TLR4-NOD2协同信号传递增强树突状细胞活性的作用

Role of Toll-like receptor 4-NOD2 synergistic signal transduction in enhancing the activity of dendritic cells

目的探讨Toll样受体4(Toll-like receptor 4,TLR4)-核苷酸结合寡聚化结构域样受体2(nucleotide-binding oligomerization domain 2,NOD2)协同信号(T4N2)传递在增强树突状细胞(dendritic cell,DC)活性中的作用。方法对数生长期DC随机分为空白对照1组(RPMI-1640培养基1mL)、脂多糖(lipopolysaccharide,LPS)1组(RPMI-1640培养基1mL+LPS 10μg)、胞壁酰二肽(muramyl dipeptide,MDP)1组(RPMI-1640培养基1mL+MDP 5ng)、LPS+MDP1组(RPMI-1640培养基1mL+LPS 10μg+MDP 5ng),培养24h,采用ELISA法检测4组细胞上清中白细胞介素-6(interleukin-6,IL-6)水平;体外设计并合成针对NOD2基因的慢病毒shRNA干扰载体,转染DC 48h后,将DC分为空白对照2组、LPS2组、MDP2组,LPS+MDP2组,采用ELISA法检测培养24h时细胞上清中IL-6水平。结果培养24h,IL-6水平在LPS1组[(221.00±5.00)ng/L]、MDP1组[(213.3±6.11)ng/L]、LPS+MDP1组[(469.00±3.60)ng/L]均高于空白对照1组[(125.66±4.50)ng/L](P<0.05),且LPS+MDP1组高于LPS1组、MDP1组(P<0.05);沉默NOD2基因后,LPS+MDP2组及MDP2组IL-6水平[(238.66±7.02)、(125.66±7.67)ng/L]低于LPS+MDP1组和MDP1组(P<0.05);空白对照2组及LPS2组IL-6水平[(116.30±5.68)、(207.00±8.54)ng/L]与空白对照1组及LPS1组比较差异无统计学意义(P>0.05)。结论 TLR4、NOD2间存在协同作用并可促进DC活化。

Objective To investigate the role of Toll-like receptor 4（TRL4）-nucleotide-binding oligomerization domain 2（NOD2）synergistic signal transduction in enhancing the activity of the dendritic cell（DC）.Methods The DCs in logarithmic growth phase were divided into blank control group 1（1 mL RPMI-1640 medium）,lipopolysaccharide（LPS）group 1（1 mL RPMI-1640 medium ＋ 10μg LPS）,muramyl dipeptide（MDP）group 1（1 mL RPMI-1640 medium ＋5 ng MDP）,and LPS＋MDP group 1（1 mL RPMI-1640 medium ＋ 10μg LPS ＋ 5 ng MDP）,cultured for 24 h.The level of interleukin-6（IL-6）in the cell supernatant was detected by ELISA.The lentivirus shRNA interfering vector was designed in vitro and synthesized for NOD2 gene.After being transfected for 48 h,DCs were divided into blank control group 2,LPS group 2,MDP group 2 and LPS＋MDP group 2.The IL-6 level in cell supernatant was detected by ELISA after 24 hculture.Results After 24 hculture,the IL-6 level was significantly higher in LPS group 1（（221.00±5.00）ng/L）,MDP group 1（（213.3±6.11）ng/L）,and LPS＋MDP group 1（（469.00±3.60）ng/L）than that in blank control group 1（（125.66±4.50）ng/L）（P〈0.05）,and LPS＋MDP group 1 than that in LPS group 1 and MDP group 1（P〈0.05）.After silencing NOD2 gene,the IL-6 level was significantly lower in LPS＋MDP group 2（（238.66±7.02）ng/L）and MDP group 2（（125.66±7.67）ng/L）than that in LPS＋MDP group 1 and MDP group 1（P〈0.05）,and there were no significant differences between blank control group 2（（116.30±5.68）ng/L）and blank control group 1 as well as between LPS group 2（（207.00±8.54）ng/L and LPS group 1（P〉0.05）.Conclusion There is a synergistic effect between TLR4 and NOD2,which can promote the activation of DCs.