ATM基因变异与多环芳烃暴露致染色体损伤遗传易感性及分子机制研究

发布时间：2018-05-11 12:24

本文选题：多环芳烃 + 遗传易感性　；参考：《中国疾病预防控制中心》2010年博士论文

【摘要】： 焦炉逸散物已被国际癌症研究署定义为1类致癌物,即确定的人类致癌物。焦炉逸散物(Coke oven emission, COE)中的致癌性多环芳烃(Polycyclic aromatic hydrocarbons, PAH)是导致焦炉工肺癌的主要原因,在我国焦炉工肺癌被列为八种法定职业肿瘤之一。研究表明,基因组不稳定性是多环芳烃暴露到肺癌发生的重要生物学事件。多环芳烃暴露工人染色体损伤水平较非暴露组明显升高,但在相同暴露条件下,其损伤水平不尽相同,提示人群易感性的存在。本研究探讨共济失调毛细血管扩张症突变基因(ATM)多态性与多环芳烃暴露致染色体损伤遗传易感性及相关分子机制。结果如下：一、对140名多环芳烃暴露工人和66名非暴露工人外周血淋巴细胞微核率与共济失调毛细血管扩张症突变基因多态性关系的分析显示,在校正性别、年龄、吸烟和自然对数转换的尿1-OHP后,PAH暴露组中ATM基因rs600931 GA和GA+AA基因型携带者外周血淋巴细胞微核率(11.14±6.92‰和10.57±6.82‰)明显高于GG基因型携带者(7.66±5.69‰,P=0.015和0.038)；rs189037 GA和GA+AA基因型个体外周血淋巴细胞微核率(10.99±6.90‰和10.51±6.77‰)明显高于GG基因型携带者(7.72±5.83‰,P=0.018和0.035)；rs624366 GC和GC+CC基因型个体外周血淋巴细胞微核率(11.34±6.74‰和10.73±6.63‰)明显高于GG基因型携带者(7.61±6.07‰,P=0.001和0.003)；rs227092 GT基因型个体外周血淋巴细胞微核率(10.78±6.60‰)明显高于GG基因型携带者(7.91±6.30‰,P=0.025)。单体型对GGGGTGC/AAACATT携带者外周血淋巴细胞微核率(12.05±7.40‰)明显高于GGGGTGC/GGGGTGC携带者(7.51±6.20‰,P=0.007)。二、利用胞质阻滞细胞微核(Cytokinesis-block micronucleus, CBMN)实验探讨ATM干扰细胞和空载质粒细胞对B(a)P诱导所致遗传损伤的差异。结果显示,随着B(a)P作用浓度的增加,ATM干扰细胞和空载质粒细胞CBMN率均逐渐升高,呈明显的剂量反应关系。在4μM和8μM作用浓度时,ATM干扰细胞CBMN率明显高于空载质粒细胞CBMN率,差异具有统计学意义(P0.05)。采用单细胞凝胶电泳技术探讨ATM干扰细胞和空载质粒细胞对博莱霉素诱导的DNA修复能力(DNA repair capacity, DRC)差异。结果显示,在10μg/ml博莱霉素诱导下16HBE-siATM细胞的DRC(56.664±0.78%)明显低于16HBE-siGFP细胞(70.13±3.82%),差异具有统计学意义(P=0.039)；与16HBE细胞的表现相似,HEK-siATM细胞DRC(63.86±2.17%)亦明显低于HEK-siGFP细胞(73.904±1.67%),差异具有统计学意义(P=0.044)。三、采用流式细胞技术探讨ATM干扰细胞和空载质粒细胞对B(a)P诱导的细胞周期变化。结果显示,与16HBE-siGFP细胞相比,4μM B(a)P使16HBE-siATM细胞G1期明显缩短,G2期明显延长；与HEK-siGFP相比,4μM B(a)P使HEK-siATM细胞G1期和G2期均明显缩短,S期明显延长。采用Western-blotting技术探讨B(a)P作用下ATN、P53、NBS1和rH2AX蛋白表达情况。结果显示,与对应的溶剂对照组比较,8μM B(a)P作用24h后,16HBE-siGFP和HEK-siGFP细胞ATM总蛋白表达无明显变化；与对应的溶剂对照组比较,8μMB(a)P可使ATM干扰细胞及空载质粒细胞P53、NBS1和rH2AX表达水平均升高。四、利用双荧光素酶报告基因实验探讨ATM基因5'UTR (rs189037GA)和3'UTR (rs227092 GT)变异体外驱动报告基因表达的差异。结果显示,在三种肺组织来源的细胞系(16HBE细胞、CCC-HPF-1细胞和MRC-5细胞)中,含rs189037G等位基因的5'UTR和含rs189037A等位基因的5'UTR调控报告基因表达的差异均没有统计学意义(P0.05)；含rs227092G等位基因的3'UTR和含rs227092T等位基因的3'UTR调控报告基因表达的差异亦均没有统计学意义(P0.05)。总之,本研究探讨了ATM基因多态性与多环芳烃暴露致染色体损伤遗传易感性关系及易感性差异的分子机制,揭示了ATM基因变异可能是多环芳烃暴露致染色体损伤的易感因素之一,证实了ATM在B(a)P致染色体损伤修复及细胞周期调控中起重要作用。
[Abstract]:Coke oven escapes have been defined by the international cancer research agency as 1 types of carcinogens, known as human carcinogens. Carcinogenic polycyclic aromatic hydrocarbons (Polycyclic aromatic hydrocarbons, PAH) in Coke oven emission (COE) are the main causes of lung cancer in coke oven workers. Lung cancer in coke oven workers in China is listed as eight legal occupational tumors. The study shows that genomic instability is an important biological event of polycyclic aromatic hydrocarbons (PAHs) exposure to lung cancer. The levels of chromosomal damage in workers exposed to polycyclic aromatic hydrocarbons are significantly higher than those in non exposed groups, but under the same exposure conditions, the level of damage is not the same, suggesting the presence of susceptibility to the population.
In this study, the genetic susceptibility and molecular mechanisms of chromosomal damage induced by ataxia telangiectasia (ATM) and polycyclic aromatic hydrocarbons (PAH) exposure were investigated. The results were as follows:
The analysis of the relationship between the micronucleus rate of peripheral lymphocyte micronucleus and ataxia telangiectasia in 140 polycyclic aromatic exposed workers and 66 non exposed workers showed that the ATM gene rs600931 GA and GA+AA genotype carriers in the PAH exposure group were after the correction of sex, age, smoking and natural logarithmic conversion of urine 1-OHP. The micronucleus rate of lymphocyte in peripheral blood (11.14 + 6.92% and 10.57 + 6.82 per 1000) was significantly higher than that of GG genotype (7.66 + 5.69%, P=0.015 and 0.038). The micronucleus rate of lymphocytes in rs189037 GA and GA+AA genotype (10.99 + 6.90% and 10.51 +%) was significantly higher than that of GG based carriers (7.72 + 5.83 per thousand, P=0.018 and 0.035); rs624366 G The micronucleus rate of lymphocytes in C and GC+CC genotypes (11.34 + 6.74% and 10.73 + 6.63%) was significantly higher than that of GG genotype carriers (7.61 + 6.07 per 1000, P=0.001 and 0.003), and the micronucleus rate (10.78 + 6.60%) in rs227092 GT genotype was significantly higher than that of GG genotype (7.91 + 6.30%, P=0.025). Monopal to GGGGTG The rate of micronucleus in peripheral blood lymphocytes of C/AAACATT carriers (12.05 + 7.40 per thousand) was significantly higher than that of GGGGTGC/GGGGTGC carriers (7.51 + 6.20 per thousand, P=0.007).
Two, Cytokinesis-block micronucleus (CBMN) was used to investigate the difference between B (a) P induced genetic damage induced by ATM interference cells and empty plasmid cells. The results showed that the CBMN rates of ATM and no-load granulocytes increased with the increase of B (a) P concentration, and showed a significant dose response relationship. At the concentration of 4 M and 8 M, the CBMN rate of ATM interference cells was significantly higher than the CBMN rate of empty plasmid cells, the difference was statistically significant (P0.05).
The difference between the DNA repair ability (DNA repair capacity, DRC) induced by bleomycin induced by ATM interference cells and empty plasmid cells was investigated by single cell gel electrophoresis. The results showed that the DRC (56.664 + 0.78%) of 16HBE-siATM cells was significantly lower than that of 16HBE-siGFP cells (70.13 + 3.82%) under the induction of bleomycin in 10 mu g/ml, and the difference was statistically significant. Meaning (P=0.039); similar to the expression of 16HBE cells, DRC (63.86 + 2.17%) of HEK-siATM cells was also significantly lower than that of HEK-siGFP cells (73.904 + 1.67%), and the difference was statistically significant (P=0.044).
Three, the cell cycle of B (a) P induced by ATM and unloaded plasmid cells was investigated by flow cytometry. The results showed that compared with 16HBE-siGFP cells, 4 M B (a) P significantly shortened the G1 phase of 16HBE-siATM cells and prolonged the G2 phase obviously. Lengthening.
The expression of ATN, P53, NBS1 and rH2AX protein under the action of B (a) P was investigated by Western-blotting technique. The results showed that there was no obvious change in the total protein expression of 8 u M B (a) P as compared with that of the corresponding solvent control group. The expression level of P53, NBS1 and rH2AX in granulocyte increased.
Four, using the double luciferase reporter gene experiment to investigate the differences in the expression of the ATM gene 5'UTR (rs189037GA) and 3'UTR (rs227092 GT) variation in vitro driven reporter gene. The results showed that the 5'UTR and rs189037A alleles containing the rs189037G allele were found in the cell lines of the three lung tissue sources (16HBE, CCC-HPF-1, and MRC-5). There was no significant difference in the expression of 5'UTR regulatory report gene (P0.05), and there was no statistical difference between the 3'UTR and the rs227092T alleles containing the rs227092G allele and the 3'UTR regulation of the rs227092T allele. (P0.05).
In conclusion, this study explored the genetic polymorphisms of ATM gene and the molecular mechanism of susceptibility to chromosomal damage induced by polycyclic aromatic hydrocarbons (PAH) exposure, and revealed that the ATM gene mutation may be one of the susceptible factors of chromosomal damage induced by polycyclic aromatic hydrocarbons (PAH) exposure, which confirms that ATM plays a role in the repair of chromosome damage induced by B (a) P and the regulation of cell cycle. Important role.

【学位授予单位】：中国疾病预防控制中心
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R394

【参考文献】