蒋 0 摘要.docx

1、蒋 0 摘要ERCC1及其剪切变异体与卵巢癌耐药关系的研究摘 要卵巢解剖上隐匿于盆腔深处，在发病早期缺乏特异性症状和体征，很难在早期发现和诊断。早期卵巢癌患者缺乏典型的临床症状，因此仅有约25%的患者在期时能被确诊。卵巢癌是严重危胁妇女生命健康的恶性肿瘤，死亡率在全部女性恶性肿瘤中排第四位，在女性生殖道恶性肿瘤中居首位，并有上升趋势。期卵巢癌患者经过恰当的治疗5年生存率可达95%，当病变发展到晚期（期）时5年生存率降至约2025%。近30年来，虽然经过全世界妇科肿瘤专家的不懈努力，不断改进手术方式，发现更新更好的化疗药物，增加新的化疗方案，但是5年生存率一直未有明显上升。除手术外，化学治疗是卵

2、巢癌的主要辅助治疗方法，多使用以顺铂为基础的联合化疗，常用一线方案为顺铂联合环磷酰胺或顺铂联合紫杉醇，多采用静脉化疗或腹腔化疗或者两途径联合用药。术后及时化疗，既可预防术后的复发，也可用于手术未能全部切除者，反应良好的部分患者可获暂时缓解，甚至长期存活。然而有20%到30% 的患者对一线化疗药物始终无反应，初治有反应的患者仍有部分会发生继发耐药，大大降低了化疗的效果，患者生存质量差，预后不佳。卵巢癌化疗耐药是卵巢癌复发和治疗失败的最主要原因之一。如何预测耐药，如何避免发生耐药，以及如何克服和逆转耐药，不仅是当前研究的热点，也将是今后卵巢癌研究中的长期任务。核酸切除修复系统的修复能力增强是铂类药

3、物产生耐药的主要原因，切除修复交叉互补基因1是核酸切除修复系统的关键基因，本次研究拟详细探讨切除修复交叉互补基因1在卵巢癌以铂类为基础的化疗中的意义。第一章文献回顾第二章TaqMan实时荧光定量RT-PCR检测变异剪接体 目的：建立应用TaqMan实时荧光定量RT-PCR技术对基因mRNA的各个变异剪接体分别进行定量检测的技术。方法：以对ERCC1的变异剪接体进行检测为例。使用TIRzol裂解新鲜手术切除的卵巢癌组织后提取总RNA，逆转录为cDNA后，PCR扩增ERCC1基因以及beta-actin基因，琼脂糖电泳分离PCR产物，纯化后连接到pMD18-T载体，转化入感受态大肠杆菌，提取并纯化

4、质粒，稀释成不同浓度作为标准品以便制作标准曲线。设计针对ERCC1基因mRNA的不同剪接体的TaqMan探针和对应的引物，使用TaqMan实时荧光定量技术检测ERCC1基因mRNA各个变异剪接体的表达水平。结果：成功构建了缺失第VIII外显子的ERCC1质粒以及beta-actin质粒，设计了两套针针对ERCC1的TaqMan探针和引物，建立了稳定的定量检测ERCC1 mRNA变异剪接体表达水平的平台。结论：TaqMan实时荧光定量RT-PCR方法可以成功地分别对基因的变异剪接定体进行定量检测。关键词 TaqMan探针；实时荧光定量RT-PCR；ERCC1基因；变异剪接，卵巢癌第三章切除修复交

5、叉互补基因1 mRNA表达水平与卵巢上皮癌患者化疗敏感性和生存期的关系 目的：研究切除修复交叉互补基因1(ERCC1)在卵巢癌组织中的mRNA表达水平与患者接受铂类药物化疗的敏感性及生存期的关系，并探讨ERCC1基因缺失第VIII外显子变异剪接体的临床意义。方法：收集手术切除卵巢癌组织63例，使用TRIZol试剂提取总RNA，TaqMan实时荧光定量RT-PCR技术分别检测ERCC1基因的总量及缺失第VIII外显子变异剪接体的mRNA表达水平，分别分析ERCC1基因的总量及变异剪接体与患者的化疗敏感性和术后生存期的关系。结果：ERCC1基因总量与全长mRNA表达水平与化疗敏感性（P=0.043

6、,P=0.023）和术后生存期(P=0.02,P0.01)均相关。缺失第VIII外显子变异剪接体的mRNA表达水平与化疗敏感性和术后生存期均无关（P=0.08, P=0.07）。结论：卵巢癌组织中ERCC1基因mRNA表达水平可预测患者的化疗敏感性与术后生存期，缺失第VIII外显子变异剪接体无临床意义，与患者的化疗反应和术后生存期均无关，是无功能的转录产物。在ERCC1表达总量中剔除此剪接体的量，则能更好的预测患者的化疗敏感性与术后生存期。关键词 卵巢肿瘤；抗肿瘤联合化疗方案；逆转录聚合酶链反应；选择性剪接；切除修复交叉互补基因1；第四章ERCC1基因单核苷酸多态性与卵巢上皮癌患者化疗反应性和

