【作者】 王丽霞；

【导师】 吴逸明；

【作者基本信息】 郑州大学 ， 卫生毒理学， 2010， 硕士

【摘要】 煤焦沥青(Coal tar pitch, CTP)是以多环芳烃类物质为其主要成分,已经被确认为人类致癌物,其致癌机制还不清楚,需要进一步研究。染色不稳定性(Chromosomal Instability, CIN)学说越来越引起人们的密切关注。研究发现,染色体不稳定性是肿瘤细胞的一个普遍特征,与肿瘤的发生机制有关。除了染色体不稳定性外,绝大多数实体肿瘤还存在中心体异常的现象。中心体异常是染色体不稳定形成的主要原因,具体机制需要进一步的研究。许多抑癌基因及其产物和激酶,如P53、BRCA1/2、CDK2/CyclinE复合物等都参与了中心体复制的调控。P53失活或表达降低和CyclinE过度表达,可导致中心体扩增进而导致染色体不稳定,对肿瘤的发生产生深远的影响。目的该课题以人支气管上皮细胞BEAS-2B细胞系作为研究对象,用煤焦沥青烟提取物诱导其发生恶性转化,探讨BEAS-2B细胞恶性转化过程中染色体核型和中心体的改变情况,以及恶性转化过程中中心体异常的可能分子生物学机制,为探索煤焦沥青致肺癌的发病机制提供科学的理论依据。方法1.用气相色谱-质谱联用技术分析煤焦沥青烟提取物溶液的成分。实验分3组：即煤焦沥青组(煤焦沥青烟提取物溶液)、DMSO组、正常对照组(生理盐水)。2.以煤焦沥青烟提取物作为诱导剂,MTT法测定BEAS-2B细胞的LC50。以近20% LC50对细胞进行诱导,观察细胞增殖和细胞形态的变化。3.油镜下观察第10、20和30代细胞染色体,计数染色体数目。采用间接免疫荧光法检测中心体的变化。4.提取第10、20和30代细胞的总RNA,用实时定量PCR检测P53、P21和CyclinE mRNA表达水平。免疫组化方法检测P53、P21和CyclinE蛋白表达水平。5.采用统计软件SPSS12.0进行数据分析。测得的数据用均数±标准差(x±s)表示。检验水准a=0.05。结果1.煤焦沥青烟提取物溶液经气相色谱-质谱联用技术分析,鉴定出主要成分是多环芳烃类化合物(87.91%)。计算出半数致死浓度LC50为8.64 mg/L,确定最终的染毒剂量为2 mg/L。2.诱导后,细胞从第10代到第30代,二倍体核型比例呈下降趋势,出现了大量非整倍体,表明细胞在生长繁殖过程中存在染色体不稳定。3.诱导后,第10代细胞中心体总异常率在3组间比较差异无统计学意义(P>0.05)；到第20代时,中心体总异常率升高,主要表现为数目异常(3.41%)；到第30代时,中心体总异常率进一步升高,表现为中心体数目增多,体积增大和数目及体积同时异常。4.诱导后细胞中心体异常和染色体不稳定性呈正相关(r=0.75,P=0.02),表明中心体异常与染色体不稳定性间有密切关系。5.诱导后,第10代细胞P53、P21和CyclinE mRNA和蛋白质的表达3组间比较差异无统计学意义(P>0.05)；诱导后第20代、30代细胞P53和P21 mRNA和蛋白质的表达量均降低,而CyclinE mRNA和蛋白质的表达量均升高,差异有统计学意义(P<0.05)；蛋白P53和P21间存在正相关,而CyclinE与P53和P21分别呈负相关。从第10代到第30代,蛋白P53和P21表达逐步下降,而蛋白CyclinE表达升高。6.诱导后第20和30代细胞中心体异常率和蛋白质P53、P21和CyclinE相关性分析发现,中心体总异常率与P53和P21蛋白间均呈负相关性(r=-0.57,P=0.01；r=-0.65,P=0.00),而中心体总异常率与CyclinE呈正相关性(r=0.57,P=0.01)。结论在煤焦沥青烟提取物诱导BEAS-2B细胞恶性转化过程中,细胞可通过P53/P21通路调控中心体复制过程。中心体异常在细胞染色体不稳定性发生中可起重要作用。更多还原

