB522602)~~

摘　　要：目的研究促肾上腺皮质激素释放激素(corticotrophin-releasing hormone,CRH)对大鼠腹腔巨噬细胞分泌白介素-10(interleukin-10,IL-10)的影响及其信号转导机制。方法将原代培养的大鼠腹腔巨噬细胞分为正常对照组、CRH组、CP+CRH组、HL+CRH组、H89+CRH组以及PKAi+CRH组。其中正常对照组仅加入新鲜RPMIl640培养液2ml;CRH处理组,加入含CRH终浓度为10-9mol/L的培养液2ml;后4组在分别用CRHR1选择性阻断剂CP-154526(5×10-4mol/L)、CRHR1/2非选择性阻断剂α-helical CRH9-41(10-6mol/L)、AC抑制剂H89(10-6mol/L)和PKA抑制剂PKAi(6-22)amide(10-4mol/L)预孵育2h后,加入含终浓度为10-9mol/L的CRH培养液,再分别加入CRH(终浓度为109mol/L)刺激。各组分别于CRH刺激后0.5、1、2、4、8h收集培养皿中的上清液,应用ELISA方法检测IL-10的含量。结果正常对照组巨噬细胞分泌IL-10的含量为(29.41±3.10)μg/g。CRH组IL-10含量呈现双峰,在1、4h达到高峰,分别为(53.15±7.98)、(46.01±1.70)μg/g,与CRH组0.5h相比差异均非常显著(P<0.01),接近正常对照组的2倍(P<0.01)。CP+CRH组与HL+CRH组IL-10含量在1、4h均明显低于CRH组(P<0.01),并接近正常对照组水平。但在8h时明显增加,显著高于正常对照组(P<0.05)和CRH组(P<0.05)。且两组之间IL-10含量在0.5～4h差异不显著,但在8h时HL+CRH组显著低于CP+CRH组。H89预孵育后细胞IL-10含量在1、4、8h显著下降,与CRH组比较差异显著(P<0.01);在4、8h低于正常对照组水平(P<0.05)。PKAi(6-22)amide预孵育细胞后,IL-10含量在0.5～8h下降,与正常对照组以及CRH组比较差异非常显著(P<0.01)。结论CRH可通过CRHR1促进巨噬细胞分泌产生抗炎因子IL-10,其与cAMP/PKA信号通路密切相关。Objective To investigate the effects of corticotrophin-releasing hormone（CRH） on production of interleukin-10（IL-10） from macrophages and to further elucidate its signal transduction mechanism.Methods Rat peritoneal macrophages cultured were divided into 6 groups：control group,CRH group,CP＋CRH group,HL＋CRH group,H89＋CRH group and PKAi＋CRH group.The control group was cultured with 2 ml RPMI1640 medium.The CHR group was stimulated with 2 ml CRH at a final concentration of 10^9 mol/L.The other four groups were respectively pretreated with CRHR1 antagonist CP154526 at 5 × 10-4 tool/L, CRHR1/2 nonselective antagonist eL-helical CRH9-41 at 10 6 mol/L, AC inhibitor H89 compound at 10-6 mol/L or PKA inhibitor PKAi （6-22） amide （10-4 tool/L） for 2 h before stimulation with CRH. The concentration of IL-10 in the supernatant after stimulation for 0.5, 1, 2, 4 and 8 h were measured by ELISA method. Results In the normal control group, the average concentration of IL-10 kept at （29.41± 3.10）μg/g. As macrophages exposed to CRH, the content of IL-10 increased quickly and reached its first peak at 1 h compared with that in CRH group at O. 5 h （P 〈0. Ol ） , nearly two folds of that in normal control group （P 〈0. 01 ）, then its level decreased by degrees. There existed a second peak of IL-10 expression at 4 h, which was significantly higher than that in CRH group at 0. 5 h （P 〈0.01 ） and that in control group （P 〈0. 01 ）. The concentration of IL-10 in CP ＋ CRH group and HL ＋ CRH group at 1 and 4 h were significantly lower than that in CRH group （P 〈0. 01 ）, and were similar to that in control group （P 〉0. 05）. At 8 h, the concentration of IL-10 in the 2 groups were significantly higher than that in normal control group （ P 〈 0.01 ） or CRH group （ P 〈 0.01 ）. There was no statistical difference of IL-10 concentration between CP ＋ CRH group and HL ＋ CRH group （P 〉 0. 05 ） at 0.5 to 4 h, however that in HL ＋ CRH group was significantl

关 键 词：促肾上腺皮质激素释放激素 巨噬细胞 IL-10 CRHR CAMP/PKA 

分 类 号：R392.33[医药卫生—免疫学] R392.12[医药卫生—基础医学]