7、生存期的关系目的：研究切除修复交叉互补基因1(ERCC1)处于编码区第4号外显子Asn118Asn C/T 单核苷酸多态性位点与患者接受铂类药物化疗的敏感性及生存期的关系。方法：收集手术切除卵巢癌组织67例，使用酚和氯仿进行抽提外周血有核细胞的基因组DNA，使用PCR-RFLP技术检测ERCC1Asn118Asn C/T 单核苷酸多态性位点，分别分析ERCC1基因的ERCC1Asn118Asn C/T 单核苷酸多态性位点与患者的化疗敏感性和术后生存期的关系。结果：ERCC1Asn118Asn C/T 单核苷酸多态性位点与患者的化疗敏感性存在相关性（P=0.0420.05），位点为C的提示患者对

8、以铂类药物为基础的化疗敏感，位点为T的提示患者更有可能会耐药。但是生存分析并没有显示出ERCC1Asn118Asn C/T 单核苷酸多态性位点与患者的生存时间存在相关性。结论：卵巢癌患者ERCC1基因Asn118Asn C/T 单核苷酸多态性位点可预测患者的化疗敏感性，但是却与生存期不存在相关性。关键词 卵巢肿瘤；抗肿瘤联合化疗方案；逆转录聚合酶链反应；单核苷酸多态性；切除修复交叉互补基因1；限制性片段长度多态性第五章顺铂对卵巢上皮癌细胞系A2780中ERCC1及其剪接变异体表达影响的体外实验目的：探讨ERCC1基因在细胞对顺铂抵抗中的作用，明确顺铂对ERCC1基因表达的影响，同探明在顺铂作用

9、下ERCC1缺失第8外显子的变异剪接体的表达水平有无变化。方法：以不同浓度的顺铂（共计9个浓度）作用于卵巢癌细胞系A2780 1小时后弃去顺铂继续培养48小时后提取RNA,使用TaqMan实时荧光定量RT-PCR技术检测ERCC1基因的mRNA表达水平变化；以近IC50的固定浓度顺铂25g/ml作用于A2780细胞1小时后，在不同的时点（共设定了6个时点）提取RNA检测ERCC1基因的表达水平变化。结果：ERCC1基因的表达在顺铂作用后随时间和顺铂浓度的增长而增长，但是在高浓度的顺铂（高达100g/ml）作用后，ERCC1的表达增高不十分明显。缺失第8外显子的变异剪接体在ERCC1 mRNA总

10、量中的比例是一个定值，几乎不受顺铂的影响。结论：顺铂可以刺激细胞ERC1基因的表达，ERCC1基因与细胞对顺铂的药物抵抗相关。缺失第8外显子的变异剪接体在ERCC1 mRNA总量中的比例是一个定值，剪接的发生可能是一个偶然的现象。关键词 顺铂；逆转录聚合酶链反应；切除修复交叉互补基因1；抗药性, 肿瘤THE ASSOCIATION OF ERCC1 GENE AND ALTERNATIVE SPLICING VARIANTS WITH OVARIAN CANCER CHEMOTHERAPY RESISTANCEABSTRACTOvarian cancer is a cancerous grow

11、th arising from an ovary. Ovarian cancer ranks as the fifth leading cause of female cancer death, and it is diagnosed primarily in women aged 55 and older. Early stages of the cancer cause somewhat vague symptoms and many malignancies go undetected until other organs and tissues are involved. Surgic

12、al treatment may be sufficient for malignant tumors that are well-differentiated and confined to the ovary. Addition of chemotherapy may be required for more aggressive tumors that are confined to the ovary. For patients with advanced disease a combination of surgical reduction with a combination ch

13、emotherapy regimen is standard. Chemotherapy has been a general standard of care for ovarian cancer for decades, although with highly variable protocols. Chemotherapy is used after surgery to treat any residual disease, if appropriate. This depends on the histology of the tumor; some kinds of tumor

14、(particularly teratoma) are not sensitive to chemotherapy. In some cases, there may be reason to perform chemotherapy first, followed by surgery.Cisplatin combination chemotherapy is the cornerstone of treatment of many cancers, ovarian cancer included. Initial platinum responsiveness is high but th

15、e majority of cancer patients will eventually relapse with cisplatin-resistant disease. Many mechanisms of cisplatin resistance have been proposed including changes in cellular uptake and efflux of the drug, increased detoxification of the drug, inhibition of apoptosis and increased DNA repair. Oxal

16、iplatin is active in highly cisplatin-resistant cancer cells in the laboratory, however there is little evidence for its activity in the clinical treatment of patients with cisplatin-resistant cancer. The drug Paclitaxel may be useful in the treatment of cisplatin-resistant cancer; the mechanism for

