【作者】 石思恩；

【导师】 王建军；

【作者基本信息】 华中科技大学 ， 胸外科， 2010， 博士

【摘要】 背景：肺癌已经成为我国发病率和死亡率最高的恶性肿瘤,大约70%的患者在诊断时已发展至晚期,无法通过手术治愈,化疗成为肺癌综合治疗中的重要部分,但是肿瘤细胞对化疗药物发生耐药常导致化疗失败。近年来,人们对肿瘤细胞的耐药机制进行了深入的研究。一直以来,顺铂被认为是肺癌化疗的基石,含铂方案是晚期NSCLC化疗的标准方案,几乎所有一线NSCLC化疗方案都是与顺铂联合应用的方案。顺铂的作用机理是进入肺癌细胞后,与DNA交叉联结,形成稳定的顺铂-DNA复合物,阻断DNA复制,引起肺癌细胞死亡。近年来国内外研究表明,肺癌细胞对顺铂发生耐药的机制,除了早先认为的肿瘤细胞内药物浓度降低、药物在细胞内代谢降解外,更是与DNA损伤修复增强密切相关,是一个多基因参与的复杂事件,涉及了多种蛋白质与数条途径,如已发现由跨膜转运蛋白、多药耐药相关蛋白和肺耐药相关蛋白介导的典型耐药途径,和由其它非跨膜机制如凋亡、酶活性、细胞内pH等变化介导的非典型耐药途径。但肺癌耐药的机制至今仍未完全阐明。因此不断寻找新的耐药相关基因并进行深入研究,对阐明肺癌细胞的耐药机制,进而寻找逆转耐药的途径,具有重要意义。目的：诱导人肺腺癌细胞A549对顺铂耐药,研究A549细胞产生耐药性前后的蛋白表达差异,明确这些差异蛋白与肺癌耐药的关系,以期为肺癌的治疗发现新的靶点,为揭示肺癌的耐药机制提供线索,并为寻找逆转耐药途径提供基础。方法：以顺铂为诱导药物,人肺腺癌细胞系A549为诱导对象,采用逐步增加剂量与大剂量冲击相结合的方法,诱导建立耐顺铂细胞株A549/CDDP。对A549与A549/CDDP进行蛋白组学研究,即利用双向凝胶电泳(two-dimensional polyacrylamide electrophoresis,2D-PAGE)分离两组总蛋白后,通过图像分析寻找表达差异的蛋白点,对其进行MALDI-TOF质谱分析。对经质谱鉴定的蛋白质,通过慢病毒介导的RNA干扰技术沉默A549和A549/CDDP细胞内目标蛋白基因的表达,应用蛋白印迹法(western blot)和四甲基偶氮唑蓝比色法(MTT)探讨沉默该基因表达对肺癌细胞生物学行为的影响,阐明其功能。结果：建立了稳定的耐顺铂细胞系A549/CDDP。比较A549和A549/CDDP两者的2D-PAGE图谱,得到差异蛋白点82个。对其中6个差异蛋白点进行肽质量指纹图分析,鉴定出葡萄糖调节蛋白75、核糖体蛋白S4、线粒体F1-ATP合酶p亚单位、免疫球蛋白重链可变区。以上差异蛋白均在耐药株中过表达,而在对照组细胞中低表达或不表达。A549和A549/CDDP细胞成功转染GRP75 siRNA,转染后A549/CDDP细胞GRP75蛋白明显下调,bcl-2蛋白下调,p53蛋白上调,转染后A549/CDDP细胞对顺铂敏感性有所增强。结论：成功地通过逐步增加剂量与大剂量冲击相结合的诱导方法建立肺癌耐药株。运用蛋白组学技术鉴定的差异蛋白为阐明肺癌细胞对顺铂耐药性产生的机制提供线索,也为筛选具体潜在价值的肺癌化疗靶位提供理论依据。证实了GRP75高表达与肺癌细胞耐药性的产生相关,为克服肺癌化疗耐药提供了新的突破口。更多还原

