【作者】 张淑红；

【导师】 张向宁；

【作者基本信息】 山东大学 ， 妇产科学， 2008， 硕士

【摘要】 卵巢恶性肿瘤中以上皮性癌最为多见,其起病隐匿,易于广泛扩散和转移,就诊时三分之一已属晚期,且多见有腹水及盆腹腔广泛的种植转移,使之始终占据卵巢恶性肿瘤死亡率的首位。目前认为肿瘤是一种基因性疾病,原癌基因激活、抑癌基因功能丧失以及一些修饰基因功能的改变导致肿瘤的发生和发展。对抑癌基因的研究是目前肿瘤研究领域内的热点。而脆性组氨酸三联体（fragile histidine triad,FHIT）基因和抑癌基因WW的结构域（WWdomain-containing oxidaseredurase,WWOX）分别是活跃的第一和第二位脆性位点的抑癌基因,它们在肺癌、乳腺癌等多种肿瘤中都有不同程度的表达缺失且具有相关性。FHIT基因是1996年被分离出来的一种新的肿瘤抑制基因,定位于染色体3P^14.2,全长500Kb,其编码序列含组氨酸三联体,属于HIT家族,又因为此基因跨越脆性位点,故将其命名为脆性组氨酸三联体。FHIT蛋白具有三腺苷三磷酸（AP3A）水解酶的活性,可以将其水解为ADP和AMP。目前FHIT基因的功能主要与它表达产物的AP3A水解酶活性有关。WWOX基因位于染色体16q23.3-24.1区域,跨越了整个常见染色体脆性位点FRA16D,而FRA16D是第二位发现的较活跃的脆性位点。WWOX蛋白的氨基酸末端的两个WW结构域主要与蛋白之间的相互作用有关,而蛋白之间的相互作用是机体抑癌基因通过信号转导途径抑制肿瘤生长所必需。大量研究表明,FHIT基因与WWOX基因表达的减少或缺失参与肿瘤的恶性进展,与肿瘤的预后密切相关。本文旨在探讨FHIT基因与WWOX基因在人卵巢上皮癌组织中的表达特点及其表达水平的变化与上皮性卵巢癌临床、病理等因素的关系,从而为临床治疗、随诊以及预后判断提供理论依据。目的:通过对不同年龄、组织类型、临床分期、病理分级、雌孕激素受体、淋巴结（或远处）转移以及有无癌性腹水的卵巢上皮性癌组织中FHIT基因和WWOX基因表达进行研究,探讨FHIT基因和WWOX基因表达与卵巢癌临床病理各相关因素的关系,了解它们在卵巢上皮性癌发生发展中的临床病理意义,并预测价值。方法:1、β-actin为内参照,应用逆转录聚合酶链反应（RT-PCR）法检测卵巢上皮性癌和正常卵巢组织中脆性位点抑癌基因FHITmRNA与WWOXmRNA的表达,半定量分析电泳结果。以免疫组化法检测卵巢上皮性癌石蜡标本中雌激素受体（ER）和孕激素受体（PR）的表达情况。基因表达与临床病理各因素之间关系的分析采用秩和检验;Kaplan meier生存分析法分析FHIT基因和WWOX基因表达与卵巢癌患者预后的关系。2、用免疫组织化学SP法检测卵巢上皮性癌组织中脆性位点抑癌基因FHIT和WWOX蛋白的表达情况,并收集各临床检验指标。基因表达与临床病理各因素之间的比较采用x²检验和Fisher’s精确检验进行统计分析。结果:1、①在上皮性卵巢癌中,FHITmRNA与WWOXmRNA的弱表达或无表达率分别为31.3%（15/48）、39.6%（19/48）,正常卵巢组织FHITmRNA与WWOXmRNA均为正常表达。②FHITmRNA与WWOXmRNA在上皮性卵巢癌组织中的低表达有明显相关性（P＜0.05）。③FHITmRNA与WWOXmRNA的表达与临床分期呈负相关（P＜0.05）。④FHITmRNA与WWOXmRNA的表达与肿瘤的组织学分级呈负相关（P＜0.05）。⑤FHITmRNA与WWOXmRNA的表达与与卵巢癌转移和癌性腹水统计学意义显著（P＜0.05）⑥WWOXmRNA的表达缺失与PR表达阴性有相关性（P＜0.05）。⑦WWOX基因的表达与患者预后有显著相关性。2、①上皮性卵巢癌组织中,FHIT蛋白和WWOX蛋白的阴性表达率分别为56.25%（27/48）和52.08%（25/48）,FHTI蛋白在正常卵巢组织中均阳性表达,WWOX蛋白在正常卵巢组织中有1例阴性表达,差异具有统计学意义（P＜0.05）。②上皮性卵巢癌组织中FHIT和WWOX蛋白的阴性表达率随着肿瘤临床分期的增加而增加,二者呈正相关,统计学意义显著（P＜0.05）。③上皮性卵巢癌组织中FHIT和WWOX蛋白的阴性表达率随着肿瘤组织学分级的增加而增加,二者呈正相关,统计学意义显著（P＜0.05）。④上皮性卵巢癌组织中FHIT和WWOX蛋白的阴性表达率与卵巢癌转移及癌性腹水呈正相关,统计学意义显著（P＜0.05）。⑤上皮性卵巢癌组织中FHIT和WWOX蛋白的阴性表达与患者年龄和肿瘤的组织学类型不具统计学意义（P＞0.05）。⑥FHIT和WWOX蛋白在上皮性卵巢癌组织中的低表达具有明显相关性（P＜0.05）。结论:1.FHIT和WWOX基因的表达缺失与上皮性卵巢癌的发生和发展密切相关,检测其表达水平对卵巢癌的早期诊断、临床进展及估计预后有一定的参考价值。2.FHITmRNA和WWOXmRNA的低表达或不表达及其它们蛋白的表达缺失分别与卵巢癌的临床分期、病理分级、卵巢癌转移与癌性腹水形成具有明显的相关性,与年龄、组织学类型无明显相关性。更多还原

