【作者】 夏颖；

【导师】 毕勇毅；

【作者基本信息】 武汉大学 ， 劳动卫生与环境卫生学， 2005， 硕士

【摘要】 苯是一种在工业上用途极其广泛的有机溶剂,它作为重要的生产原料及有机溶剂广泛应用于多个生产领域,长期接触苯可致苯中毒、再生障碍性贫血和白血病,但是迄今对苯中毒、苯致白血病的发病机理尚未完全阐明。近年来随着分子生物学技术的发展,苯致白血病机制得以从基因的水平进行探讨,但至今尚未有关于苯中毒人群基因差异表达的系统研究。 目的:研究不同程度苯中毒工人外周血白细胞肿瘤相关基因差异表达改变的情况,探讨接触苯导致肿瘤发生的分子机制。 方法:选择被法定职业病诊断机构确诊的6名制鞋作业苯中毒工人和1名疑似苯中毒工人作为实验组,6名苯中毒工人分别为慢性轻度苯中毒2例、慢性中度苯中毒2例、慢性重度苯中毒1例、再生障碍性贫血1例;将未接触毒物的非苯作业工人作为对照。实验组和对照组在我们跟踪调查的3个月内均无服用药物史,且实验组与对照组按年龄(差别3岁以内)、性别(均为女性)、文化程度、吸烟史、饮酒史等一比一配对。 用白细胞分离液分离出外周血白细胞,Trizol法提取RNA,琼脂糖凝胶电泳鉴定RNA的质量,纯化RNA,随后进行逆转录杂交探针标记,实验组样品采用反转录Cy3荧光标记cDNA(呈红色),对照组样品采用反转录Cy5荧光标记cDNA(呈绿色)。利用七张含有2780个cDNA克隆的微阵列肿瘤相关基因芯片进行杂交,杂交信号用扫描仪进行扫描,然后将图像转化为基于荧光强度的数字信号,运用聚类分析软件,对筛选出的具有特异性表达的肿瘤相关基因进行分析。 实验结果:在测试的七张2780条肿瘤相关基因芯片中共发现特异性差异表达基因44个,这些基因出现上调或下调趋势。所有基因均已在GENEBANK中登录。上调表达的癌基因按蛋白产物的功能分为:① 生长因子类:GRO1、GRO2、TGFBR3基因;② 蛋白激酶类:RAF-1、PIM2、LYN基因;③ 线粒体膜因子类:BCL2基因;④ RAS家族类:RAP1A、RALB基因;以及mRNA干扰素诱导的跨更多还原

【Abstract】 Benzene is a heavily used industrial chemical, a hematotoxin and carcinogen, is ubiquitous in the environment. Exposure to benzene may result in benzene poisoning, aplastic anaemia, and leukemia.The mechanism of benzene poisoning is not known completely. Recent years, it is the development of the molecular biological technology that bring us hopes to know more about the molecule mechanism of the illness induced by benzene. But up to today, there is no report about the research to explore the systematical gene differential expression in the patient exposed to benzene.Objective: To study and research the change of tumor related genes in peripheral blood leucocyte of workers which have different levels of getting poisoned and investigate the mechanism of benzene induced leukemia.Method: Seven women workers were choosed from shoe-factories who diagnosed as benzene poisoning by authority occupational disease diagnose groups, including one doubtful benzene poisoning, two chronical light degree, two chronical moderate degree, one chronical heavy degree and one aplastic anemia. Seven female workers were selected as control who are examined as healthy people and not contact poison. The experimental groups and control are one-to-one matched by their ages (varying of three years), gender (all female), degree of education, smoking habits and drinking habits.Peripheral mononuclear cells were isolated, the RNA were extracted through Trizol and purified, then identified by agarose gel electrophoresis. The RNA were reverse transcription to cDNAs with concomitant incorporation of fluorescent dCTP (Cy3 or Cy5). Cy3 was used to fluorescent labeling experimental samples, Cy5 for fluorescent labeling control samples. The cDNAs were used as probes in microarray of 2780 cloned cDNA. Fluorescent signals were scanned to detect the genes differentially expression in patients and normal subjects. The gene expression profiles of peripheral white blood cell in benzene poisoning were analysis by cluster analysis software.Result: Among 7 cDNA microarray of 2780 tumour related genes, the expression of 16 genes increased, whereas that of 28 genes decreased. All genes had been registered in GENEBANK. Up-regulation oncogenes are classified according to the function of protein production: ?growth factor, such as GRO1, GRO2 and TGFBR3; ?protein kinase, for example RAF-1, PIM2 and LYN; ?mitochondria membrane factor, BCL2; ?RAS family, RAP1A, RALB; and interferon induced transmembrane protein l(IFITMl), malignant cell expression-enhanced gene/ tumor progression-enhanced gene (LENG4) ,myeloid cell nuclear differentiation antigen (MNDA) , chromosome 20 open reading frame 3 (C20ORF3) , SET translocation (myeloid leukemia-associated). Anti-oncogene, such as FAU, DOC1, ING1, BRCA2, TP53, VHL expression decreased.The results of cluster analysis is that part of differential expression gene clustering together according to gene expression profiles, 2 benzene poisoning patients of chronical light degree clustering together, chronical moderate degree is similar to light degree, but doubtful benzene poisoning, chronical heavy degree and aplastic anemia not obviously regularity.Conclusion: Study showed that the increased expression oncogene such as GRO1, GRO2, TGFBR3, RAF-1, PIM2, BCL2, LYN, RAP1A, RALB, IFITM1, LENG4, MNDA, C20ORF3, SET which maybe the early step and the significance signal of the cell canceration. The decreased expression of anti-oncogene FAU, DOC1, ING1, BRCA2, TP53, VHL suggest the deactivation, absence or mutation of the function. In this situation, cell has a malignant reverse and forms to be tumor. The decreased expression of oncogenes, such as FOSB, MCT-1, DJ-1, JUN, MAP3K8, JUNB.etc maybe cause of the decrease of leucocyte.Cluster analysis classified the samples into different disease types depend on gene expression profiles. Concerning the clinical analysis is connected to the gene differential expression.更多还原