【作者】 张颖；

【导师】 辛晓燕； 王建；

【作者基本信息】 第四军医大学 ， 妇产科学， 2008， 硕士

【摘要】 卵巢癌是严重威胁妇女健康的常见恶性肿瘤,死亡率在女性生殖器恶性肿瘤中占第1位。由于难于早期发现,近15年来,在手术和辅助化疗的综合治疗下,卵巢癌患者5年生存率一直徘徊于30%～50%之间。卵巢癌对铂类化疗药物的耐药是影响化疗效果,造成肿瘤复发、转移及死亡率高居不下的重要原因。明确卵巢癌耐药的分子机制,寻找有效的耐药逆转剂或有效的治疗靶标已成为国内外妇科肿瘤学界密切关注的研究热点。脱嘌呤/脱嘧啶核酸内切酶(apurinic/apyrimidinic endonuclease,APE1)又称氧化还原因子-1(redox factor-1,ref-1)它是人类细胞中唯一修复DNA AP位点的核酸内切酶,并能通过氧化还原机制调节多种转录因子的DNA结合活性及下游靶基因表达。受其调控的转录因子包括与细胞增殖、分化、转化和凋亡相关的重要因子,如NF-κB,AP-1,p53等。而这些转录因子与肿瘤放化疗抵抗密切相关。本研究应用免疫组织化学技术检测卵巢上皮性癌初治患者手术切除标本和卵巢癌顺铂敏感株A2780细胞、顺铂耐药株CP70细胞中APE1的表达,分析APE1与卵巢癌铂类耐药之间的关系,应用siRNA技术,观察抑制APE1表达对卵巢癌细胞化疗敏感性的影响,探讨APE1参与卵巢癌细胞耐药的机制,为卵巢癌逆转铂类耐药性提供有价值的实验依据。研究目的1.探讨APE1基因在卵巢上皮性癌中的表达与临床病理因素及铂类耐药之间的关系,以确证APE1为卵巢癌治疗潜在的分子靶点;2.探讨以APE1基因与卵巢癌铂类耐药的相关性及其在卵巢癌铂类耐药机制中的作用;3. APE1基因“敲除”对卵巢癌细胞耐药性的影响及其作用机制。研究内容和方法1.采用免疫组化检测卵巢上皮性癌组织APE1蛋白表达,分析APE1表达与卵巢癌临床病理因素的关系。2.免疫组化、western blot检测卵巢癌顺铂敏感株A2780细胞、顺铂耐药株CP70细胞中APE1蛋白表达及定位情况;western blot检测顺铂处理后A2780和CP70细胞中APE1蛋白表达及定位的变化,分析APE1表达与卵巢癌铂类耐药性的相关性。3. MTT法检测Ad5/F35-APE1 siRNA瞬时转染抑制APE1蛋白表达后A2780和CP70细胞对顺铂化疗敏感性的改变;TUNEL法检测抑制APE1蛋白表达对顺铂诱导的A2780和CP70细胞凋亡影响。研究结果1.卵巢上皮癌组织APE1表达与病理类型、细胞学分级等肿瘤的恶性生物学行为有关(P<0.05)。APE1表达强度和亚细胞定位均与以铂类为基础化疗的药物敏感性相关。2. CP70细胞的APE1蛋白表达的水平较A2780细胞高,A2780细胞中APE1表达以核表达为主,而在CP70细胞中呈核浆共表达。顺铂处理A2780和CP70细胞后,APE1蛋白表达呈剂量和时间依赖性增强。3. Ad5/F35-APE1 siRNA对卵巢上皮性癌顺铂化疗敏感性的影响:(1)感染Ad5/F35-APE1 siRNA后,两种卵巢癌细胞对顺铂的敏感性明显增强。40MOI的Ad5/F35-APE1 siRNA感染细胞后,A2780细胞和CP70细胞IC50分别为6.60μM和39.73μM,较对照组分别下降了61.89%和41.47%。(2)Ad5/F35-APE1 siRNA感染A2780细胞和CP70细胞后,顺铂诱导的卵巢癌细胞凋亡明显增多。Ad5/F35-APE1 siRNA 40MOI时,A2780细胞和CP70细胞凋亡指数为(42.16±3.61)%和(36.11±3.81)%,分别是DDP组的3.57倍、3.87倍,有显著差异(p<0.01)。结论1. APE1表达水平和亚细胞定位与卵巢癌的发生发展密切相关, APE1对卵巢癌的铂类化疗敏感性和预后有一定提示作用。2. APE1在顺铂敏感细胞和耐药细胞中蛋白表达强度和亚细胞定位不同,顺铂诱导卵巢癌细胞APE1蛋白表达呈剂量和时间依赖性增强,提示APE1在卵巢癌顺铂耐药形成机制中发挥重要作用。3.抑制APE1蛋白表达能一定程度增强卵巢癌细胞对顺铂化疗的敏感性,其机制与抑制癌细胞DNA修复和通过氧化还原调控凋亡途径调控有关。APE1可能为逆转卵巢癌铂类耐药的有效靶点。更多还原

