PTEN对巨噬细胞炎性细胞因子分泌的影响及其机制研究

Effect of PTEN on TNF-alpha secretion in RAW264.7 cells and its mechanism

目的:PTEN/PI-3K/Akt通路在LPS介导的炎症和内毒素血症中可能具有重要的调控作用,PTEN与LPS刺激引起的炎性介质增多之间的关系尚不明确。文中观察PTEN对LPS攻击所致RAW 264.7细胞TNF-α分泌水平的影响并探讨其可能的机制。方法:①以RAW 264.7细胞为研究对象,第1组为对照组,第2组和第3组为PTEN抑制剂组,分别提前1 h加入PTEN抑制剂200、100 nmol/L,以LPS刺激6 h后收集细胞培养上清,用ELISA法检测上清中TNF-α的水平。②将NF-κB-LUC质粒与pRL-CMV质粒共转染RAW 264.7细胞,以LPS处理细胞6 h后通过检测NF-κB反应性荧光素酶报告基因表达,观察PTEN在LPS攻击巨噬细胞时对NF-κB转录激活的影响。结果:与正常细胞相比,抑制PTEN可使LPS诱导的TNF-α分泌减少,NF-κB活性减弱。结论:PTEN可上调LPS诱导的巨噬细胞TNF-α分泌水平,可能是通过激活NF-κB的转录活性增加TNF-α的分泌水平,从而在内毒素所诱导的炎症反应中发挥重要的调控作用。

Objective： The PTEN/PI3K/Akt pathway plays an important role in regulating inflammation and sepsis induced by lipopolysaccharide（LPS）.However,the correlation of PTEN with LPS-induced secretion of inflammatory cytokines is not clear.The purpose of this study was to explore the effect of PTEN on TNF-α secretion in the RAW264.7 cells treated with LPS and its mechanism.Methods： RAW264.7 cells were left untreated or treated with the PTEN inhibitor for 1 hour and then stimulated with 1μg/ml LPS for 6 hours.The supernatants were collected,and the levels of TNF-α measured by ELISA.The NF-κB plasmids containing the LUC reporter gene and control plasmid pRL-CMV were cotransfected into the RAW264.7 cells using LipofectamineTM2000.Transfected cells were left untreated or treated with the inhibitor of PTEN for 1 hour followed by stimulation with 1μg/ml LPS for 6 hours.Then the cells were lysed in 100μl of 5×Passive Lysis Buffer.Dual-Luciferase activity was measured using the Dual-Luciferase Reporter Assay System.Results： Compared with the untreated group,the cells treated with the PTEN inhibitor showed an obvious decrease in the LPS-induced secretion of TNF-α and the transcription activity of NF-κB.Conclusion： PTEN upregulates the LPS-induced secretion of TNF-α,and plays an important role in regulating LPS-induced inflammation,probably by increasing the transcription activity of NF-κB in macrophages.