【Abstract】 Background:Lung cancer has highest incidence rate and mortality rate of all malignancy cancers in China. About 70% of the patients have been developed to advanced stage at their diagnosis, and have lost the opportunity of surgical resection. Chemotherapy has become a very important part in the comprehensive treatment of lung cancer, but the resistance of tumor cells to chemotherapy drug often fails the chemotherapy. In recent years, many research has been done to reveal the drug-resistant mechanism of tumor cells.Cisplatin has long been the foundation stone of the chemotherapy of lung cancer. Platinum-based regime is the standard of advanced NSCLC chemotherapy. Almost all first-line NSCLC chemotherapy regime is cisplatin application solutions. The mechanism of cisplatin is cross-coupling with DNA after entering lung cancer cells, founding stable cisplatin-DNA complex, blocking DNA duplication, and causing lung cancer cells death. Recent research has shown that the cisplatin-resistant mechanism of lung cancer cells includes lower drug concentration and higher degradation metabolism in tumor cells, and more importantly the enhancement of DNA damage repair. The mechanism is a complex and multi-gene involved event, coming down to several pathways and many proteins. It involves the classical pathways mediated by transmembrane transporters, multidrug resistance associated protein, lung resistance protein, and non-classical pathways mediated by non-transmembrane mechanism such as apoptosis, enzyme activity, intracellular PH. However, the cisplatin-resistant mechanism of lung cancer cells is still not fully understood. It’s of great importance to keep looking for new drug resistance related genes and continue in-depth research to elucidate the resistant mechanism of lung cancer cells, and then we shall find ways of reversing the resistance. Objective:Our study is to identify differently expressed proteins before and after cisplatin resistance of human lung adenocarcinoma cell A549 by using proteome analysis, so as to find new targets for the treatment of lung cancer, reveal drug-resistant mechanism, and provide clues for ways to reverse resistance.Methods:Cisplatin-resistant cell strain A549/CDDP was established from their parental human lung adenocarcinoma cell line A549 by combining gradually increasing concentration of cisplatin with big dosage impact. Comparative proteome analysis of A549 and A549/CDDP were carried out by means of two-dimensional gel electrophoresis. The differentially expressed proteins were analyzed and identified by MALDI-TOF mass spectrometry. The siRNA for the gene of the identified protein was transfect to A549 and A549/CDDP cells by lentivirus. Western blot and methyl thiazolyl tetrazolium (MTT) assay were applied to explore the influence of silencing the gene expression on lung cancer cell biological behavior, and elucidate the function of the gene.Results:82 differentially expressed proteins were screened by analyzing the electrophoretic maps of A549 and A549/CDDP.6 differential proteins were analyzed by peptide mass fingerprinting. Glusose regulating protein 75 (GRP75), ribosomal protein S4, mitochondrial ATP synthase Fl complex beta subunit, and immunoglobulin heavy chain variable region were identified. All four differentially expressed proteins were over-expressed in A549/CDDP, whereas low-expressed or no-expressed in A549. GRP75siRNA was successfully transfected to A549 and A549/CDDP. After transfection GRP75 protein in A549/CDDP dramatically decreased, bcl-2 protein decreased, p53 protein increased. And the sensitivity to cisplatin of the transfected cells was increased compared with A549/CDDP.Conclusion:Our study Successfully established cisplatin-resistant A549/CDDP cell strain by combining gradually increasing concentration of cisplatin with big dosage impact. The differentially expressed proteins give some clues to elucidate the mechanism of lung cancer cell resistant of cisplatin, and provide the basis of searching for potential target of chemotherapy of lung cancer. The fact that cisplatin resistance of lung cancer cells may be associated with the over-expression of GRP75 gene gives priority to overcome the drug resistance of lung cancer cells.更多还原