【Abstract】 Ovarian carcinoma is one of the most frequent malignancies in women,and the leading cause of death from gynecological cancer. Early diagnosis is very difficult because of hiding symptom in early stage.Even with the combined use of surgery and chemotherapy,the five year survival rate of the patients is about 30%～50%.The major reason for the high mortality of ovarian carcinoma is the high metastatic ability and the resistance to platinum-based chemotherapy. The drug-resistant patients more tend to high relapse,high metastases and high mortality eventually.Identifying the molecules mechanism of the drug-resistance in tumor cells is a hot field for exploring the effective target for new eversal agent of drug resistance and new avaliable chemotherapeutics.The human apurinic/apyrimidinic endonuclease (APE1) is a ubiquitous multifunctional protein which has both DNA repair activity and redox regulatory activity. APE1 participates in some crucial cellular processes, including the response to oxidative stress, regulation of transcription factors. The transcription factors particpate with cellular proliferation,cell differentiation and apoptosis,such as NF-κB,p53,AP-1 etc.Meanwhile,these transcription factors are associated with chemoresistance and radioresistance. In this study, we detected the expression of APE1 protein in 120 cases ovarian carcinoma patients and two ovarian carcinoma cell lines.A2780 cell line is sensitive to cisplatin,and CP70 cell line is resistance to cisplatin.We investigated the relationship between the expression of APE1 and clinic-pathological parameters especially cisplatin- resistance of ovarian carcinoma. Then we used chimeric adenoviral vector Ad5/F35 carrying human APE1 siRNA to investigate its effect on the cispatin sensitivity in human ovarian carcinoma cells. After suppression APE1 protein,we investigated the cell proliferation inhibition and apoptosis induced by cisplatin in two kinds of human ovarian carcinoma cells lines in vitro, and presume that the APE1 protein may contribute to the cisplatin-resistance in ovarian carcinama.As a result,we offered the experiment evidence that the inhibiton of APE1 by chimeric adenoviral vector Ad5/F35 carrying human APE1 siRNA can influence the chemoresistance of ovarian carcinoma cells to cisplatin. Therefore,APE1 siRNA is a potiential new eversal agent of chemoresistance.OBJECTIVES:1. To investigate the expression of APE1 protein in ovarian carcinoma cases.To identify APE1 gene is potential molecules target of gene therapy against cancer.2. To investigate the relationship between APE1 expression and the clinical cisplatin resistance cases in ovarian carcinoma. To investigate the expression of APE1 protein in ovarian carcinoma A2780 and CP70 cell lines, and analysis the role of APE1 gene in platinum-resistance of ovarian carcinoma.3. To investigate the inhibitory effects of Ad5/F35-APE1 siRNA on human ovarian carcinoma in vitro, and explore the effect of enhancing chemosensitivity of ovarian carcinoma by inducing APE1 gene silence. METHODS:1. Expression of APE1 was examined by SP immunohistochemical technique in 120 cases of ovarian carcinoma. The medical records of the ovarian carcinoma patients were collected. The data was analyzed to explore the correlation between APE1 expression and clinical pathological parameters.2. Expression of APE1 in A2780 and CP70 cells was detected by western blot and SP immunohistochemical technique. Then we detected the change of APE1 protein expression of A2780 and CP70 cells which were pretreated with cisplatin by western blot and immunocytochemical staining.It was analyzed that relationship between APE1 expression and cisplatin resistance in ovarian carcinoma.3. Cells were treated with cisplatin after Ad5/F35-APE1 siRNA transient transfection into A2780 and CP70 cells, and the cellular proliferation capacity was observed with MTT assay. To further verify that the effect of Ad5/F35-APE1 siRNA,cellular apoptosis was determined by TUNEL .RESULTS:1. Statistic analysis showed that APE1 expression was related with maligancy biological behaviour such as pathologic classification ,FIGO stage and cell differentiated classify (P<0.05). The intensity and subcellular of APE1 protein expression is related with platinum-based chemotherapy sensetivity.2. We found that APE1 almost localized in cytoplasmic in A2780 cell, but in cytoplasmic and nucleus in CP70 cell,and the APE1 expression in CP70 cell is higher than A2780 cell. The result of Western blot showed that expression of APE1 gene was high in both of the two cell lines pretreated by DDP, the degree of expression of APE1 protein was a prositive corretation with DDP dose and treating time. 3. The influence of Ad5/F35-APE1 siRNA to platinum sensitivity in human ovarian carcinoma cells:(1)The result of MTT showed that Ad5/F35-APE1 siRNA enhanced platinum sensitivity of A2780 and CP70 cell lines.Pretreatmented with 40MOI Ad5/F35-APE1 siRNA ,the 50% inhibitory concentration (IC50) value of cisplatin in A2780 and CP70 cells was 6.60μM and 39.73μM, respectively, decreased by 61.89% and 41.47% compared by pretreatmented with control group.(2)Ad5/F35-APE1 siRNA increased cell apoptosis induction by DDP. Apoptotic index of A2780 and CP70 cell lines respectively significantly increased by transfecting APE1siRNA. it had significant statistics difference(P<0.01).CONCLUSION1.Expression of APE1 and subcellular location was correlated with ovrian carcinogenesis. To detect gene APE1 will be helpful to judge the therapeutic effect and prognostic of ovarian carcinoma.2.The level of APE1 expression and the subcellular location were notable different between A2780 and CP70 cell line.After DDP treated, APE1 expression was increased remarkably in both A2780 and CP70 cell lines.We presumed APE1 may partially contribute to drugresistance of ovarian carcinoma cells.3. Inhibiting APE1 expression improved cisplatin chemotherapy sensitivity remarkably and increase apoptosis in ovarian carcinoma cells. We hypothesis that the main molecular mechanisms involved may be downregulation of base excision repair capacity , regulate the cell generation cycle by its oxidoreduction function, and induce transcription activity of apoptosis pathway of ovarian carcinoma cells. Targeting inhibition of APE1 may be a promising approach to a new eversal platinum resistance agent in ovarian carcinoma.更多还原