低氧对人成熟树突状细胞MMP9/TIMP1及CCR7表达的影响

Effects of hypoxia on expression of MMP9/TIMP1 and CCR7 in human monocyte-derived mature dendritic cells

目的:观察低氧对人单核细胞来源成熟树突状细胞(mDCs)趋化因子受体(CCR7),基质金属蛋白酶-9(MMP-9)及其组织抑制剂(TIMP-1)表达的影响,以期为改进DCs疫苗体内迁移能力低下提供新策略。方法:分离制备人外周血单核细胞(PBMCs),采用体外常规培养体系[粒-巨噬细胞集落刺激因子(GM-CSF)与白细胞介素-4(IL-4)共同刺激]诱导DCs,于诱导后第5d加入TNF-α促成熟,48h后将mDCs置低氧环境下(1%O2、5%CO2、94%N2)分别继续培养6h、12h,设常氧对照组(21%O2、5%CO2),收获细胞及培养上清;采用RT-PCR技术检测MMP-9、TIMP-1及CCR7的表达;明胶酶谱法检测培养上清中MMP-9的水平与活性,流式细胞术检测mDCs表面CCR7的表达。结果:RT-PCR及酶谱实验结果显示,低氧6h、12h处理组mDCsMMP-9水平显著下降;与对照组比较,各低氧处理组mDCs均未检测到TIMP-1mRNA表达的变化;转录及细胞表面表达分析结果显示,与常氧对照组比较,低氧6h处理组趋化因子受体CCR7表达显著降低,而12h处理组其表达显著回升至近常氧组水平。结论:低氧下调mDCsMMP-9表达,破坏MMP-9/TIMP-1平衡,可能是DCs疫苗体内迁移能力低下的主要因素之一。趋化因子受体CCR7在mDCs对低氧应答中可能起重要作用。

AIM： To investigate the effect of acute hypoxia on the expression of CCR7, MMP- 9 and TIMP- 1 by mature dendritic cells （DCs）. METHODS： Immature DCs were generated from human peripheral monocytes and induced by treating the cells with TNF - α for 48 h to facilitate maturation. The mature DCs （mDCs） were cultured under hypoxic （ 1% 02 ） or normoxic conditions for 6 h and 12 h, respectively. The gene expressions of MMP - 9, TIMP - 1 and CCR7 in mDCs under hypoxia and normoxia were analyzed by RT - PCR. The enzymatic activity of MMP - 9 was detected in mDCs under the conditions of hypoxia or normoxia using gelatin zymography assay. CCR7 expression on the surface of the mDCs was analyzed using flow cytometry. RESULTS： Compared to normoxic mDCs, MMP9 expression and the level of active - MMP9 in mDCs under hypoxic condition for 6 h or 12 h were downregulated significantly. No change was detected on the expression of TIMP - 1. Interestingly, CCR7 expression by mDCs under hypoxia for 6 h was inhibited notably both on transcription and on the surface expression, whereas CCR7 expression under hypoxic condition for 12 b recovered approximately to the level of the normal control using RT - PCR and flow cytometric analysis. CONCLUSION： Inhibition of MMP9 expression and the disturbance of the balance of MMP - 9/TIMP - 1 on hypoxic condition may play an important role in decreasing the migration activity of DCs - based vaccine in vivo. The changes of CCR7 expression in mDCs under hypoxia suggest that it may contribute to DCs survival on hypoxic microenvironment.