17、 this activity is unknown. The goal of present study was to investigate the role of ERCC1. excision repair cross-complementation group 1, in the cisplatin resistance in ovarian cancer patients.CHAPTER ONELITERATURE REVIEWSCHAPTER TWOQUANTIFICATION OF SPLICING VARIANTS BY TAQMAN REAL-TIME RT-PCRObjec

18、tive: The goal of this study was to illustrate a TaqMan real-time quantity PCR method that can determine the absolute quantity of alternative splicing variants. Methods：Total RNA was isolated and purified from fresh ovarian cancer tissue using TRIzol reagent and was reverse transcribed into cDNA. ER

19、CC1 and beta-actin were amplified using PCR and the products were separated by electrophoresis with a horizontal agarose gel and purified. The purified PCR products were cloned into pMD18-T vector and then transformed into competent Escherichia coli DH5 and cultured. The recombinant vectors were pur

20、ified from DH5 and diluted as standard templates to establish standard curves. Results: We successfully constructed two recombinant vectors, one containing the ERCC1 alternative splicing variant lacking exon VIII and the other containing beta-actin fragment. Both vectors can be used as standard temp

21、late. Conclusions: TaqMan real-time quantity PCR assay is a reliable method for measuring the expression levels of spliced variants.Key Words: TaqMan probe; real-time quantity RT-PCR; ERCC1; alternative splicing，ovarian cancerCHAPTER THREEERCC1 mRNA EXPRESSION LEVEL AS A PROGNOSTIC FACTOR FOR EPITHE

22、LIAL OVARIAN CANCERObjective: The goal of this study was to investigate the effect of ERCC1 (excision repair cross-complementation group 1) messenger RNA expression level on chemotherapy response and survival time of epithelial ovarian cancer patients. And the alternative splicing variant lacking ex

23、on VIII was especially investigated.Method: Total RNA was isolated using TRIZol reagent from 63 epithelial ovarian cancer tissues and then reverse-transcripted into cDNA. ERCC1 mRNA expression level was evaluated by TaqMan real-time quantitative RT-PCR and the alternative splicing variant lacking ex

24、on VIII was especially investigated determined. All patients were follow-up to determine the survival time after surgery.Results: ERCC1 mRNA expression level was associated with chemotherapy response and survival time (P=0.043, P=0.02, respectively).The alternative splicing variant lacking exon VIII

25、 had no association with chemotherapy response and survival time (P=0.08, P=0.07, respectively). Conclusion: ERCC1 mRNA expression level was a prognostic factors and it seemed that the full length transcript variant maybe can more precisely to predict chemotherapy response and survival time than tha

26、t of total ERCC1 mRNA expression level. The alternative splicing variant lacking exon VIII could not predict chemotherapy response and survival time and was not a prognostic factor for epithelial ovarian cancer patients. 【Key Words】Ovarian Neoplasms, Antineoplastic Combined Chemotherapy Protocols; R

27、everse Transcriptase Polymerase Chain Reaction；Alternative Splicing；Excision Repair Cross-Complementation Group 1CHAPTER FOURERCC1 CODON 118 POLYMORPHISM AND CLINICAL OUTCOME OF PLATINUM-BASED CHEMOTHERAPY IN PATIENTS WITH EPITHELIAL OVARIAN CANCERObjective: The goal of this study was to investigate

28、 the effect of ERCC1 (excision repair cross-complementation group 1) single nucleotide polymorphisms of codon 118 C/T on chemotherapy response and survival time of epithelial ovarian cancer patients.Method: Genome DNA was isolated from peripheral nucleated cells of 67 epithelial ovarian cancer patie

29、nts and then PCR-RFLP was applied to determine the polymorphism of codon 118 C/T. All patients were follow-up to determine the survival time after surgery.Results: ERCC1 codon 118 C/T was associated with chemotherapy response. But codon 118 C/T had not shown association with survival interval.Conclu

30、sion: ERCC1 codon 118 C suggests that patient maybe sensitive to platinum-based chemotherapy while T suggest resistant. ERCC1 codon 118 polymorphism can not predict survival time for epithelial ovarian cancer patients. 【Key Words】Ovarian Neoplasms, Antineoplastic Combined Chemotherapy Protocols; pol

31、ymerase chain reaction；Restriction fragment length polymorphism；single nucleotide polymorphism；Excision Repair Cross-Complementation Group 1CHAPTER FIVECISPLATIN MODULATION EXCISION REPAIR CROSS-COMPLEMENTATION GROUP 1 AND ALTERNATIVE SPLICING IN HUMAN OVARIAN CARCINOMA CELL LINE A2780Objective: The

32、 goal of this study was to investigate the effect of cisplatin on the mRNA and alternative splicing expression of ERCC1 (excision repair cross-complementation group 1) in a human ovarian carcinoma cell line A2780.Method: A human ovarian carcinoma cell line A2780 was cultured in RPMI 1640 medium and then was exposed to different concentration of cisplatin for indicated time. Then the cells were lysed by TRIZol reagent and total RNA was isolated. The mRNA and alternative splicing express