【作者】 张智弘；

【导师】 范钦和； Jinsong Liu；

【作者基本信息】 南京医科大学 ， 病理学， 2007， 博士

【摘要】 目的：明确BRCA1／2杂合性突变卵巢表面上皮细胞形态学的改变，在预防性卵巢切除标本中BTAK和p53的表达水平，进一步探讨BRCA杂合性突变的卵巢上皮细胞中BTAK与p53的关系以及卵巢癌的发生机制。方法：1．携带BRCA杂合性突变的卵巢表面上皮细胞形态学的改变：选取32例BRCA1／2杂合性突变病人的卵巢、27例正常卵巢，HE染色后，在显微镜下观察其病理形态学改变。观察指标：表面上皮内陷，上皮包涵囊肿，皮质间质增生，砂粒体，子宫内膜异位，表面乳头状突起，乳头状瘤病，上皮增生(专指上皮细胞假复层，即有多层核)，上皮细胞的异型性(专指在上皮增生基础上有不典型性、多形性和极性消失)。2．携带BRCA杂合性突变的卵巢表面上皮细胞BTAK的表达：32例BRCA1／2杂合性突变病人的卵巢、27例正常卵巢以及194例卵巢癌的组织芯片，免疫组织化学标记BTAK蛋白，图像分析检测光密度值，统计分析表达水平的差异；1个BRCA1突变的细胞株OSE76和3个没有突变的细胞株OSE72，OSE103和OSE137，用Western Blot方法检测BTAK蛋白表达的差异。3．携带BRCA杂合性突变的卵巢表面上皮细胞p53的表达及卵巢癌的发生机制的研究：32例BRCA1／2杂合性突变病人的卵巢、27例正常卵巢以及194例卵巢癌的组织芯片，用免疫组织化学方法标记p53蛋白，统计分析表达水平的差异以及BTAK与p53表达的关系；用逆转录病毒载体方法构建含p53siRNA的表达载体，阻断p53基因，来研究BTAK的表达情况，从而进一步掌握BTAK与p53的相互关系，认识其在卵巢癌发生机制中的作用。结果：1．BRCA突变组和正常卵巢组的表面上皮细胞，两者的病理形态学改变有显著差异，表现在上皮增生和上皮细胞的异型性(p=0.0418和p=0.0289)，BRCA突变组的病变的发生率比正常卵巢组病变的发生率显著增高；而正常卵巢组无病理改变，比BRCA突变组显著减低(p=0.0341)。同时BRCA突变组与正常卵巢组比较，出现表面上皮内陷、间质增生和表面乳头状突起的，BRCA突变组的发生率比正常卵巢组的发生率高，但是统计学没有显著意义。2．在正常卵巢、BRCA突变卵巢和卵巢癌中BTAK的表达：组织学标本中BTAK在21／32(66％)的杂合性BRCA突变携带者的卵巢中表达，在正常卵巢4／27(15％)中表达(p＜0.05)。杂合性BRCA突变携带者卵巢与卵巢癌表达194／194(100％)相比，两者之间有显著差异(p＜0.05)。在携带或不携带BRCA1杂合性突变的原代卵巢上皮细胞株中，Western blot检查结果显示，3个不携带BRCA突变的上皮细胞株(OSE72，OSE103和OSE137)没有检测到BTAK的条带，而携带BRCA1突变的上皮细胞株(OSE76)有特异性的BTAK蛋白的强表达(46 kDa)。3．在正常卵巢、BRCA突变卵巢和卵巢癌中p53的表达：组织学标本中p53仅在正常卵巢的1例中(4％)表达，在BRCA突变携带者的卵巢中有10／32(31％)的表达，这两者之间无统计学差异(p＞0.05)。卵巢癌表达最高，有139／194(72％)表达，与杂合性BRCA突变携带者卵巢之间存在显著差异(p＜0.05)。用秩和检验证实BTAK表达与p53表达有直接的相互关系(r=0.306，p＜0.001)。4．我们建立了一个稳定的SiRNA沉默p53基因。Western blot检测了OSE76和OSE76／p53i的原代卵巢上皮细胞中BTAK的表达水平显示，感染p53siRNA OSE76细胞没有检测到BTAK的条带，而携带BRCA1突变的上皮细胞株(OSE76)有特异性的BTAK蛋白的强表达(46kDa)。结论：1．证明了BRCA杂合性突变的卵巢表面上皮细胞更易于显示不典型增生；卵巢癌发生存在早期病变：表面上皮的不典型增生；预防性卵巢切除标本是研究卵巢癌发病机制和早期病变的最佳标本。2．人类携带杂合性BRCA胚细胞突变的卵巢表面上皮细胞BTAK的蛋白表达水平增高，且在卵巢表面上皮细胞内可以由杂合性BRCA1或BRCA2突变激活；提出BTAK基因在卵巢癌发生的早期阶段扮演重要角色，BTAK表达水平升高可能是早期卵巢癌发生的信号，它可能是潜在的诊断指标和基因治疗的靶标。3．携带杂合性BRCA胚细胞突变的卵巢表面上皮细胞p53表达水平升高，且在卵巢表面上皮细胞内可以由杂合性BRCA1或BRCA2突变激活；证实BTAK高表达和p53高表达密切相关且可能在卵巢癌的发生机制中两者相互作用，与BRCA基因一起共同在卵巢癌的发生机制中扮演重要角色。更多还原

【Abstract】 Purpose:This study was to determine the morphologic changes in ovariansurface epithelial cells, and BTAK & p53 expression patterns inprophylactically removed ovaries with heterozygous BRCA1/2 mutationas compared with normal ovaries and ovarian cancer controls. We soughtto determine the relationship between BTAK and p53 so as to understandthe tumorigenesis of ovarian carcinoma.Methods:1. Morphologie features in ovarian surface epithelial cells withheterozygous BRCA1/2 mutation:Two pathologists (ZZ and DGR) observed the morphologic changesunder microscopy independently in a blinded manner in ovaries of 32patients with known BRCA1/2 mutation that underwent prophylacticoophorectomy and 27 normal ovaries from patients without any knownmutation. The evaluation of the following morphologic