摘要
目的探讨雄激素对RAW264．7巨噬细胞肿瘤坏死因子（TNF）-α和单核细胞趋化蛋白（MCP）-1表达的影响及其分子机制。方法（1）用不同浓度睾酮处理小鼠RAW264．7巨噬细胞不同时间，分别用RT-PCR和ELISA法检测细胞TNF-α和MCP-1 mRNA和培养上清液中的表达；（2）10^-7mol／L睾酮处理细胞0、10、30、60、180、360min后，Western印迹检测磷酸化NF—κB（p—NF—κB）的表达。（3）NF—κB的抑制剂PDTC处理细胞1h后再用不同浓度睾酮处理，用RT—PCR和ELISA法检测TNF-α和MCP-1的表达。结果（1）①10^-9和10^-7mol／L睾酮处理细胞6h后，TNF-α mRNA表达分别增加1．78倍和1．87倍（均P〈0．05），MCP-1 mRNA的表达也显著增加（均P〈0．01）；②睾酮处理6h后TNF-α和MCP-1的分泌量的变化差异均无统计学意义；睾酮处理24h后，TNF-α和MCP-1的分泌量显著增加（均P〈0．05）；10^-7mol/L睾酮处理组的分泌量显著高于10^-9mol/L睾酮处理组（均P〈0．05）。（2）随着睾酮处理时间的增加，p-NF—κB的表达增加，60min达峰值（2．49±0．40）倍，P〈0．05，以后逐渐降低。（3）PDTC预处理后再用睾酮处理6h，TNF-α和MCP-1 mRNA的表达均低于同浓度单纯睾酮处理组（均P〈0．05），但仍高于空白对照组（均P〈0．05）。PDTC预处理后再用睾酮处理24h，TNF-α和MCP-1的分泌量均低于同浓度单纯睾酮处理组（除10^-9mol/L睾酮组外，均P〈0．05），但仍高于空白对照组（均P〈0．05）。结论睾酮能促进离体的小鼠RAW264．7巨噬细胞TNF-α和MCP-1表达。NF—κB的活化是介导睾酮这一效应的分子机制之一。
Objective To investigate the role of androgen in TNF-α and MCP-1 expression in RAW264. 7 macrophage and its molecular mechanism. Methods （ 1 ） RAW264. 7 macrophage was treated with 10^-9 mol/L or 10^-7 moL/L testosterone （T） and then subjected to the measurement of：（1）the cellular expression of TNF-α and MCP-1 mRNA by RT-PCR ; （2）the expression of TNF-α and MCP-1 in cell culture supernatant by ELISA; （2） The expression of phospho-NF-κB after treatment with T was measured by Western blot. （3） Cells were pre-incnbated with 10-4 moL/L PDTC （an inhibitor of NF-κB） for 1 hour, followed by T treatment. Expression of mRNA and supernatant levels of TNF-α and MCP-1 were measured by RT-PCR and ELISA. Results （1）（1） After a 6 h treatment with 10^-9 mol/L or 10^-7 mol/L T, the expression of TNF-α mRNA increased by 1.78 and 1.87 folds, MCP-1 by 1.58 and 1.66 folds respectively （all P 〈 0. 05 ）. （2） Incubated with both concentration of T for 6 h showed no significant changes of supernatant levels of TNF-α and MCP-1. After a 24 h treatment, the levels of TNF-α and MCP-1 increased significantly （all P 〈0. 05） while more significant increase was found in 10-7 mol/L T group （P 〈0.05）. （2） The expression of phospho-NF-κB （set276） increased significantly after cells were treated with 10^-7 mol/L T for 30 rain （P〈0. 05） and peaked at 60 rain. （3） With 1 h PDTC pre-incubation, T （10^-9mol/L or 10^-7 mol/L） treatment for 6 h led to a lower mRNA expression and 24 h led to lower supernatant levels of TNF-α and MCP-1 than those without （P 〈 0.05 ）. However, both cellular and supernatant expression of TNF-α and MCP-1 with PDTC pre-incubation were still higher than those of blank controls （ all P 〈 0. 05, except for TNF-α in 10-9 mol/L T treatment）. Conclusion Testosterone can increase TNF-α and MCP-I expression in RAW264. 7 macrophage in vitro. Activation of cellular NF-κB by testosterone may be one of underlying molecular mechanisms.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2009年第35期2500-2503,共4页
National Medical Journal of China
基金
国家自然科学基金（30672223）
