【作者】 卢媛；

【导师】 刘惜时； 宋后燕； 李子庭； 卢大儒；

【作者基本信息】 复旦大学 ， 妇产科学， 2006， 博士

【摘要】 目的：卵巢癌是死亡率最高的生殖系统肿瘤，其中90％为上皮性卵巢癌，其发生发展是一个多因素参与的多阶段、多步骤的演变过程。卵巢癌是成年妇女中最常见的具有遗传因素的肿瘤之一，也是HNPCC最常见的伴发肿瘤之一。本课题通过检测新鲜卵巢癌组织标本中的微卫星不稳定性及错配修复基因mRNA的表达，来了解卵巢癌中是否存在基因组不稳定性及错配修复基因的失活，并了解微卫星不稳定性与错配修复基因表达与卵巢癌的分期，分级，分型的关系。同时通过检测错配修复基因启动子区域的甲基化从DNA水平来探讨卵巢癌中错配修复基因失活的机制，有助于阐明卵巢癌的发生机制。第一部分上皮性卵巢癌和微卫星不稳定性方法：收集2004年6月-2005年1月复旦大学附属肿瘤医院和附属妇产科医院的90例上皮性卵巢癌新鲜切除标本及同一患者的5ml术前外周静脉血作为判断微卫星不稳定性的正常对照，并同时收集20例同期手术的良性卵巢囊肿患者的卵巢病灶新鲜组织和5ml术前外周静脉血作为对照组：分别抽提病例组和对照组组织和外周血基因组DNA，选取国际合作HNPCC小组推荐的五个检测位点是：BAT25、BAT26、D2S123、D5S346、D17S250，进行毛细管凝胶电泳，分析软件自动分析得出结果，用SPSS11.0统计软件分析，P＜0.05提示差别有统计学意义。结果：1)：病例组中和对照组MSI-H的发生率为20％、0，差异具有显著。(P＜0.05)；2)：上皮性卵巢癌中MSI-H的发生率与分期显著显著相关(P=0.003)，Ⅰ期发生率较高，为：45％；3)：上皮性卵巢癌中MSI-H的发生率在低分化的肿瘤、粘液性腺癌、和透明细胞癌中的发生率较其他类型的卵巢癌高，但是MSI—H的发生率与患者年龄、肿瘤的分化、病理分型没有显著相关性(P＞0.05)。结论：微卫星不稳定性在上皮性卵巢癌中是一重要现象；在不同亚型的上皮性卵巢癌中的作用不同：多发生于疾病的早期，在上皮性卵巢癌的发病中起一定的作用，并推动疾病的恶变进程。第二部分上皮性卵巢癌中错配修复基因表达和微卫星不稳定性方法：收集2004年6月-2005年1月复旦大学附属肿瘤医院和附属妇产科医院的90例上皮性卵巢癌新鲜切除标本并同时收集20例同期手术的良性卵巢囊肿患者的卵巢病灶新鲜组织做为对照，提取mRNA；用实时荧光定量PCR仪相对定量分析对照组和病例组中hMLH1、hMSH2、hMSH3、hMSH6、hPMS1、hPMS2的表达；结果用SPSS11.0统计软件分析，P＜0.05提示差别有统计学意义。结果：1)：hMLH1的mRNA的表达在良性囊肿的表达明显高于上皮性卵巢癌组织(P＜0.05)；在微卫星稳定性的病例中明显高于微卫星不稳定性的病例(P＜0.05)；在分化低的病例中明显低于分化高的病例(P＜0.05)：与肿瘤的分期、分型没有显著性差异(P＞0.05)。2)：hMSH2的mRNA表达在良性囊肿和肿瘤组织没有显著性差异(P=0.987)；其表达与肿瘤的分期、分级、分型及微卫星状态没有显著性差异(P＞0.05)。3)：hMSH3、hMSH6、hPMS1、hPMS2的mRNA的表达在良性囊肿和肿瘤组织有显著性差异(P＜0.05)；其表达与肿瘤的分期、分级、分型及微卫星状念没有显著性差异(P＞0.05)。结论：人体错配修复系统中，hMLH1基因对DNA碱基错配修复具有重要的作用；错配修复基因表达的缺失尤其是hMLH1基因在上皮性卵巢癌的发生中起一定的作用：且促进疾病的恶性演进：并导致上皮性卵巢癌中的微卫星不稳定性。第三部分上皮性卵巢癌中hMLH1基因表达缺失和启动子甲基化方法：用甲基化特异PCR(MSP)的方法分析病例组和对照组中hMLH1基因启动子甲基化的状态，并分析病例组中hMLH1基因启动子甲基化的状态与肿瘤的临床病理特征和微卫星状态的关系。结果用SPSS11.0统计软件分析，P＜0.05提示差别有统计学意义。结果：1)：良性卵巢囊肿，上皮性卵巢癌中hMLH1基因启动子区甲基化的发生率分别为0、16.6％，差异有统计学意义(P＜0.05)；2)：hMLH1基因启动予区甲基化的发生率在不同分期、分级、及分型的上皮性卵巢癌中没有显著性差异(P＞0.05)；3)：hMLH1基因启动子区甲基化的发生与上皮性卵巢癌中微卫星不稳定性的发生及hMLH1基因的表达缺失显著相关(P＜0.05)。结论：hMLH1基因启动子区甲基化是上皮性卵巢癌中hMLHl基因的表达缺失的主要机制，也可能是导致上皮性卵巢癌中微卫星不稳定性的主要机制。更多还原

