【作者】 潘泓；

【导师】 唐步坚；

【作者基本信息】 广西医科大学 ， 肿瘤学， 2007， 博士

【摘要】 研究背景恶性肿瘤是目前危害人类健康最严重疾病之一。近二十年全国性死亡原因调查显示,死亡率上升幅度最大的是肺癌。目前肺癌的治疗一般采用手术、化疗、放疗、免疫治疗等综合措施,其中化疗作为全身性治疗手段,在恶性肿瘤的治疗中具有手术和放疗不能替代的地位。经过长期的临床经验总结和一些大规模临床随机对照研究,已经达成共识,对完全性切除术后的晚期非小细胞肺癌给予以铂为主的术后辅助化疗能够提高患者的生存率,但对于早期的非小细胞肺癌是否给予术后辅助化疗尚存在争议。一些研究认为对于早期的非小细胞肺癌给予术后辅助化疗可以使3年生存率提高3%,使5年生存率提高5%,建议对完全性切除术后的早期非小细胞肺癌给予以铂为主的术后辅助化疗。而另外一些研究发现对完全性切除术后的早期非小细胞肺癌给予术后辅助化疗,不能提高患者的生存率,反而增加了毒性反应和经济负担。我们认为影响非小细胞肺癌术后辅助化疗效果的原因是患者本身的分子机制不同所致。本研究重心是寻找影响非小细胞肺癌术后辅助化疗效果的分子标志物,建立一个判断非小细胞肺癌术后辅助化疗效果的分子模型以指导早期非小细胞肺癌的术后治疗。并且针对一个有意义的分子指标进行深入研究,探索其作为非小细胞肺癌靶向治疗的潜能。本研究许多内容在国内尚未有相关报道,为探索性实验,本研究主要由5部分组成。研究内容第一部分非小细胞肺癌术后辅助化疗的疗效观察目的:通过本院近10年的临床资料,分析术后辅助化疗在完全性切除术后的非小细胞肺癌中的意义,并用循证医学方法了解术后辅助化疗在完全性切除术后的早期非小细胞肺癌中的意义。方法:回顾性分析近10年在本院行根治性切除的542例非小细胞肺癌的临床资料,其中268例是Ⅲ期NSCLC,274例是Ⅰ、Ⅱ期NSCLC。通过Medline、CNKI、VIP数据库查找关于早期NSCLC行术后辅助化疗临床随机对照研究,用Review Manager4.2软件进行统计学分析。结果:从本院资料分析显示:对切除术后Ⅲ期NSCLC患者进行3～4周期,以顺铂为基础的化疗方案的辅助化疗,可以提高长期生存率;对根治切除术后Ⅰ、Ⅱ期NSCLC患者不需常规进行辅助化疗。Meta分析显示:共16项研究纳入分析,早期NSCLC完全切除术后加用含铂的辅助化疗方案,不能显著提高患者的生存率。结论:寻找分子标志物以指导早期非小细胞肺癌的术后治疗是一个有意义且亟待解决的研究课题。第二部分多基因蛋白表达判断早期非小细胞肺癌术后化疗疗效的探讨目的:目前研究认为与癌症化疗相关的基因有以下六类:(1)药物动力学相关基因;(2)各种生物酶相关基因;(3)DNA损伤与修复相关基因;(4)凋亡相关基因;(5)致癌、抑癌基因;(6)细胞增殖、转移相关基因等,本部分是分析其中的30个基因在早期NSCLC中表达情况,寻找与早期NSCLC术后化疗效果密切相关的基因,通过Logistic回归分析获得一组分子指标以指导早期非小细胞肺癌的术后治疗。方法:利用组织芯片(tissue microarray,TMA)技术和免疫组织化学二步法,对86例行术后辅助化疗的早期非小细胞肺癌标本进行Caspase-3、Fas、Bax、Bcl-2、Survivin、PCNA、Ki67、MGMT、P53、P63、P73、P16、P27、VEGF、nm23、P-gp、MRP、LRP、GST-π、TopoⅡ、C-myc、Cyclin-D1、Her-2、Cox-2、Ku70、Ku80、DNA-PKcs、ERCC1、MSH2、BCRP30种化疗相关蛋白检测。结果:30种化疗相关蛋白在肺癌组织中表达的阳性率为27.9%至91.9%不等。经过单因素分析,与早期NSCLC术后化疗效果密切相关的基因有8个,Survivin、P-gp、LRP、Ki67、P53、ERCC1高表达,NSCLC术后化疗效果差;Bax、VEGF低表达,NSCLC术后化疗效果差。通过Logistic回归分析ERCC1、Survivin、Bax、VEGF4个因子进入方程,方程分类能力达69.8%;方程有效性经卡方检验X~2=29.15,P=0.000。结论:在单因素分析中ERCC1是判断非小细胞肺癌术后辅助化疗效果最敏感的指标,可以进一步深入研究。通过多因素分析ERCC1、Survivin、Bax and VEGF是一组判断非小细胞肺癌术后辅助化疗效果,指导早期非小细胞肺癌的术后治疗的分子指标。对于其可靠性有待于扩大病例进一步研究。第三部分ERCC1基因在肺癌组织及肺癌细胞系中表达分析目的:ERCC1是判断非小细胞肺癌术后辅助化疗效果最敏感的指标,本部分首先观察ERCC1基因在肺癌细胞系、肺癌组织与癌旁正常肺组织中表达情况,对ERCC1基因的表达特性有一定了解。然后通过耐药细胞系与亲本细胞系中ERCC1mRNA差别及分析单独行化疗的肺癌中ERCC1基因表达情况,进一步证实ERCC1基因表达与肺癌化疗的关系。方法:首先应用半定量RT-PCR方法检测6株肺癌细胞及30例肺癌组织与癌旁正常肺组织中表达情况。第二使用高浓度反复间歇诱导法建立了对多种化疗药物耐药的细胞系A549/DDP,并用RT-PCR方法检测耐药的细胞系和亲本细胞系中ERCC1表达差别。最后利用免疫组织化学法检测65例单独行化疗的NSCLC中ERCC1表达。结果:与癌旁正常肺组织相比ERCC1在肺癌组织表达明显降低。成功建立了肺癌耐药的细胞系A549/DDP,与亲本细胞A549相比其对常规化疗药物的耐药指数增加了4-5倍,ERCC1在A549/DDP表达明显高于A549细胞。在单独行化疗的NSCLC中,ERCC1表达阳性患者的化疗效果明显差于ERCC1表达阴性患者。结论:ERCC1表达降低可能与肺癌的发生有一定联系,体外细胞实验和体内临床病理分析证实ERCC1基因表达与肺癌化疗密切相关,ERCC1基因表达增高,化疗效果不佳。第四部分ERCC1基因的初步功能分析目的:ERCC1是与肺癌化疗密切相关的基因,本部分主要通过基因克隆,细胞转染的技术进一步了解ERCC1基因的生物学功能并初步探讨其引起癌细胞化疗耐受的机制。方法:首先采用基因克隆的方法构建ERCC1基因真核表达载体,用脂质体将载体转染致肺癌细胞H1299中,用G418筛选稳定转染细胞克隆,采用RT-PCR和Western-Blot技术证实ERCC1基因转染成功。第二采用细胞生长曲线,克隆形成实验,细胞侵袭实验,流式细胞仪分析细胞生长周期等方法了解ERCC1基因转染对以上细胞生物学行为的影响。