摘要目的 研究吸烟促进小鼠肺癌生长的作用机制.方法 使用髓系细胞RelA/p65敲除小鼠(RelA/p65-/-)和对照WT小鼠建立小鼠转移性肺癌模型,分为吸烟组和空气组,计数小鼠肺表面肿瘤数并对肿瘤大小进行评价,利用Kaplan-Meier生存曲线分析小鼠的生存情况.采用免疫组化法检测小鼠肿瘤增殖抗原Ki-67、肿瘤坏死因子α(TNF-α)和CD68的表达,采用Western blot法检测肿瘤细胞cyclin D1和c-myc的表达,采用原位末端转移酶标记技术(TUNEL)检测肿瘤细胞的凋亡,采用酶联免疫吸附(ELISA)试验检测肺肿瘤组织中TNF-α、白细胞介素6(IL-6)和KC的浓度.结果 WT小鼠在受到香烟刺激后,肺重量、肿瘤个数和最大肿瘤直径分别为(1.5±0.1)g、(64.8±4.1)个和(7.6±0.2)mm,均明显高于未受香烟刺激的WT小鼠(均P＜0.05);而RelA/p65-/-小鼠的肺重量、肿瘤个数和最大肿瘤直径在香烟刺激前后无明显变化(均P ＞0.05).生存分析的结果显示,WT吸烟组小鼠的生存时间明显低于WT空气组(P＜0.05),但RelA/p65-/-小鼠的生存时间均明显长于WT小鼠(均P＜0.05).WT空气组、WT吸烟组、RelA/p65-/-空气组和RelA/p65-/-吸烟组小鼠Ki-67阳性肿瘤细胞的百分比分别为(43.4±2.9)％、(60.6±5.4)％、(12.8±3.6)％和(15.0±4.2)％,香烟刺激后,WT小鼠Ki-67阳性肿瘤细胞百分比明显增高(P＜0.05),而RelA/p65-/-小鼠Ki-67的表达无明显变化(P＞0.05).WT空气组、WT吸烟组、RelA/p65-/-空气组和RelA/p65-/-吸烟组小鼠肿瘤细胞的凋亡率分别为(11.6±1.7)％、(13.0±2.0)％、(13.2±2.0)％和(11.0±1.4)％,差异均无统计学意义(均P＞0.05).WT小鼠受到香烟刺激后,肿瘤细胞中cyclin D1和c-myc蛋白的表达明显增强;而RelA/p65-/-小鼠受到香烟刺激后,肿瘤细胞中cyclin D1和c-myc蛋白的表达没有明显变化.香烟刺激后,渗入到肿瘤组织中的巨噬细胞的数量无论在WT还是RelA/p65-/-小鼠中均明显增加(P＜0.05).WT小鼠在香烟刺激后,肿瘤组织中TNF-α、IL-6和KC的表达均明显升高(P＜0.05).结论 吸烟促进了小鼠肺癌的生长,髓系细胞的RelA/p65基因介导了吸烟的促肿瘤生长效应,由RelA/p65基因调控的TNF-α表达可能参与了肺癌的发展.

abstractsObjective The aim of this study was to investigate the mechanism of cigarette smoking (CS)-induced lung cancer growth in mice.Methods RelA/p65-/-mice and WT mice were used to establish mouse models of lung cancer.Both mice were divided into two groups:air group and CS group,respectively.Tumor number on the lung surface was counted and maximal tumor size was evaluated using HE staining.Kaplan Meier (K-M) survival curve was used to analyze the survival rate of the mice.Expression of Ki-67,TNF-α and CD68 in the tumor tissue was determined by immunohistochemical analysis,and cyclin D1 and c-myc proteins were examined by Western blot.Apoptosis of tumor cells was analyzed using TUNEL staining.The concentrations of inflammatory cytokines TNF-α,[L-6 and KC in the mouse lung tissues were evaluated by ELISA.Results Compared with the WT air group,the lung weight,lung tumor multiplicity,as well as maximum tumor size in the WT mice exposed to CS were (1.5 ±0.1)g,(64.8 ±4.1) and (7.6 ± 0.2) mm,respectively,significantly increased than those in the WT mice not exposed to CS (P ＜ 0.05 for all).However,there were no statistically significant differences between RelA/p65-/-mice before and after CS exposure (P ＞ 0.05 for all).Kaplan-Meier survival analysis showed that CS exposure significantly shortened the life time of WT mice (P ＜ 0.05),and deletion of RelA/p65 in myeloid cells resulted in an increased survival compared with that of the WT mice (P ＜0.05 for all).The ratios of Ki-67 positive tumor cells were (43.4 ±2.9)％,(60.6 ±5.4)％,(12.8 ±3.6)％ and (15.0 ±4.2)％ in the WT air group,WT CS groups,RelA/p65-/-air groups and RelA/p65-/-CS groups,respectively.After smoking,the number of Ki-67-positive cells was significantly increased in the WT mice (P ＜ 0.05).However,there was no significant difference between the RelA/p65 /-groups before and after smoking (P ＞ 0.05).The apoptosis rate of WT air,WT CS,RelA/p65-/-air and RelA/p65-/-CS groups were (11.6 ± 1.7)％,(13.0 ± 2.0) ％,(13.2 ± 2.0) ％ and (11.0 ± 1.4) ％,respectively,with no significant difference among them (P ＞ 0.05).Expression of cyclin D1 and c-myc was induced in response to CS exposure in lung tumor cells of WT mice.In contrast,their expressions were not significantly changed in the RelA/p65-/-mice after smoke exposure.CS exposure was associated with an increased number of macrophages infiltrating inthe tumor tissue,in both WT and RelA/p65-/-mice (P ＜ 0.05).The concentrations of IL-6,KC and TNF-α were significantly increased after CS exposure in the lungs of WT mice (P ＜ 0.05).Conclusions Cigarette smoking promotes the lung cancer growth in mice.Myeloid cell RelA/p65 mediates CS-induced tumor growth.TNFα regulated by RelA/p65 may be involved in the lung cancer development.