【作者】 张少峰；

【导师】 吴逸明；

【作者基本信息】 郑州大学 ， 劳动卫生与环境卫生学， 2011， 硕士

【摘要】 背景流行病学资料和遗传学资料证明慢性炎症能够促进肿瘤发生发展的进程。肿瘤相关巨噬细胞(Tumor-associated macrophage, TAM)是炎症与癌症之间联系的主要调节因素。肿瘤微环境在造就TAM的同时,TAM也表现出促进肿瘤发生发展的功能。核转录因子kB(Nuclear factor-kappa B, NF-kB)是炎症和癌症之间关键的连接点之一。煤焦沥青(coal tar pitch, CTP)引发的肺癌是焦炉工人职业肿瘤中的主要组成部分。炎症在煤焦沥青这一混合物致癌过程中可能作用的机制如何,少有报道,需要进一步研究。研究利用中温煤焦沥青烟提取物(Coal tar pitch smoke extract, CTP)诱导永生化人支气管上皮细胞(Human Bronchial Epithelial Cells, BEAS-2B)恶性转化,建立BEAS-2B细胞与人单核细胞系THP-1细胞共培养模型,观察BEAS-2B细胞在恶性转化过程中的恶性变指标,以期在体外实验中探索肿瘤相关巨噬细胞在肺癌发生过程中可能发挥的作用,之后通过研究染毒细胞NF-kB基因及蛋白的改变,探讨煤焦沥青致细胞癌变过程中NF-kB的作用,以期为肺癌提供早期诊断的生物标识及预防煤焦沥青导致的职业性肺癌提供理论基础。方法以BEAS-2B细胞为研究对象,设立煤焦沥青烟提取物染毒(煤焦沥青,CTP)组、苯并(a)芘(B(a)P)组、二甲基亚砜(DMSO)对照组、BEAS-2B与THP-1细胞共同培养组,建立癌变模型,应用软琼脂集落形成实验、染色体数目畸变分析、流式细胞仪测定细胞周期分析细胞的恶变情况,采用RT-PCR检测NF-Kb p65 mRNA的表达变化,Western Blot检测NF-kB p65蛋白表达。结果1.细胞周期测定：细胞周期测定显示10代、20代、30代细胞煤焦沥青提取物染毒组、苯并(a)芘组、共培养组较DMSO组细胞G1期细胞减少,S期细胞增加,表明染毒的3组细胞生长有加速倾向。20代时共培养组细胞S期细胞多于煤焦沥青提取物染毒组(P=0.001)和苯并(a)芘组(P=0.001),差异有统计学意义,而到30代时这3组细胞S期细胞所占比例差别更加明显。2.染色体数目畸变分析：染色体数目异常在实验的早期已经显现,表现为非整倍体和多倍体比例增加,二倍体数目减少。3.软琼脂克隆形成：10代各组细胞形成克隆数在20个以内。20代时共培养组克隆形成率高于煤焦沥青提取物染毒组和苯并(a)芘组,差异有统计学意义(P=0.001,P=0.001),煤焦沥青提取物染毒组克隆形成率与苯并(a)芘组相比较差异无统计学意义(P=0.090)。30代细胞组,苯并(a)芘组、煤焦沥青提取物染毒组克隆形成率低于共培养组,差异有统计学意义(P=0.001,P=0.001),而煤焦沥青提取物染毒组克隆形成率高于苯并(a)芘组,差异有统计学意义(P=0.032)。4. NF-kB p65基因与蛋白表达：第10代细胞即表现出暴露组NF-kB p65表达水平高于DMSO组,而暴露组之间NF-kB p65表达无差异。第20代细胞共培养组表达较煤焦沥青组、苯并(a)芘组NF-kB p65表达升高,苯并(a)芘组与煤焦沥青组相比NF-kB p65表达无差异。30代细胞中,苯并(a)芘组与煤焦沥青组NF-kB p65表达低于共培养组,煤焦沥青组NF-kB p65表达水平高于苯并(a)芘组。结论单核巨噬细胞能够加速煤焦沥青诱导的人支气管上皮细胞恶变,且NF-kB在此过程中可能起重要作用。更多还原