第三采用MTT法检测ERCC1基因转染对各种化疗药物毒性反应的影响。最后利用流式细胞术检测细胞膜蛋白P-gp、MRP的表达,利用高效液相色谱仪检测细胞内药物浓度,探讨ERCC1基因转染引起癌细胞化疗耐受的机制。结果:成功构建ERCC1基因真核表达载体PCDNA3.1/ERCC1,通过筛选建立了稳定转染细胞系H1299/ERCC1和H1299/Vecter。生长曲线、克隆形成实验、细胞侵袭实验、流式细胞仪分析细胞生长周期等实验显示:H1299/ERCC1和H1299/Vecter在以上实验中无差别。化疗药物毒性反应实验显示H1299/ERCC1较H1299/Vecter对顺铂的耐药指数增加4.23倍。流式细胞术检测细胞膜蛋白P-gp、MRP的表达及高效液相色谱仪检测细胞内药物浓度结果显示H1299/ERCC1和H1299/Vecter无明显差异。结论:ERCC1表达与细胞增殖、细胞侵袭、细胞周期失调等特性关系不大。ERCC1基因表达增高,细胞可以出现化疗耐药,初步研究显示ERCC1基因导致的化疗耐药并非传统的耐药机制(如膜转运蛋白表达差异)使药物摄入减少及外排增加引起,可能与DNA损伤与修复机制改变有关。第五部分RNA干扰抑制肺癌细胞ERCC1基因表达的初步功能分析目的:ERCC1基因在肺癌细胞的各种生物学行为中并不扮演重要角色,其改变主要是引起细胞对化疗药物的敏感性改变,是功能比较单一的基因,可能是提高非小细胞肺癌化疗疗效的一个有潜质的新靶点。本部分主要通过RNA干扰技术抑制肺癌细胞ERCC1基因表达,进行初步功能实验。方法:首先通过构建ERCC1基因及β-actin基因的克隆载体作为标准品,建立ERCC1基因荧光实时定量PCR检测平台。第二构建针对ERCC1基因的SiRNA真核表达载体,瞬时转染肺癌细胞A549,采用RT-PCR和Western-Blot技术证实转染细胞中ERCC1基因沉默效果良好。最后采用MTT法检测构建真核表达载体转染细胞对顺铂药物毒性反应的影响。结果:荧光实时定量PCR能够准确检测样本中ERCC1基因的mRNA表达水平。成功构建针对ERCC1基因的SiRNA真核表达载体,将其转染肺癌细胞A549,可以抑制细胞中ERCC1基因表达。RNA干扰的A549细胞较亲本细胞对顺铂的耐药指数降低了2.93倍。结论:RNA干扰是一种沉默ERCC1表达的理想方法。沉默ERCC1表达可以提高肺癌细胞的化疗敏感性,它可能是一种有潜质的肺癌辅助治疗新方法。更多还原

【Abstract】 [Background]The malignant tumor is one of the severe diseases which are harmful to the health of human beings.The investigation of the death causes shows that the death rate of lung cancer is more rapidly increasing in last twenty years,Now lung cancer is usually treated with local measures including surgery,chemotherapy,radiotherapy and immunotherapy.Chemotherapy as a systemic therapy means can not be replaced by surgery and radiotherapy as for the therapies of malignant tumors.Several randomized trials now have confirmed the survival benefit with adjuvant platinum-based chemotherapy for patients with late-stage NSCLC.The role of adjuvant chemotherapy in patients with completely resected Early-Stage NSCLC is unclear.A recent meta-analysis of all randomised trials showed that the absolute risk of death was reduced by 3%at 3 years and 5%at 5 years for patients who were treated with postoperative cisplatin-containing regimens compared to patients who were treated with surgery alone.On these bases,some institutions,in spite of drug toxicity,proposed the introduction of adjuvant chemotherapy even after presumed radical surgery at stage I NSCLC.Nevertheless,according to another multicentric study,there was no significant effect on survival;hence the toxicity induced by chemotherapy appeared to radically operated patients,especially for those at stage I NSCLC,to be too high cost to pay.our researches focused on the molecular mechnism of adjuvant chemotherapy in patients with completely resected Early-Stage NSCLC.we try to develop methods to judge the effect of adjuvant chemotherapy and guide the treatment to this patient.No documents has been published on such a study till now.This provided vacuum for our expriment.So the present study proceed five parts.