肿瘤坏死因子-α和NF-κB在单核巨噬细胞促煤焦沥青烟气提取物诱导BEAS-2B细胞恶性转化中的作用

Tumor necrosis factro-α and NF-KB play a role in macrophage-like THP-1 cells promoting coal tarpitch extract-induced tumorigenic transformation of human bronchial epithelial cells

目的探讨肿瘤坏死因子-α（TNF-α）和NF．KB在人单核巨噬细胞系（THP-1）细胞促煤焦沥青烟气提取物（CTPE）诱导人支气管上皮细胞系（BEAS-2B）细胞恶性转化中的作用。方法在第10代BEAS-2B和THP-1细胞共培养组中分别加入NF—KB的抑制剂一吡咯烷二硫代氨基甲酸（PDTC）和TNF-α中和抗体，用细胞周期检测、染色体核型分析和软琼脂集落形成实验检测第20代PDTC组和TNF-α中和抗体组BEAS-2B细胞的恶性转化情况，并与第10代、第20代共培养组进行比较；采用实时定量PCR和免疫印迹法（Westernblot）法检测各组BEAS．2B细胞中TNFR相关因子2（TRAF2）和细胞周期蛋白D1（CyclinDl）基因及其蛋白的表达。结果第20代PDTC组和TNF-α中和抗体组BEAS．2B细胞中S期细胞的比例分别为（33．97％±2．16％）和（34．29％±2．04％），明显低于第20代共培养组（44．46％±0．83％％），差异有统计学意义（P〈0．05）。第20代PDTC组和TNF-α中和抗体组100个细胞中出现异常染色体核型的细胞数量为40个和37个，而第20代共培养组为75个，差异有统计学意义（P〈0．05）。第20代PDTC组软琼脂集落形成数（15．17±2．48）个，集落形成率为（1．51‰±0．25‰），第20代TNF—α中和抗体组集落形成数为（13．33±2．58）个；集落形成率为（1．33‰±0．26‰），均明显低于20代共培养组[集落形成数：（172．33±12．09）个和集落形成率：（17．23‰±1．20‰）]，差异有统计学意义（P〈0．05）；PDTC组和TNF．d中和抗体组TRAF2和CyclinD1基因及其蛋白的表达明显低于第20代共培养组，差异有统计学意义（P〈0．05）。结论TNF．仅和NF-KB在THP-1细胞促进煤焦沥青烟气提取物诱导BEAS-2B细胞恶性转化的早期阶段发挥重要的作用，并可能通过影响TRAF2和CyelinD1的表达促使细胞恶性变发生。

Objective To characterize the role of tumor necrosis factro-ot （TNF-α） and NF-KB play a role in macrophage-like THP-1 cells promoting coal tar pitch extract （CTPE）-induced tumorigenic transformation of human bronchial epithelial cells （BEAS-2B）. Methods From passage 10, CTPE-induced BEAS-2B cells co- cultured with THP-1 cells were treated with NF-KB inhibitor-Pyrrolidine dithiocarbamate （PDTC） every 3 passages and TNF-α antibody every passage. Alterations of cell cycle, karyotype and colony formation in soft agar of BEAS-2B cells at passages 20, indicative of tumorigenecity, were determined, respectively. In addition, mRNA and protein levels of TNF receptor associated factor2 （TRAF2） and Cyclin D 1 in BEAS-2B cells were measured with Real Time-PCR and Western blot, respectively. Results The percentages of S-phase BEAS-2B cells at passage 20 in PDTC group and TNF-α antibody group were （33.97±2.16）% and （34.29±2.04）% respectively, which were less than that in Co-euhure＋CTPE group of 20th passage [（44.46±0.83）%], P〈O.05; The number of cells with aneuploidy in 100 cells in 20th passage PDTC group and TNF-α antibody group were 40 and 37, and there were significantly different when comparing to that of 20th passage Co-euhure＋CTPE group （75）; The number of colony formation and the rate of colony formation of BEAS-2B cells in soft agar at passage 20 in PDTC group were （ 15.17 ±2.48） and （ 1.51‰±0.25‰）, （ 13.33±2.58） and （ 1.33‰±0.26‰） in TNF-α antibodygroup, which were less that those in 20th passage Co-cuhure＋CTPE group [（172.33±12.09） and （17.23‰± 1.20‰）], P〈0.05; at the same time, the mRNA and protein levels of TRAF2 and Cyclin D1 in BEAS-2B cells were decreased after PDTC and TNF-α antibody treatment. Conclusion TNF-α and NF-KB could play an important role in THP-1 cells promoting coal tar pitch extract-induced tumorigenic transformation of BEAS-2B cells by influencing the expression of TRAF2 and Cyelin D1.