【Abstract】 As one of the most malignant gynecologic tumors,the early symptoms of epithelial ovarian carcinoma are very secret and very easy to metastasize,almost thirty percent patients have cancerous ascites and extensive implantation metastasis when they are diagnosed.The epithelial ovarian carcinoma was characterized by highest mortality rate。Nowadays we consider that tumor is gene disease,the oncogene activation,anti-oncogene inactivation and the change of some modificator genes are benefit to the occurrence and development of tumors.It is becoming an attractive spot to study the anti-oncogene.Fragile histidine triad（FHIT） gene and WWOX gene are first and second anti-oncogenes loading in fragile site,there were some extent expression deletion in lung cancer,breast cancer and so on.Fragile histidine triad（FHIT） was discovered in 1996 as a new anti-oncogene,mapping chromosome 3P^14.2,500Kb,the exon was histidine triad. FHIT protein has AP 3A hydrolytic enzymatic active,which is relate to the function of FHIT protein.WWOX gene maps chromosome16q23.3-24.1, spanning the whole fragile site FRA16D,the two WW structures are relate to interaction of proteins,which is necessary for anti-oncogenes to inhibit tumor growth.The study has showed that the decrease or deletion of FHIT and WWOX genes had significant relationship to tumor progression and prignosis,Our study aimed to approach the expression of FHIT and WWOX in EOC,and further to explore the relationship between expression lever and clinical and pathologic characteristics,then offer the theory basement for treatment,follow-up and prognosis judgement.Objective:To investigate the expressions of FHITand WWOX in epithelial ovarian carcinomas and their relations with clinicopathological and other molecular parameters.To help determine whether the expression of the genes FHIT and WWOX are associated with EOC progression and metastasis.Methods:1、With reference to the expression ofβ-actin,the expressions of FHITmRNA and WWOXmRNA in epithelial ovarian carcinomas and normal ovarian epithelial tissues were determined by reverse transcription-polymerase chainreaction（RT-PCR）,and semi-quantification of band densities was also performed.The expressions of estrogen receptor（ER） and progesterone receptor（PR） in the 48 ovarian cancer lesions were detected by immunohistochemical method.The relationship between the expressions of the genes and the prognosis of patients was also studied..2、Immunohistochemistry SP method was performed to examine the expression level of FHIT and WWOX in normal ovary tissues and various grades of primary and metastatic EOC.Comparisons were made between expression patterns in normal ovary and EOC.The expression of FHIT and WWOX among groups were initially compared by x² analyses and Fisher exact probabilities test.Results:1、19 and 15 cases of epithelial ovarian carcinomas（39.6%and 31.3%） showed respectively loss or absence of WWOX and FHIT genes,but there was no loss of WWOX and FHIT genes in normal ovary tissues.The expressions of FHITmRNA and WWOXmRNA in 48 epithelial ovarian tumors were correlative（P＜0.05）,but were not related with patients’ ages,tumor histologictype,ER,metastasis and ascites.The expression of WWOXmRNA was related to PR（P＜0.05）.There was reverse correlation between the expressions of FHIT and WWOX and the clinical stages.In addition,WWOX was closely connected with the prognosis of patients.2、The study revealed a close relationship between tumor phenotype and FHIT and WWOX expressionlevels,characterized by high expression in normal ovary,while low expression（27/48,25/48） in primary invasive and metstatic tumors. Statistically siginificant differences were noted between normal ovary and EOC（P＜0.05）.The statistical evaluation showed that decrease of FHIT and WWOX expression was associated with the advanced stage of EOC（P＜0.05）.It also revealed an inverse relationship between histological grade and FHIT and WWOX（P＜0.05）.The statistical evaluation also showed that the expression of FHIT and WWOX had a correlation with lymph node metastasis（or distant metastasis）（P＜0.O5）.There is no significant relationship among FHIT and WWOX expression and age and tumor histologic type（P＞0.05）.The expressions of FHI Tand WWOX in 48 epithelial ovarian tumors were correlative（P＜0.05）.Conclusions:1.FHIT and WWOX expression are closely correlated with the malignant progression and metastasis.It may be an early event in the progression of this tumor.Detecting the expression of FHIT and WWOX probably possesses clinical significance in evaluating the differentiation,tumor progression and predicting the prognosis.2.The expressions of FHIT and WWOX in 48 epithelial ovarian tumors are related with the advanced stage of EOC and lymph node metastasis（or distant metastasis）,but no relationship with patients’ ages,tumor grades,tumor histologic type.更多还原