【作者】 陈连生；

【导师】 王东；

【作者基本信息】 第三军医大学 ， 肿瘤学， 2007， 硕士

【摘要】 非小细胞肺癌(Non-small-cell lung cancer,NSCLC)约占肺癌的80%,已经成为全球第一癌症杀手。晚期NSCLC的化疗主要是以铂类为主,多采用铂类加泰素(paclitaxel),泰素帝(docetaxel),健择(gemcitabine)或去甲长春花碱(NVB)为主的联合化疗方案。但是这几种联合化疗方案临床疗效并没有显著差异,表明以铂类为主的化疗目前已经达到瓶颈。为了突破NSCLC的化疗瓶颈,进一步探讨铂类抗癌作用机制及其耐药机制具有非常重要的临床意义。研究表明,铂类抗癌作用的主要靶点为DNA,阻止其复制及转录,造成细胞死亡。铂类耐药的机制仍未完全阐明,可能是由多种因素引起,包括药物积聚减少;药物解毒增加(如谷胱甘肽和金属硫蛋白);DNA修复能力增强及顺铂-DNA加合物增加等。DNA损伤修复系统作为机体抵抗各种损伤的分子基础,对于维护基因组的稳定与完整起着至关重要的作用,然而对于通过损伤DNA杀死癌细胞的铂类化疗,这一过程无疑削弱了治疗效果。因此,DNA修复能力增强可能是铂类耐药的主要机制之一。DNA损伤修复基因脱嘌呤/脱嘧啶核酸内切酶(apurinic/apyrimidinic endonuclease,AP,1具有核酸内切酶及氧化还原双功能,是细胞放射性损伤和基因毒性药物烷化剂致伤的重要修复因子,并通过氧化还原机制调节多种转录因子及下游靶基因表达,而这些转录因子与肿瘤放化疗抵抗密切相关。因此本研究检测NSCLC患者APE1蛋白的表达特点,分析APEl与NSCLC术后复发和铂类化疗敏感性的关系,并用Ad5/F35-APE1 siRNA重组腺病毒载体特异性“敲除”APE1基因表达,研究其体外对NSCLC细胞顺铂化疗敏感性的影响。研究目的:1、研究APE1蛋白在非小细胞肺癌组织细胞中表达情况,分析APE1表达特点与NSCLC术后复发和铂类抵抗的关系,以确证APE1为NSCLC治疗有效的靶基因。2、探讨诱导APE1基因沉默对NSCLC含铂类化疗的增敏作用。研究内容和方法:1、APE1蛋白在NSCLC细胞中的表达特点及NSCLC术后复发和含铂类药物抵抗的关系收集临床NSCLC患者的病理标本及完整临床病历资料,并且设计随访问卷;同时采用免疫细胞化学检测NSCLC患者组织标本中APE1蛋白的表达,并分析其表达与NSCLCI临床分型、术后复发和含铂类药物化疗等的关系;2、Ad5/F35-APE1 siRNA重组腺病毒增强肺腺癌顺铂化疗敏感性的实验研究不同剂量顺铂和/或Ad5/F35-APE1 siRNA重组腺病毒处AA549细胞,MTT法检测其存活率的改变;TUNEL法检测A549细胞凋亡;Western blot检测APE1蛋白表达。研究结果l、APE1蛋白在NSCLC组织细胞的表达特点及NSCLC术后复发和含铂类药物抵抗的关系正常肺组织及肺癌组织均有APE1表达,但正常肺组织以胞核表达为主,肺癌组织主要在胞浆表达;APE1的表达与患者的年龄、性别、肿瘤大小、有无淋巴结转移及组织类型无明显关系;APE1高表达组和APE1低表达组患者的1年生存率差异无统计学意义(χ~2=53.03,P＞0.05),而前者的2年和3年生存率低于后者,差异有统计学意义(P＜0.05)。24例铂类敏感组中,APE1强表达仅为2例,占8.33%,12例铂类耐药组APE1表达强阳性为10例,占83.33%,两两比较均有有统计学意义(χ~2=9.06,P＜0.01)。APE1基因检测有助于NSCLC疗效及预后的判断。2、Ad5/F35-APEl siRNA重组腺病毒显著增强肺腺癌细胞顺铂化疗敏感性顺铂呈剂量依赖性诱导A549细胞APE1蛋白表达增强,Ad5/F35-APE1 siRNA能显著抑制A549细胞APE1蛋白表达,且呈剂量依赖性。感染Ad5/F35-APE1 siRNA腺病毒比感染对照腺病毒的A549细胞对顺铂的敏感性明显增强,IC50分别为0.26μg/ml和1.54μg/ml。Ad5/F35-APE1 siRNA加强顺铂诱导的A549细胞凋亡。结论1、APE1胞浆异位表达可能与非小细胞肺癌的发生、侵袭和转移有关,同时APEl的表达强度与含铂类药物抵抗相关。APE1是非小细胞肺癌治疗潜在的分子靶点,具有重要的临床应用价值。2、铂类化疗药物处理后肺癌细胞APE1蛋白表达水平明显升高,表明化疗药物在杀伤癌细胞的同时,癌细胞合成表达APE1增强DNA损伤修复,这可能是NSCLC癌细胞发生耐药的机制之一。3、阻断NSCLC细胞APE1基因表达,可显著提高NSCLC细胞化疗敏感性,诱导NSCLC细胞发生凋亡。基于APEl基因的RNAi技术靶向增强NSCLC细胞化疗敏感性、诱导NSCLC细胞凋亡对提高NSCLC化疗疗效可能具有一定的临床应用前景。更多还原

