摘要
先分离培养小鼠腹腔巨噬细胞,经差速贴壁法纯化后,随机分为6组:空白对照组、0.5mg/L脂多糖(LPS)组、10-6 mol/L孕酮(P4)组、LPS+10-5 mol/L P4组、LPS+10-6 mol/L P4组、LPS+10-7 mol/L P4组。各组在处理12、24h分别提取上清液,ELISA法测TNF-α和IL-1β的含量;各组在处理24h分别提取细胞总RNA,用RT-PCR法测TLR4、CD14、MD2mRNA的表达。结果显示,处理12、24h,0.5mg/L LPS组TNF-α和IL-1β的含量均极显著高于对照组(P<0.01);10-6 mol/L P4组与对照组差异不显著(P>0.05);LPS+10-5 mol/L P4组极显著低于对照组(P<0.01);LPS+10-6 mol/L P4组显著低于对照组(P<0.05);而LPS+10-7 mol/L P4组TNF-α的表达差异不显著(P>0.05),IL-1β的表达差异显著(P<0.05)。说明P4可降低LPS刺激小鼠腹腔巨噬细胞TNF-α和IL-1β的分泌,且呈剂量依赖关系。LPS单独处理,TLR4和CD14mRNA的表达极显著高于对照组(P<0.01);10-6 mol/L P4单独处理与对照组无显著差异(P>0.05);分别添加1-5、10-6、10-7 mol/L P4组均极显著降低LPS诱导TLR4和CD14mRNA的表达(P<0.01),而MD2mRNA的表达差异不显著(P>0.05)。说明P4可极显著降低LPS刺激小鼠腹腔巨噬细胞TLR4和CD14mRNA表达,但对MD2mRNA表达影响不显著。结果显示,P4能抑制LPS刺激的小鼠腹腔巨噬细胞TNF-α和IL-1β的分泌,此过程与细胞TLR4和CD14表达下降相关,而与MD2的表达无关。
The objective of this experiment was to evaluate the effects of progesterone with different concentrations on the expression of TNF-α,IL-1β,TLR4,CD14,MD2 in mouse peritoneal macrophages stimulated by LPS.Then the mechanism of progesterone interferes in the expression of inflammatory cytokines regulated by TLR4,CD14 and MD2 gene was revealed.Cultured mouse peritoneal macrophages were separated and purified by differential adhesion method.After that,mouse peritoneal macrophages were randomly divided into six groups：blank control group,0.5 mg/L LPS treated group,10-6 mol/L progesterone treated group,LPS＋10-5 mol/L progesterone treated group,LPS＋10-6 mol/L progesterone treated group,and LPS＋10-7 mol/L progesterone treated group.Supernatants of these groups were respectively extracted at 12 h and 24 h after differential treatment,and the concentrations of TNF-α,IL-1β here were examined by ELISA.Total RNA of macrophages was extracted at 24 h after different treated and examined the mRNA expression of TLR4,CD14 and MD2 by RT-PCR.The results showed that at 12 h and 24 h,compared with blank control group the concentrations of TNF-α and IL-1β in 0.5 mg/L LPS treated group increased significantly（P0.01）;the concentrations of TNF-α and IL-1β in 10-6 mol/L progesterone treated group had no significant change（P0.05）.LPS＋10-5 mol/L progesterone treated group extremely significantly lower than blank control group（P0.01）;LPS＋10-6 mol/L progesterone treated group significantly lower than blank control group（P0.05）;the expression of TNF-α in LPS＋10-7 mol/L progesterone treated group had no significant difference（P0.05）,while the expression of IL-1β had significant difference（P0.05）.These above illustrate that progesterone can reduce the secretion TNF-α and IL-1β in mouse peritoneal macrophage stimulated by LPS with a dose-dependent relationship.The mRNA expression of TLR4 and CD14 significantly higher than blank control group by LPS treated（P0.01）,while 10-6 mol/L progesterone treated group had no significant difference compared with blank control group（P0.05）.10-5,10-6,10-7 mol/L progesterone treated group significantly decreased the mRNA expression of TLR4 and CD14 stimulated by LPS（P0.01）,while the mRNA expression of MD2 had no significant difference（P0.05）.The results above illustrate that progesterone can significantly reduce the mRNA expression of TLR4 and CD14 in mouse peritoneal macrophages stimulated by LPS,but the effect on the mRNA expression of MD2 is not significant.The results indicated that progesterone could inhibits the secretion of TNF-α,IL-1β in mouse peritoneal macrophages stimulated by LPS.This procedure is associated with the decrease of the expression of TLR4 and CD1,while it is not related do with the expression of MD2.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2012年第10期1526-1531,共6页
Chinese Journal of Veterinary Science
基金
山东省教育厅高等学校科技计划资助项目(J10LC03)
