【作者】 罗树立；

【导师】 张兴义；

【作者基本信息】 吉林大学 ， 外科学， 2011， 博士

【摘要】 肺癌是世界上最常见的恶性肿瘤,是癌症引起患者特别是男性患者死亡的主要原因。世界卫生组织(WHO)公布的资料显示,肺癌无论从发病数量还是死亡数量来看,均为全球最主要的癌症。同期预测,我国将成为21世纪的肺癌大国。肺癌几乎包括来源于气管、支气管或肺上皮的肿瘤,其基本病理类型包括鳞状细胞癌、腺癌,大细胞癌(统称非小细胞肺癌,NSCLC)及小细胞肺癌。除了吸烟,肺癌阳性家族史被认为是一个危险因素。基因改变包括p53基因的频繁突变,激活癌基因KRAS的点突变,FHIT基因杂合子频繁丢失和异常转录,CDK抑制子P16纯合子缺失和转录沉默。肺癌风险的增加与特定的细胞色素P450基因的多态性和DNA修复能力缺陷包括DNA碱基切除修复基因XRCC1,PARP-1和ERCC4相关。DNA是生命体中最重要的遗传物质,DNA损伤引起的细胞应激反应对于保证遗传物质的稳定性、抑制基因突变的产生及维持细胞寿命具有重要意义。如同其它恶性肿瘤的发生一样,NSCLC的发生也是一个多因素、多阶段、多步骤的复杂的过程。其主要机制是各种因素导致的原癌基因激活或抑癌基因失活,DNA损伤修复基因功能低下或缺失,以及一些信号通路共同参与的结果。DNA损伤修复基因在维护遗传基因的完整性和稳定性,防止细胞癌变方面具有非常重要的作用。本实验主要探讨参与DNA损伤修复蛋白Mre11、Ku80的表达与肺癌患者相关性,并构建DNA损伤修复蛋白PARP-1缺失的小鼠肺癌细胞系,为进一步临床抗肿瘤药物研究提供细胞评价平台。实验方法：(1)采用免疫组织化学的方法检测DNA损伤修复蛋白在人肺癌组织中的表达情况,进行统计学分析,运用非参数检验的方法,进而评价肺癌组织中Mre-11、Ki67蛋白的表达与患者性别、年龄、临床分期及病理分型的相关性。(2)应用PARP-1敲除小鼠肺肿瘤模型进行原代培养,通过形态学观察、免疫荧光及致瘤性分析,以此鉴定构建PARP-1缺失的小鼠肺腺癌细胞系。实验结果：(1)通过免疫组化学及统计学分析表明,虽然Mre-11蛋白表达量与患者年龄、性别、病理分型、肿瘤大小(TNM分期)无明显关系(P>0.05),但其在Ⅰ期肺腺癌组织中表达较低,随着肿瘤的进展表达上升,在Ⅱ期标本中表达达到峰值,在Ⅲ期标本中表达又回落至较低水平,统计学上具有显著差异(P<0.05),提示DNA损伤修复蛋白Mre11与肺癌的发生具有相关性。(2)建立了有效的小鼠原代肺癌细胞培养方法。(3)成功的获得了两例原代小鼠肺腺癌细胞株,光镜和电镜下具有典型的上皮特征。裸鼠移植瘤接种检测表明这些上皮来源的细胞具有致瘤性。结论：(1)通过免疫组织化学结果分析表明DNA损伤修复蛋白Mre-11的表达与人肺腺癌组织临床分期有显著关联性(P<0.05),根据在各期表达的规律,提示Mre-11在肺腺癌进展过程中可能起到抑制作用；而Ki67蛋白的表达则与肺腺癌患者性别、年龄、临床分期、TNM分期及病理分化程度均无明显关系(P>0.05)。(2)成功建立了PARP-1缺失的小鼠肺腺癌原代培养细胞株,并证明导致肿瘤的发生；此细胞系的确立,为临床抗肿瘤药物提供了一个研究平台。更多还原