features in H&Estained slides includes deep surface epithelial invaginations, epithelialinclusion cysts, cortical stromal hyperplasia, psammoma bodies,endometriosis, surface papillations, papillomatosis, epithelial hyperplasia (defined by epithelial nuclear stratification), epithelial dysplasia (definedby epithelial hyperplasia with atypia, pleomorphism, and loss of polarity).2. Expression of BTAK in primary ovarian epithelial cells with orwithout BRCA1 heterozygous mutation:We performed immunohistochemical staining of morphologically normalovaries in a cohort of 32 patients who had prophylactic surgery andcompared these with 27 normal ovaries and 194 ovarian cancer controls.The BTAK intensity of staining was analyzed by computerized imageanalysis. The mean relative optical density was expressed as arbitraryunits of intensity and used for analysis. For statistical purposes opticaldensity values were grouped in a 4 score grading system as the meanoptical density±SD (177.46±15.10). Absence of staining was defined asnegative and given a score of 0, weak expression a score of 1, moderateexpression a score of 2 and strong expression a score of 3. Western blotanalysis of BTAK was performed in a primary cell culture carryingheterozygous BRCA1 mutation (OSE76) and three normal ovariansurface epithelial cell cultures (OSE72, OSE103, OSE137).3. Expression of p53 in primary ovarian epithelial cells with orwithout BRCA1 heterozygous mutation and study oftumorigenesis on ovarian carcinoma:We performed immunohistochemical staining in 32 patients with BRCAmutation compared these with 27 normal ovaries and 194 ovarian cancercontrols. Evaluation of the average expression for p53 expression wasperformed visually by two pathologists (ZZ and DGR) as follows: 0, lessthan 10% nuclear staining; 1, more than10%, less than25% nuclear staining; 2, more than 25%, less than 50% nuclear staining; 3, more than50% nuclear staining, discrepancy was resolved by third pathologist (JL).The relationship between expression of BTAK and p53 was analyzedwith Spearman Rank Order Correlation test. We developed aretrovirus-based system for stably expressing siRNA against p53 geneand then analyzed the BTAK expression by Western Blot. Withunderstanding of the relationship between BTAK expression and p53expression, the carcinogenesis of ovarian carcinoma was analyzed.Results:1. Morphologic Change:There was significantly difference in histological alterations betweencases and controls, especially including hyperplastic or dysplastic lesions(p=0.0418 and p=0.0289) on hematoxylin and eosin-stained sections.Normal ovaries have less frequency of pathologic lesions than BRCAmutation group (p=0.0341). The two groups both have deep surfaceepithelial invaginations, cortical stromal hyperplasia, surface papillations,but there were