[Study]PartⅠClinical Analysis of adjuvant chemotherapy in patients with completely resected NSCLC in 10 Years and meta-analysis of Adjuvant Chemotherapy with Complete Resection for Early-Stage NSCLCObjective.To evaluate the effect of adjuvant chemotherapy after radical surgery for NSCLC and analyze the factor that effect the adjuvant chemotherapy.Methods:we analysed clinical data in 542 cases of NSCLC undergone resectional surgery in our hospitol.268 patients isⅢNSCLC and 274 patients isⅠ、ⅡNSCLC.Domestic and overseas literatures were reviewed to collecte correlative study data of adjuvant chemotherapy or solitary opration for Early-Stage NSCLC.3-year survival times of the two treatment methods were meta analyzed with Review manager 4.2 according to the data of literatures.Results:1\To stageⅢNSCLC patients administer adjuvant chemotherapy with platinum-based regimen for 3 or 4 cycles can improve survival rate.2\ To stageⅠ,ⅡNSCLC patients administer adjuvant chemotherapy is not a recommended therapy methods.3\ The effectiveness of some adjuvant chemotherapy regimens with complete resection in patients with NSCLC has been improved.Conclusion:it is a important work to looking for molecular marker guide the treatment the patient with completely resected Early-Stage NSCLC.PartⅡStudy on the Relationship between Combined Multigene Detection and Response to Chemotherapy and Prognosis in Early-Stage non-small cell lung cancer tissuesobjective:Many studies have show that 30 genes expression associated to chemotherapy in cancer.in this study we detect the 30 genes expression in the patient with completely resected Early-Stage NSCLC follow by adjuvant chemotherapy.in order to fine the molecular marker relation to adjuvant chemotherapy Methods:The tissue microarray technique was used to prepare for tissue microarray in 86 cases of Early-Stage NSCLC that received adjuvant chemotherapy after undergone radical surgery.The expressions of Caspase-3、Fas、Bax、Bcl-2、Survivin、PCNA、Ki67、MGMT、P53、P63、P73、P16、P27、VEGF、nm23、P-gp、MRP、LRP、GST-π、TopoⅡ、C-myc、Cyclin-D1、Her-2、Cox-2、Ku70、Ku80、DNA-PKcs、ERCC1、MSH2、BCRP proteins were detected using immunohistochemical two-step method.Results:The positive rate of 30 genes in lung cancer tissue were 27.9%-91.9%respectively.There are 8 genes expression was related to NSCLC adjuvant chemotherapy.The higher expressing intensity of Survivin、P-gp、LRP、Ki67、P53 and ERCC1,the lower 3-year survival rate in NSCLC,there was significant discrepancy(P＞0.05). The higher expressing intensity of Bax and VEGF,the higher 3-year survival rate in NSCLC,there was significant