【Abstract】 Lung cancer, one of the most common malignant tumor in the world, is the main reason of deaths caused by cancers in patients, especially in males. Datas published by WHO show that lung cancer is a globally important cancer either in morbility or in mortality. Also our country is predicted to be a leading country of lung cancers in the 21st country. Lung cancers include tumors derived from tracheas, bronchias and pulmonary epithelium, and its pathological types include squamous carcinoma, adenocarcinoma, large cell carcinoma (generally called non-small cell lung carcinoma, NSCLC) and small cell lung carcinoma. Besides smoking, lung cancer positive family history is considered to be a risk factor. The transformations of genes include the frequent mutation of p53 gene, the activating point mutation of KRAS oncogene, the frequent deletion and abnormal transcription of FHIT gene heterozygote, also the deletion and transcriptional silence of P16 homozygote, which is a CDK repressor. The increasing risk of lung cancers is related to the polymorphism of specific cytochrome P450 genes, the defect of DNA repair ability including DNA bases excision repair genes XRCC1, PARP-1 and ERCC4.DNA is the most important hereditary substance in living creatures. The cellular stress response caused by DNA damage has a significance for protecting DNA stability, repressing gene mutations and maintaining cells’ life span. Like other malignant tumors, the generating process of NSCLC is a complex process with mutiple factors, mutiple stages and mutiple steps. Its main machanisms result from the combined action of the activation of oncogenes and the inactivation of tumor suppressor genes, the hypofunction and deletion of DNA damage repair genes, and some signaling pathways caused by a variety of factors.DNA damage repair genes play an important role in maintaining the integrity and stability of hereditary genes, and also preventing cell cancerization. Our study mainly discusses the relativity between lung cancer patients and the expressions of DNA damage repair proteins such as Mre11 and Ku80. Moreover, we construct a mouse lung cancer cell line deleting DNA damage repair protein PARP-1 and then develop a cell evaluating platform for further studies of clinically anticarcinoma drugs.Methods:1. We test the expressions of DNA damage repair proteins in human lung cancer tissues by immunohistochemistry, do the statistical analysis by the non-parametric test and then evaluate the relativity between the expressions of protein Mre11, Ki67 and the patients’ sexes, ages, clinical stages and pathological classifications.2. We use the PARP-1 knocked-off mouse lung cancer model to do the primary culture, and then identify this PARP-1 knocked-off cell line by morphological observation, the analysis of immunofluorescence and tumorigenicity.Results:1. By the analysis of immunohistochemistry and statistics, there is no apparent relativity (P>0.05) between the expressions of protein Mrell, Ki67 and the patients’ sexes, ages, pathological classifications and tumor sizes(TNM classification). However, the expressions of those proteins become lower in stage I lung adenocarcinoma organization, and then their expressions become higher with the progression of the tumor. The peak values of the expression of protein Mre11 and Ki67 are at stage II lung adenocarcinoma organization, and then their expressions become lower again in stage III lung adenocarcinoma organization. These datas show a significant difference in statistics (P<0.05), and suggest that DNA damage repair protein Mre11 is related to the generating process of lung cancers.2. We establish an effective method of primary culture in mouse lung cancer cells.3. We successfully aquire two primary lung adenocarcinoma cell lines in mice, with a typical character of epithelium in both light and electron microscope. Transplant tumor inoculation test in nude mice shows that those epithelial origin of cells have tumorigenicity.Conclusions:1. By the analysis of immunohistochemistry, it indicates that there is a notable relativity (P<0.05) between the expression of DNA damage repair protein Mrell and the clinical stages in human lung adenocarcinoma. According to the expression rule in every stage, it’s suggested that Mre11 may repress the process of lung adenocarcinoma; however, there is no apparent relativity (P>0.05) between the expressions of protein Ki67 and the patients’ sexes, ages, clinical stages, TNM classification and pathological differentiation degree.2. We successfully establish a primary lung adenocarcinoma cell line deleted PARP-1 in mice, and prove the cause of tumorigenicity. The establishment of this cell line provides a platform for studying clinically anticarcinoma drugs.更多还原