【Abstract】 Ovarian cancer represents the most lethal malignancy among gynecological tumours in China, in which 90% are epithelial ovarian tumors. Etiology is yet unknown. Multistep process, with accumul ation of genetic alterations concerning factors with key role in cell regulation -oncogenes, tumor-supressor genes and mismatch-repair genes is supposed. This study was conducted to evaluate the frequency of MSI and the expression of the mismatch repair gene in epithelial ovarian tumors and their relationship with clinicopathologic features, in order to know whether there is genomic instability and losing expression of mismatch repair gene. In this study we have explored the mechanism of hMLH1 gene silencing by analyzing the aberrant methylation of CpG islands in its promoter. It helps us to understand the molecular events involved in the development of ovarian tumors.Part I: Study of microsatellite instability and its relationship withclinicopathologic features in epithelial ovarian tumorsObjective: This study was conducted to evaluate the frequency of MSI inepithelial ovarian tumors and its relationship with clinicopathologicfeatures.Methods: ninety fresh specimens of epithelial ovarian tumors (primary 74,secondary 16) and twenty fresh specimens of ovarian cyst as control werecollected from The Obstetrics and Gynecology Hospital of Medical Centerof Fudan University from 2004 to 2005. Microsatellite analysis was carriedout using 5 mono- and dinucleotide markers from the National CancerInstitute Consensus Panel by fluorescence-labeled polymerase chainreaction. The results were auto-analyzed by software of GeneScanAnalysis3. 7 and Genotyper.Results: 1): Of the 20 ovarian cysts analyzed, all were demonstrated MSS.Of the 90 epithelial ovarian tumors analyzed, 18 were demonstrated a highlevel of MSI (MSI-H), 30 were demonstrated a low level of MSI (MSI-L),and the remaining 42 exhibited microsatellite stability (MSS).2): Frequency of MSI at loci BAT-25 was higher than that at any other loci. No correlation was found between MSI level and patient age, tumor type, tumor differentiation (P >0.05).3): The microsatellite instability-high phenotype correlate with clinical stage, it tended to occur more frequently in early-stage tumors (P=0.03).Conclusions: Our results indicate that there are frequent MSI in epithelial ovarian tumors. It is an early event and it is involved in the development of epithelial ovarian tumors.Part II: Study of expression of mismatch repair genes and its relationship with clinicopathologic features in epithelial ovarian tumors Objective: This study was conducted to evaluate the expression of mismatch repair genes in epithelial ovarian tumors and its relationship with clinicopathologic features. Investigate the correlation between the MSI and the expression of mismatch repair genes.Methods: Ninety fresh specimens of epithelial ovarian tumors (primary 74, secondary 16) and twenty fresh specimens of ovarian cyst as control were collected from The Obstetrics and Gynecology Hospital of Medical Center of Fudan University from 2004 to 2005. Comparatively quantitative analysis of expression of MMR genes (hMLH1、 hMSH2、 hMSH3、 hMSH6、 hPMS1、 hPMS2) were conducted by real-time polymerase chain reaction. Results:1): The quantity of expression of mRNA of the hMLH1 gene in the ovarian cyst is significantly higher than that in the epithelial ovarian tumors (P<0.05) . The quantity of expression of mRNA of the hMLHl gene in high grade tumor is significantly higher than that in the low grade tumor (P<0.05) .There is a significant correlation between the quantity of expression of mRNA of the hMLHl gene and the status of the microsatellite(P<0.05). No correlation was found between the quantity of expression of mRNA of the hMLHl gene and tumor type, tumor stage (P>0.05). 2) : The quantity of expression of mRNA of the hMSH2 gene in the ovarian cyst is not significantly higher than that in the epithelial ovarian tumors (P>0.05) . No correlation was found between the quantity of expression of mRNA of the hMSH2 gene and tumor type, tumor stage, tumordifferentiation and the status of the microsatellite. (P>0.05).3): The quantity of expression of mRNA of the hMSH3、 hMSH6、 hPMS1、 hPMS2 gene in the ovarian cyst is significantly higher than that in the epithelial ovarian tumors (P<0.05) . No correlation was found between the quantity of expression of mRNA of the hMSH3、 hMSH6、 hPMS1、 hPMS2 gene and tumor type, tumor stage, tumor differentiation and the status of the microsatellite (P>0.05).Conclusions: The losing expression of MMR genes, especially hMLH1 gene have a key effect in the tumorgenesis of the epithelial ovarian tumors, which can result in MSI in the tumor.Part III: Study of aberrant methylation of promoter CpG islands of hMLHlgene in epithelial ovarian tumorsObjective: To analyze the aberrant methylation of CpG islands in hMLHlgene promoter by using MSP (methylation specific polymerase chainreaction), and then to investigate its relationship withclinicopathologic features, the expression of hMLHl gene and the statusof the microsatellite.Results: 1): The frequency of the methylation of CpG island in the hMLHlgene in control group (0%) were significantly lower than that inepithelial ovarian tumors (16.6%) (P<0.05).2): No correlation was found between the frequency of the methylation of CpG island in the hMLHl gene and tumor type, tumor stage, tumor differentiation (P>0. 05) .3) : There was a highly significant negative correlation among methylation of CpG island in hMLHl gene, mRNA expression of hMLHl gene and the status of the microsatellite(P>0.05).Conclusions: Methylation of CpG island in hMLHl is the main cause of its loss expression. It may be also the key mechanism which induce the MSI-H in the epithelial ovarian tumors.更多还原