discrepancy(P＞0.05).by logistic regression analysis the ERCC1、Survivin、Bax and VEGF are good group for predict the effective of adjuvant chemotherapy in Early-Stage NSCLC.Conclusion.ERCC1 is the most sensitivity marker to judge effect of adjuvant chemotherapy and it is ideal marker to be deeply research. Survivin、ERCC1、Bax and VEGF are ideal group factor that effect the adjuvant chemotherapy and their must be comfirn by deeply research.PartⅢA study on the expression of ERCC1 gene in human non-small cell lung cancer tissues and cell linesobjective:The aim of this study is to study the mRNA expression of ERCC1 in human lung cancer tissues and normal lung tissues.and to investigate the relationship between ERCC1 protein expression and the effect of chemotherapy to NSCLC in vivo or vitro.Methods:1\ Semi-quantification expression analysis of ERCC1 mRNA in human lung cancer and corresponding normal lung tissues were performed by RT-PCR and electrophoresis band opacity density comparison analysis.2\ Establishment of an DDP-resistant human NSCLC cell line A549/DDP by high does interval inactivated methods.3\ Semi-quantification expression analysis of ERCC1 mRNA in A549/DDP and corresponding A549 were performed by RT-PCR.4\ The expressions of ERCC1 protein in 64 cases of NSCLC that received chemotherapy alone were detected using immunohistochemical two-step method.Results: 1\ The mRNA expression of ERCC1 gene decreased significantly in human lung cancer tissue compared withthat in normal lung tissue.2\ DDP-resistant human NSCLC cell line A549/DDP were established and the values of IC50 of resistant cells to relative drugs were elevated 4-5 times.3\ The mRNA expression of ERCC1 gene increased significantly in A549/DDP compared with A549.4\ The protein expression of ERCC1 gene in good effect group of NSCLC is significantly lower than bed effect group patients.Conclusion:the ERCC1 is lower expression gene in lung cancer compared to lung tissue,the ERCC1 protein expression is relationship to the effect of chemotherapy to NSCLC in vivo or vitro.PartⅣStudy on biological effects of ERCC1 in lung cancer cellsobjective:Study on biological effects of ERCC1 in lung cancer cells by gene clone technich.Methods:1\ the ERCC1 expression vector were established and introduced into H1299 cells by liposome transfection reagent.Positive clones were screened with G418.RT-PCR was used to confirm whether the recombinant vector DNA integrated with the genomic DNA of H1299 cells.Western-blot was used to confirm whether the clone gene expression in Positive clones cells.2\ The cell growth curves,clonogenicity efficiency in plating and the cell cycle were measured to observe the changes in cell proliferation.3\The characteristic of