【Abstract】 Non-small-cell lung cancer(NSCLC) accounts for 80%of all lung cancer cases and is the leading cause of cancer mortality.The treatment of advanced NSCLC is based on the combination of platinum and one of the following agents:paclitaxel,docetaxel, gemcitabine or NVB.There are no significant differences in efficacy among these combinations suggesting that the outcome of platinum therapy on NSCLC have reached a plateau.Therefore,the biological mechanisms of cisplatin action need to be understood in order to overcome the treatment plateau on NSCLC.The molecular target of platinum action is the cellular DNA,which hampers DNA replication and transcription,resulting in cell death.Moreover,the development of resistance is a hurdle in the use of this drug.The molecular mechanisms that underlie this chemoresistance are largely unknown.Possible mechanisms of acquired resistance to platinum include reduced intracellular accumulation of platinum,enhanced drug inactivation by metallothionein and glutathione,increased repair activity of DNA damage,and formation of cisplatin-DNA adducts,et al.DNA-repair systems,as the molecular basis of defending against environmental damage to cellular DNA,play an important role in protecting the genomic stabilization and integrity.However,an elevated DNA repair capacity in tumor cells leads to drug resistance and severely limits the efficacy of platinum.The human apurinic/apyrimidinic endonuclease (APE1),is abundant in human cells and accounts for nearly all of the abasic site cleavage activity observed in cellular extracts.In addition to its DNA repair functions,APE1 is also a multifunctional protein that participates in other crucial cellular processes,including the response to oxidative stress and regulation of transcription factors.The transcription factors are associated with chemoresistance.Therefore,targeting inhibition of APE1 display an emerging cancer therapeutic opportunity.In this study,we first investigate the expression of APE1 and its correlation with sensitivity of platinum chemotherapy in NSCLC patients. Then,we investigated the effect of adenoviral vector Ad5/F35 carrying human APE1 siRNA on the sensitivity of cisplatin in human NSCLC cells.Objective1.To research the expression of APE1 protein in NSCLC cells,analyze the relationship between APE1 expression and NSCLC clinical effect,and determinate that APE1 is an effective therapeutic target gene of NSCLC.2.To investigate the perspective of clinical application of gene therapy targeting APE1 gene enhancing the sensitivity of NSCLC cells to DDP.Materials and Methods1.The expression of APE1 protein in NSCLC cells and its significanceNSCLC patients and their medical records were collected.The expression of APE1 in NSCLC patients and NSCLC cell lines(A549) were detected by immunocytochemical staining and Western blot,then analyzed relationship between expression of APE1 and clinical classification,clinical effect of NSCLC.2.Study of Ad5/F35-APE1 siRNA enhancing sensitivity of human NSCLC cells to DDPCells were treated with DDP at various concentrations 48 hours after Ad5/F35-APE1 siRNA or Ad5/F35-EGFP was transfered into A549 cells,and the cellular proliferation capacity was observed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.Cell apoptosis was determined by TUNEL.The expression of human APE1 protein was detected by western blot analysis.Results1.The expression of APE1 protein in NSCLC cells and its significanceProtein expression of APE1 was noticed in all non-small cell lung carcinomas and normal lung tissue.The APE1 expression of normal lung tissue mainly located in the nucleus,while APE1 expression of non-small cell lung carcinomas tissue mainly located in cytoplasm.In addition,APE1 expression was not related with the age and gender of patients,tumor size,lymphonode metastasis,nor the pathologic classification.It was not statistical significance that the patient one-year survival rate between APE1- high expression group and the low expression group(x~2=53.03,P＞0.05),but in the 2 and 3-years survival rates of APE1 low expression higher than the high expression(P＜0.05).In the 12 examples platinum drug fast group,APE1- high expression was 10 examples(83.33%),and in the 24 examples platinum sensitiveness group,APE1- high expression was 2 examples (8.33%),they were also notable statistical slgnlficance(x~2=9.06,P＜0.01).To detect gene APE1 will be conduced to judge the therapeutic effect and prognostic of NSCLC.2.Study of Ad5/F35-APE1 siRNA enhancing sensitivity of human NSCLC cells to DDPThe protein expression of APE1 in A549 cells was induced by DDP in a dosedependent manner.Infection of A549 cells with Ad5/F35-APE1 siRNA resulted in a dose-dependent suppression of APE1 protein in vitro.The result of MTT showed that Ad5/F35-APE1 siRNA enhanced sensitivity of A549 cells to DDP.The 50%inhibitory concentration(IC50) value for A549 cells pretreatmented with Ad5/F35-APE1 siRNA and Ad5/35-EGFP at 72 h after DDP treatment was 0.26μg/ml and 1.54μg/ml,respectively. Ad5/F35-APE1 siRNA also increased cell apoptosis induction by DDP.Conclusion1.The shifts of APE1 from nucleus to cytoplasm might play a pivotal role in the carcinogenesis,progression and metastasis of NSCLC.High expression of APE1 protein may indicate poor prognosis,and correlated with the DDP drug resistance.It suggests that APE1 is a potential molecular therapeutic target of NSCLC.2.The protein expression of APE1 in NSCLC cells was induced by DDP in a dose-dependent manner,which suggests that elevated DNA repair capacity may partially contribute to the resistance of DDP in NSCLC cells.3.Inhibiting APE1 expression could improve sensitivityof DDP remarkably and induce apoptosis in NSCLC cells.Therefore,gene therapy targeting APE1 gene shows a promising approach in enhancing the sensitivity of NSCLC to chemotherapy.更多还原