摘要
目的探索豆蔻酸-佛波醇-乙酸酯(phorbol 12-myristate 13-acetate,PMA)对诱导分化后THP-1细胞膜表面受体髓样分化蛋白-2(MD-2)表达和分布的影响及其与炎症因子TNF-α、IL-6分泌的关系。方法采用PMA梯度剂量(0、1、5、10、50、100 ng/ml)诱导THP-1细胞48 h后观察贴壁细胞形态并计算贴壁率;实时荧光定量聚合酶链式反应(RT-qPCR)检测贴壁细胞膜表面受体MD-2、CD14 mRNA相对表达量,ELISA检测细胞培养上清中分泌性MD-2(sMD-2)、TNF-α和IL-6的分泌水平;进一步通过Western blot检测贴壁细胞的MD-2蛋白表达、激光扫描共聚焦显微镜观察MD-2在贴壁细胞的分布和定位。结果梯度PMA剂量(1~100 ng/ml)48 h成功诱导悬浮THP-1细胞贴壁;激光扫描共聚焦显微镜观察显示PMA诱导后MD-2主要表达于胞膜和胞浆,诱导质量浓度增加对MD-2的表达和分布均无显著影响;RT-qPCR检测THP-1细胞经PMA梯度剂量(1~100 ng/ml)诱导后MD-2和CD14 mRNA相对表达量较诱导前均显著升高(P<0.01),诱导剂量组之间无显著差异(P>0.05);ELISA检测THP-1细胞经PMA梯度剂量(1~100 ng/ml)诱导后,细胞培养上清中的TNF-α、IL-6分泌水平依次显著增加(P<0.01),sMD-2仅诱导前后有明显统计学差异(P<0.01);Western blot检测显示梯度剂量PMA诱导后细胞内MD-2蛋白表达较诱导前显著增加,诱导剂量组之间无显著差异。结论 1~100 ng/ml PMA均可将THP-1细胞诱导分化为成熟巨噬细胞,增加诱导质量浓度可显著提高炎症因子TNF-α、IL-6的分泌水平,但对其膜表面受体MD-2表达和分布无明显影响。
This study aimed to investigate the influence of differentiation inducer PMA on the expression and distribution of membrane surface receptor MD-2 in THP-1 cells,and the correlation of which with the levels of inflammatory factors TNF-α and IL-6 in cell supernatant.After PMA(0,1,5,10,50,100 ng/ml) inducement for 48 h,suspended THP-1 cells transferred as adherent macrophages successfully.Confocal laser scanning microscopy showed that MD-2 mainly expressed on the cellular membrane and cytoplasm,while increasing induction concentrations had no significant effects on expression and subcellular localization.Real-time fluorescence quantitative polymerase chain reaction(RT-qPCR) demonstrated that MD-2 and CD14 mRNA relative expression quantity increased to the level of undifferentiated cells(P < 0.01),but there was no significant difference between PMA-induced groups(P >0.05).By ELISA,we found that levels of IL-6 and TNF-α in the cell culture supernatant increased significantly with the increase of concentration of PMA(P < 0.01),and level of secretory MD-2(sMD-2) increased significantly just between before and after induction(P < 0.01).Furthermore,Western blot confirmed that the protein expression level of total MD-2 increased significantly between before and after induction.In conclusion,THP-1 cells could be differentiated into macrophages with PMA at concentrations of 1-100 ng/ml,and increasing induction concentration could increased inflammatory factors secretion including TNF-α and IL-6,but had no significant effects on expression and distribution of membrane surface receptor MD-2.
出处
《免疫学杂志》
CAS
CSCD
北大核心
2013年第2期121-125,共5页
Immunological Journal
基金
国家自然科学基金(81171848
30700289)
关键词
巨噬细胞
膜受体
髓样分化蛋白-2
炎症因子
Macrophage
Membrane receptor
Myeloid differentiation protein-2
Inflammatory factor