drug resistance in stable transfection cell were measured by MTT assay.4\ Intracellular concentrations of chemotherapy drugs were detected using high-performance liquid chromatography.5\The expression of resistant gene P-pg and MRP in stable transfection cell were measured by Flow Cytometry method.Results:1\ the ERCC1 expression vector were identified by the sequence analysis.2\the Positive clones H1299/ERCC1 and H1299/vecter were confirm by RT-PCR and Western-blot.3\ The characteristic of cell proliferation was not change in H1299/ERCC1 and H1299/vecter.4\ the values of IC50 of H1299/ERCC1 to chemotherapy drugs were elevated 4.23 times than H1299/vecter.5\ Intracellular concentrations of chemotherapy drugs and The expression of resistant gene P-pg and MRP were not different between H1299/ERCC1 and H1299/vecter..Conclusion:the ERCC1 function is not relactive to cell proliferation.The expression of ERCC1 can improve capitent of resistant chemotherapy drugs in lung cancer cell.The mechnism of resistant chemotherapy drugs in ERCC1 expression cell not relative to change of Intracellular concentrations of chemotherapy drugs and change of expression of resistant gene P-pg and MRP.PartⅤ:Effects of RNA interference on ERCC1 expression in non-small-cell lung cancer cells in vitroobjective.To investigate whether RNA interference(RNAi)could induce gene silencing in non-small-cell lung cancer(NSCLC)cells as well as assess the degree of ERCC1 gene silencing and its effect on functional outcome..Methods:1\ The methods of detect expression of ERCC1 gene by Fluorescence real-time quantitative PCR(FQ-PCR)are constructed satisfactorily.2\the small interference RNA(siRNA)of ERCC1 expression vector were established and introduced into lung cancer cells(A549)by liposome transfection reagent. RT-PCR and Western-blot were used to confirm whether the siRNA silencing the expression of ERCC1 in A549 cell.3\ The characteristic of drug resistance in instantaneous transfection cell were measured by MTT assay.Results:1\ the FQ-PCR can be applied to detect expression of ERCC1 gene accurately.2\ the siRNA of ERCC1 expression vector were identified by the sequence analysis.3\ RT-PCR and Western-blot assay show that the expression of ERCC1 in instantaneous transfection cell were significantly lower than not siRNA of ERCC1 transfection cell.4\ the values of IC50 of A549/siRNA to chemotherapy drugs were decrease 2.93 times than A549/vecter.Conclusion.the RNA interference to ERCC1 gene is a good method to silence expression of ERCC1 in lung cancer cell.inhibit the expression of ERCC1 can enhance sensitivity to chemotherapy drugs in lung cancer cell.it may be a available therapeutic modalities in the treatment of lung cancer.更多还原