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。 Objective To investigate the effect of phorbol 12-myristate 13-acetate (PMA) on the expression and distribution of MD-2 in the THP-1 cell surface after induced differentiation And its relationship with inflammatory cytokines TNF-α, IL-6 secretion. Methods THP-1 cells were induced by gradient dose (0, 1, 5, 10, 50 and 100 ng / ml) of PMA for 48 h, then the morphology of adherent cells was observed and the adherent rate was calculated. Real-time fluorescence quantitative polymerase chain reaction -qPCR) were used to detect the relative expression of MD-2 and CD14 mRNA on the adherent cell membrane surface. The secretion of secreted MD-2 (sMD-2), TNF-α and IL-6 in the cell culture supernatant was detected by ELISA. The expression of MD-2 protein in adherent cells was detected by Western blot. The distribution and localization of MD-2 in adherent cells were observed by laser scanning confocal microscopy. Results The suspension of THP-1 cells was successfully induced by gradient PMA (1 ~ 100 ng / ml) for 48 h. Laser scanning confocal microscopy showed that MD-2 was mainly expressed in the membrane and cytoplasm after PMA induction and the induced mass concentration was increased The expression of MD-2 and CD14 mRNA in THP-1 cells induced by PMA gradient dose (1 ~ 100 ng / ml) were significantly higher than that before induction by RT-qPCR (P <0.01). There was no significant difference between the induction dose groups (P> 0.05). After the THP-1 cells were induced by PMA gradient dose (1-100 ng / ml), the TNF- (P <0.01). There was a significant difference between before and after induction of sMD-2 (P <0.01) .Western blot analysis showed that the expression of MD-2 protein in PMA-treated group was significantly lower than that in control group There was no significant difference between the induction dose groups before induction. Conclusions 1 ~ 100 ng / ml PMA can induce THP-1 cells to differentiate into mature macrophages. Increasing the induction mass concentration can significantly increase the secretion of inflammatory cytokines TNF-α and IL-6. However, MD-2 expression and distribution had no significant effect.