【Abstract】 Background Epidemiological and genetic data revealed that chronic inflammation could promote tumorigenesis. Tumor-associated macrophages (TAM) was major regulators between inflammation and cancer. TAM which was created in the tumor microenvironment displayed the function to promote tumorigenesis. Nuclear factor kB (NF-kB) was a key connection between inflammation and cancer. Lung cancer induced by coal tar was a main component of occupational tumors in coke oven workers. There were few reports about the mechanism how inflammation performed in the process of carcinogenesis induced by the mixture of coal tar pitch.In this study, middle temperature coal tar pitch smoke extract was used to induce the malignant transformation of immortalized human bronchial epithelial cells (Human Bronchial Epithelial Cells, BEAS-2B). The BEAS-2B and THP-1 cells co-cultured model was established to explore the function and effect of TAM in the malignant transformation of the BEAS-2B cells. In order to provide early diagnosis biomarkers of lung cancer and to provide a theoretical basis for the prevention of occupational lung cancer caused by CTP, the functions of NF-kB signaling pathway in the process of carcinogenesis were explored by studying the changes of NF-kB gene and protein expression.Methods BEAS-2B cells were used as objects. Four groups were established, the extracts of coal tar pitch exposure group (CTP group), benzo(a)pyrene (B(a)P) group, dimethyl sulfoxide (DMSO) group, BEAS-2B and THP-1 co-culture (Co-culture group) group. Carcinogenesis model was established. The malignant of cells was estimated by soft agar colony formation assay, chromosome aberrations and cell cycle tests. The expression of NF-kB p65 mRNA was detected by RT-PCR and the expression of NF-kB p65 protein was detected by Western Blot.Results:1 Cell cycle tests:Cell cycle analysed by Flow cytometry showed that compared with DMSO group, the extract of coal tar pitch exposure group, benzo (a) pyrene group and co-culture group in 10th generation,20th generation and 30th generation displayed a reduction of Gl phase cells and an increase of S phase cells. That indicated the three groups of exposed cells showed tendency of accelerated growth. The S phase cells of the co-culture group in the 20th generations were more than extraction of coal tar pitch smoke exposure group and benzo(a)pyrene group, the difference was statistically significant(P=0.001, P=0.001). To the 30th generations, the difference of proportion of S phase cells were more obviously.2 Aberrations of chromosome number: The number of chromosome abnormalities could be observed in early stage of the experiment, the performance was an increase of the ratio of aneuploid and polyploid, and a decrease in the number of diploid..3 Soft agar colony formation:The number of the colonies was less than 20 in the 10th generation of each group. To the 20th generation cells, compared with the co-cultured group, the number of colonies formation in the extraction of coal tar pitch smoke exposure group and benzo(a)pyrene group and the difference were statistically significant(P=0.001, P=0.001). The colonies formation of the co-culture group was more efficiency than the other two groups. To the 30th generation cells, compared with benzo(a)pyrene group and extracts of coal tar pitch smoke exposure group, there were more colonies in the co-culture group and the difference was statistically significant(P=0.001, P=0.001). Colonies of the extracts of coal tar pitch smoke exposure group were higher than the benzo(a)pyrene group and the difference was statistically significant(P=0.032).4 NF-kB p65 mRNA and protein expression:In the 10th generation cells, compared with DMSO group, the expression of NF-kB p65 of coal tar pitch group, benzo(a)pyrene group and co-culture group was increased. However, there was no difference among benzo(a)pyrene group, the coal tar pitch group and the co-culture group. In the 20th generation cells, analysis showed exposed group expressed high levels of NF-kB p65 than DMSO group, and co-culture group expressed high levels of NF-kB p65 than the others exposed group. In the 30th generation cells, co-culture group expressed high levels of NF-kB p65 than the others exposed group.The expression of NF-kB p65 in benzo(a)pyrene group was statistically lower than the coal tar pitch group.Conclusion:The extracts of coal tar pitch smoke could induce malignant transformation of human bronchial epithelial cells. NF-kB signaling pathway involved in the malignant transformation process of BEAS-2B which was accelerated by the present of THP-1 cells.更多还原