肿瘤微环境在神经纤毛蛋白1诱导的辐射抗性中的作用机制

The mechanisms of tumor microenvironment in neuropilin 1-induced radiation resistance

目的 观察神经纤毛蛋白1（NRP1）对肺癌细胞炎性微环境及迁移微环境的影响，并探讨肿瘤微环境中的炎性因子和趋化因子在NRP1诱导的肺癌细胞辐射抗性中的作用机制。方法 建立NRP1LowA549和A549-RR细胞模型，并鉴定两种细胞模型中NRP1的表达水平。利用三维培养技术分别建立A549＋Jurkat、NRP1LowA549＋Jurkat、A549-RR＋Jurkat三维共培养模型；A549＋HLF-1、NRP1LowA549＋HLF-1、A549-RR＋HLF-1三维共培养模型，同时设立对照组和照射组。共培养2 d后，对照射组进行10 Gy X射线照射。24 h后收集各种模型的培养基上清液，利用cytometric bead array（CBA）方法通过流式细胞仪检测相关因子的表达情况。结果 在NRP1LowA549细胞中NRP1的表达明显受到抑制，而在A549-RR细胞中NRP1的表达明显升高。在炎性微环境方面，发现IL-12p70与TNF在A549-RR＋Jurkat组中，表达明显低于A549＋Jurkat组（t=3.88、5.34，P〈0.05），但经照射后明显升高（t=8.49、5.92，P〈0.05）；而IL-6及IL-8表达则相反，在A549-RR＋Jurkat组中，表达明显高于A549＋Jurkat组（t=38.30、30.02，P〈0.05），但经照射后明显降低（t=14.39、9.78，P〈0.05）。IL-1β、IL-10表达量在各组中经照射后与对照组相比均有不同程度的降低（t=2.80~11.22，P〈0.05）。在迁移微环境方面，发现与A549＋HLF-1组相比，RANTES、MCP-1在NRP1LowA549＋HLF-1组中，均呈现表达升高（t=6.07、4.04，P〈0.05），在A549-RR＋HLF-1组中表达明显降低（t=15.50、14.62，P〈0.05）的趋势。照射后，RANTES、MCP-1在各组中与对照组相比均不同程度降低（t=2.23、8.45、16.68，P〈0.05）。与其不同的是，IP-10、CXCL8（IL-8）在其他各组中表达量均低于A549＋HLF-1组，且在NRP1LowA549＋HLF-1组中降幅最大（t=31.86、29.79、6.62、3.85，P〈0.05）。结论 NRP1对肺癌炎性微环境及迁移微环境中相关炎性因子和趋化因子的表达具有明显影响，且这些因子对肺癌细胞辐射抗性的调控具有重要作用。

Objective To study the effect of neuropilin 1（NRP1） on the inflammatory and migration of tumor microenvironment and explore the role of inflammatory cytokines and chemokines in NRP1-induced radiation resistance in tumor microenvironment of lung cancer cells.Methods NRP1LowA549 and A549-RR cell models were constructed and the expression levels of NRP1 in these models were identified. The three-dimensional co-culture models of A549＋Jurkat, NRP1LowA549＋Jurkat, A549-RR＋Jurkat and A549＋HLF-1, NRP1LowA549＋HLF-1, A549-RR＋HLF-1, and control group and irradiated group were established. After co-culturing 2 days, the cells were irradiated with 10 Gy X-rays. After 24 h, the culture medium supernatant of each model was collected and the expressions of related factors were detected by flow cytometry using cytometric bead array （CBA）. Results The expression of NRP1 was inhibited in NRP1LowA549 cells but increased significantly in A549-RR cells. In the inflammatory microenvironment, the expressions of IL-12p70 and TNF in A549-RR＋Jurkat group were significantly lower than those in A549＋Jurkat group （t=3.88, 5.34, P〈0.05）. The expressions of IL-6 and IL-8 in A549-RR＋Jurkat group were higher than those in A549＋Jurkat group （t=38.30, 30.02, P〈0.05）, but decreased significantly after irradiation （t=14.39, 9.78, P〈0.05）. The expressions of IL-1β and IL-10 decreased in all groups after irradiation compared with the control group （t=2.80~11.22, P〈0.05）. Compared with A549＋HLF-1 group, the expressions of RANTES and MCP-1 in NRP1LowA549＋ HLF-1 group were significantly higher than those in A549＋HLF-1 group （t=6.07, 4.04, P〈0.05） and A549-RR＋HLF-1 group （t=15.50, 14.62, P〈0.05）. After irradiation, RANTES and MCP-1 were decreased in all groups （t=2.23, 8.45, 16.68,P〈0.05） in comparison with the control group. In contrast, the expression levels of IP-10 and CXCL8 （IL-8） were lower in A549＋HLF-1 group than those in NRP1LowA549＋HLF-1 group （t=31.86, 29.79, 6.62, 3.85, P〈0.05）. Conclusions NRP1 has significant effects on the expression of inflammatory cytokines and chemokines in inflammatory and migrating microenvironment of lung cancer, and these factors play an important role in the regulation of radiation resistance of lung cancer cells.