阻断巨噬细胞NFκB-p65通路对香烟促进非小细胞肺癌NCI-H520细胞株增殖的影响  

Study on effect of NFKB-p65 pathway inhibition in macrophages on cigarette smoke induced non-smallcell lung cancer cell NCI-H520 cells proliferation

作　　者：李冬[1] 王旋[2] 周宏[1] 万海英[1] 

机构地区：[1]同济大学附属同济医院检验科,上海200065 [2]上海市普陀区人民医院药剂科

出　　处：《肿瘤研究与临床》2012年第9期584-588,共5页Cancer Research and Clinic

基　　金：上海市普陀区卫生系统自主创新科研资助项目（PTKW09-B02）

摘　　要：研究香烟对非小细胞性肺癌（NSCLC）NCI-H520细胞株增殖的影响以及巨噬细胞在此过程中的作用。方法 采用“Transwell？ Inserts”细胞培养室培养系统共培养人原代巨噬细胞或分化为巨噬细胞的人单核巨噬细胞株U937细胞和NCI-H520细胞。应用免疫印迹法和凝胶电泳迁移实验检测NF？B通路的激活；应用NF？B -p65 shRNA干扰质粒抑制U937细胞p65 表达；利用BrdU ELISA分析香烟抽提物（CSE）对NCI-H520细胞的促增殖效应；ELISA分析炎症细胞因子TNF？和IL-6的释放。结果 共培养及香烟刺激增强了人巨噬细胞的NF？B -p65的入核，以及同靶基因结合的活力。与对照组相比， NCI-H520细胞经不同浓度CSE单独处理，并没有发现肺癌细胞增殖明显加快（p〉0.05）；而经过与人原代巨噬细胞或分化为巨噬细胞的U937细胞共培养4天，不仅发现巨噬细胞明显促进NCI-H520细胞的增殖（p〈0.01），而且观察到CSE的刺激显著增强这一效应（p〈0.01）。NF？B -p65shRNA干扰质粒明显抑制了U937细胞的NF？B -p65表达，抑制了NF？B通路的激活，减弱了CSE对NCI-H520细胞增殖的影响（p〈0.01），同时也抑制了炎性因子IL-6和TNF？释放（p〈0.01）。结论 香烟通过巨噬细胞的中介促进NCI-H520细胞的增殖。采用RNA干扰技术阻断巨噬细胞的NF？B通路显著地抑制香烟促进的肺癌细胞的增殖，削弱香烟引起的巨噬细胞的炎症因子释放可能是其作用机制。Objective To investinute the mechanism of cigarotte smoke promotes non-small cell lung cancer （NSCLC） cells NCI-H520 cells proliferation mediated by macrophages, and the related mechanisms. Methods To coculture NCI-H520 cells with primary macrophages or U937 cells, the “Transwell？ Inserts” system was used in coculture model. NF？B activation was confirmed by electrophoretic mobility shift assay （EMSA） and western blot analysis; U937 cells were transfected with NF？B -p65 shRNA plasmid to abrogate the NF？B activation; by BrdU ELISA, the effect of cigarette smoke extract （CSE） promoted NCI-H520 cells proliferation were assessed; inflammatory factors TNF？ and IL-6 were analysed by ELISA. Results Exposure of CSE enhanced NF？B -p65 nucleus translocation, and activated the NFκB pathway. CSE did not promote NCI-H520 cells proliferation alone （p〉0.05）, but after 4 days coincubation with macrophages, the proliferation of NCI-H520 cells was significantly increased （p〈0.01）, addition of CSE to the coculture much more enhanced this effect （p〈0.01）. RNAi to NF？B -p65 significantly reduced NF？B -p65 protein expression and inhibited NF？B activation in U937 p65- cells, and markedly inhibited U937cells induced proliferation of NCI-H520 cells and IL-6, TNF？ secretion （p〈0.01）. Conclusions Cigarette smoke promotes NCI-H520 cells proliferation mediated by macrophages. Blockade of NFκB pathway with RNAi in macrophages can reduced cigarette smoke induced inflammatory factors secretion in macrophages, and significantly inhibit cigarette smoke promoted tumor proliferation.

关 键 词：巨噬细胞 吸烟 癌 非小细胞肺 NF-ΚB 炎症因子 

分 类 号：R734.2[医药卫生—肿瘤]