【Abstract】 Coal tar pitch is mainly composed of polycyclic aromatic hydrocarbons and has been recognized as a human carcinogen, however, its carcinogenic mechanism remains unclear, which is needed to study and research further.chromosomal instability theory has drawn increasing attention in the field of tumor mechanism research. Chromosomal instability is a common feature of tumor cells. It is considered that chromosomal instability is related to the mechanism of tumor development. In addition to chromosomal instability, there are centrosome abnormalities that show an increase in the number of centrosome and an abnormal shape in many solid tumors. Centrosome abnormalities may be the main reason for chromosomal instability. The specific mechanism is still unclear, pending to further study. It has been found that many tumor suppressor genes and their products and some kinases, such as P53, BRCA1/2 and CDK2/CyclinE complex, are all involved in the regulation of centrosome replication. Inactivation or decreased expression of P53 and overexpression of CyclinE may result in centrosome amplification. Centrosome abnormalities can lead to chromosomal instability, which has a profound impact on the occurrence of cancer. ObjectiveThe study, which makes BEAS-2B cells (human bronchial epithelial cells) as a object of study and use coal tar pitch fume extract as an inducer, is to probe the changes of chromosomal karyotype and centrosome during the process of malignant transformation of cells as well as the molecular mechanisms of abnormal centrosome for provide possible theoretical basis to explore the mechanism of the pathogenesis of lung cancer caused by coal tar pitch. Methods1. The components of Coal tar pitch fume extract solution were analysis by Gas chromatography-mass spectrometry. There are three groups in this study, including coal tar pitch group (coal tar pitch fume extract solution), DMSO group (dimethylsulfoxide) and control group (saline).2. With coal tar pitch fume extract as an inducer of cells, the median lethal concentration of BEAS-2B cells was assessded by MTT assay. Cells were induced at 20% LC50,and then cultured conventionally to observe the changes of proliferation rate and morphology.3. Chromosomes of the 10th,20th and 30th generation cells were observed by the oil-immersion lens. Indirect immunofluorescence staining was used to observe changes of centrosome in induced BEAS-2B cells.4. Total RNA of the 10th,20th and 30th generation cells were extracted respectively to detect the gene expression of P53, P21 and CyclinE by real-time quantitative PCR. Protein expression of P53, P21 and CyclinE of the 10th,20th and 30th generation cells were semi-quantitatively detected by immunohistochemical staining.5. The data was analyzed by the statistical software SPSS 12.0 and indicated by mean and standard deviation. Test levelα=0.05.Results1. After analyzing coal tar pitch fume extracts by Gas chromatography-mass spectrometry, Polycyclic aromatic hydrocarbon compounds were the main ingredient, accounted for 87.91%. The median lethal concentration was 8.64 mg/L, and then the final concentration which was determined to induce cells was 2 mg/L.2. After induction, From 10 passages to 30 passages, the proportion of diploid karyotype of cells in coal tar pitch group was lower than that in normal group and DMSO group respectively, with a large number of aneuploid, indicating there was chromosomal instability at this passage.3. After induction:total ratio of abnormal centrosome of the 10th generation cells was 2.79%, however, there were no significant differences among the three groups. At 20 passages, the total ratio of abnormal centrosome (6.56%) increased, which mainly showed that the number of centrosome was abnormal (3.41%). The ratio of abnormal centrosome of the 30th generation cells increased further, showing number increase, volume augmentation and the number and size abnormalities simultaneously.4. There was a positive correlation between centrosome abnormalities and chromosomal instability(r=0.75, P=0.02), indicating that centrosome abnormalities was closely related to chromosomal instability.5. After induction, the gene and protein expression of P53, P21 and CyclinE of the 10th generation cells showed no significant differences among three groups. At 20 and 30 passages, the gene and protein expression of P53 and P21 reduced, while those of CyclinE increased significantly (P<0.05). There was a positive correlation between the protein expression of P53 and P21 and so was a negative correlation between the protein expression of CyclinE and P53 and P21 respectively. From 10 to 30 generations, the protein expression of P53 and P21 gradually decreased, while the protein expression of CyclinE increased.6. After induced, the analysis of correlation between the rate of centrosome abnormality and the expression of proteins P53, P21 and CyclinE in the 20th and 30th cells showed that there was a negative correlation beween the total rate of abnormal centrosome and P53 and P21 respectively (r=-0.57, P=0.01; r=-0.65, P=0.00), while there was a positive correlation between the total rate of abnormal centrosome and the expression of CyclinE (r=0.57, P=0.01).ConclusionThe induced cells regulate the replication of centrosome by P53/P21 pathway during malignant transformation of BEAS-2B cells induced by coal tar pitch extracts. Centrosome abnormal may play an important role in the formation of chromosome instability.更多还原