no significance.2. Expression of BTAK:The expression of BTAK was increased in 21 of 32 (66%) ovariescarrying a heterozygous BRCA mutation as compared with 27 normalcontrol ovaries which 4 of 27 (15%) positive for BTAK expression(p＜0.05; Mann-Whitney test). While ovarian cancer showed furtherincrease in BTAK as compared with BRCA heterozygous ovaries ina11-194 cases (p＜0.001; Mann-whitney test). The BTAK expression was significantly increased in primary culture carrying a heterozygousBRCA1 mutation (OSE76) as compared to those with no knownBRCA1/2 mutation (OSE72, OSE103 and OSE137) by Western blotanalysis. A specific band of BTAK protein (46 kDa) was detectedstrongly in OSE76 cell line, but no clear bands were found in OSE72,OSE 103 and OSE137.3. Expression of p53:The expression of p53 was positive in 1 of 27 cases (4%) in normal ovary,weakly increased in 10 of 32 (31%) positive in ovaries with knownBRCA mutation and highly expressed in 139 of 194 (72%) positive inovarian carcinomas. There were significantly difference expressionbetween ovarian carcinomas and ovaries with BRCA mutation (p＜0.05;Mann-Whitney test). Using Spearman correlation rank order test, weobserved BTAK was correlated with p53 (r=0.306, p＜0.001).4. Study of tumorigenesis:We developed a retrovirus-based system for stably expressing siRNAagainst p53 gene. SiRNA targeting p53 reduces expression of BTAK inOSE76 Cell Line. High expression of BTAK was in OSE76 cell line(46kDa) and no expression of BTAK was detected in OSE76 after p53siRNA introduction.Conclusions:1. The current study suggests pathologic evidence for the existence ofpreneoplastic changes in ovarian surface epithelium and support thepreviously proposed concept of ovarian dysplasia. The specimen of prophylactic oophorectomy is the best sample for findingpremalignant alterations in histology and early change intumorigenesis of ovarian carcinoma.2. Increased expression of BTAK is directly correlated with mutationstatus of BRCA1/2 genes, suggesting that mutation in a single alleleof either BRCA1 or 2 may be responsible for the activation of BTAK.This activation may be a key early genetic event in the developmentof hereditary ovarian cancer. Increased expression of BTAK may bean early signal in ovarian cancer and can be a potential diagnosticmarker and offer a novel target for chemoprevention.3. Increased expression of p53 is directly correlated with mutation statusof BRCA1/2 genes, suggesting that mutation in a single allele ofeither BRCA1 or 2 may be responsible for the activation of p53.BTAK expression correlates with p53 and their expressions may be anearly genetic event in the development of human ovarian cancer,especially in BRCA mutant patient. The interaction of BRCA, BTAKand p53 regulates the initiation of tumorigenesis in ovariancarcinoma.更多还原