【作者】 李艳丽；

【导师】 谢幸；

【作者基本信息】 浙江大学 ， 妇产科学， 2010， 博士

【摘要】 卵巢癌是致死率最高的妇科恶性肿瘤,严重威胁妇女的生命和健康。浆液性卵巢癌是其中最主要的病理类型。目前卵巢癌的发病、转移机理仍不清楚。近年来诸多研究提示,肿瘤是一种分子病,肿瘤的特异表现很可能是由深层次的基因改变造成的,而其产物——蛋白质是细胞功能的最终执行者,故本论文的目的在于,从蛋白质表达水平的角度来探讨卵巢癌的发生发展机制。首先,通过双向电泳技术获得了10例浆液性卵巢癌组织和10例正常卵巢上皮组织进行了总蛋白表达胶图,用PDQUEST软件进行对比分析,找出在浆液性卵巢癌组织和正常卵巢上皮组织中表达差异2倍以上并有统计学意义(P＜0.01)的蛋白质点。挖取并富集这些差异蛋白质点进行MALDI-TOF-TOF质谱鉴定。其次,选取了其中的6个蛋白质：GGCT, GMFB, HDGF, Rho GDI 2, BANF1,和Galectin-1,采用western blot的方法检测它们在20例浆液性卵巢癌组织和20正常卵巢上皮组织中的表达情况,以检验双向电泳及质谱鉴定的准确性。接着,为了观察相应的基因表达情况,又采用了实时荧光定量RT-PCR的方法检测了相应的6个基因：GGCT, GMFB, HDGF, ARHGDIB, BANF1,和Galectin-1在同样20例浆液性卵巢癌组织和20正常卵巢上皮组织中的表达情况。然后,选取了一个在人类肿瘤中研究较少的蛋白质-GMFB,扩大样本量,采用免疫组织化学的方法,检测了其在245例各种不同程度的卵巢上皮性病变(44例正常卵巢上皮组织,51例良性浆液性囊腺瘤,40例交界性浆液性腺瘤,110例浆液性卵巢癌)中的表达情况,分析了GMFB蛋白质表达与浆液性卵巢癌临床及组织病理学参数的关系,并采用了单因素和多因素模型的方法,进一步判断了GMFB蛋白质表达与浆液性卵巢癌患者预后的关系。最后,本研究基于RNAi干扰的方法,从细胞水平进一步探讨了GMFB对浆液性卵巢癌SKOV3细胞增殖的影响。本研究最终在每张胶图上平均约分离到1800个蛋白质点,找出63个差异表达的蛋白质点,并成功鉴定到其中的61个,代表54个独立的蛋白质。Western blot验证表明相对于正常卵巢上皮组织,GGCT, GMFB, HDGF, Rho GDI 2在浆液性卵巢癌组织中表达上调(P<0.001),而BANF1,和Galectin-1在浆液性卵巢癌组织中表达下调(P<0.001),其表达变化趋势与前期双向电泳及质谱结果完全吻合,一定程度上证实了前期工作的可靠性。与于正常卵巢上皮组织比较,HDGF和ARHGDIB基因在浆液性卵巢癌组织中表达上调(P＜0.0001), BANF1和Galectin-1基因在浆液性卵巢癌组织中表达下调(P<0.0001),它们的表达变化趋势与蛋白质表达变化趋势相同；然而,GGCT和GMFB基因在正常卵巢上皮组织和浆液性卵巢癌组织中的表达未发现明显差异。免疫组化结果发现,GMFB蛋白质在正常卵巢上皮组织及三种卵巢上皮性病变中表达有显著差异(P<0.0001),且从正常卵巢上皮组织,到良性浆液性囊腺瘤,交界性浆液性腺瘤,再到浆液性卵巢癌,表达逐渐上调,并与浆液性卵巢癌患者的临床分期相关(P=0.012)。生存分析提示,GMFB与浆液性卵巢癌患者的总体生存率(P=0.003)和无瘤生存率(P=0.010)相关,而且多元回归模型分析揭示GMFB是浆液性卵巢癌患者总体生存(P=0.006)和无瘤生存(P=0.026)的独立预后因子。RNAi干扰技术成功沉默GMFB在SKOV3细胞株中的表达,基因沉默组肿瘤细胞增殖力明显下降(P<0.05)。总之,蛋白质组学技术是发现及筛选肿瘤相关蛋白质的有力手段。GGCT, GMFB, HDGF, Rho GDI 2, BANF1,和Galectin-1等差异蛋白质可能与浆液性卵巢癌的发生发展相关,也可能是潜在的分子标志物。GMFB可能通过促进细胞增殖而参与浆液性卵巢癌发生发展过程,它同时也是一个新发现的浆液性卵巢癌独立预后因子,还可能是潜在的有前途的肿瘤靶向治疗靶点。第一部分上皮性浆液性卵巢癌组织和正常卵巢上皮组织中差异表达蛋白质的分离和鉴定目的：分离和鉴定浆液性卵巢癌组织和正常卵巢上皮组织之间差异表达的蛋白质。方法：提取10例浆液性卵巢癌组织和10例正常卵巢上皮组织的总蛋白,利用双向电泳技术进行蛋白分离,采用硝酸银染色法进行凝胶染色,PDQUEST软件分析电泳图谱,找出在两组中表达差异2倍以上并有统计学意义(P＜0.01)的蛋白质点。手工挖取并富集这些差异蛋白质胶点,采用硝酸银染色兼容的串联质谱方法,MALDI-TOF-TOF MS,对这些蛋白质进行鉴定。结果：共得到60张双向电泳图谱,每张约有1800个蛋白质点,找到63个差异表达的蛋白质点,成功鉴定出其中的61个,分别代表54个互不相同的蛋白质。结论：运用双向电泳结合串联质谱技术筛选到的54个在正常卵巢上皮组织和浆液性卵巢癌组织中差异表达的蛋白质可能与浆液性卵巢癌的发生、发展有关。蛋白质组学技术是发现和筛选肿瘤相关蛋白质的有效手段。第二部分部分差异表达蛋白质的验证及其基因水平表达的研究目的：进一步检验其中6个蛋白质,GGCT, GMFB, HDGF, Rho GDI 2, BANF1,和Galectin-1,在正常卵巢上皮组织和浆液性卵巢癌组织中的差异表达情况,进而验证双向电泳及质谱技术的准确性；检测相应的6个基因在两组组织中的表达情况,并与蛋白质表达改变趋势进行比较。方法：收集20例正常卵巢上皮新鲜组织和20例浆液性卵巢癌新鲜组织,分别提取总蛋白和总RNA, Western blot方法检测蛋白质表达,real time RT-PCR方法检测mRNA表达。结果：GGCT, GMFB, HDGF, Rho GDI 2在浆液性卵巢癌组织中表达上调,BANF1和Galectin-1在浆液性卵巢癌组织中表达下调。HDGF, ARHGDIB基因在浆液性卵巢癌组织中表达上调,BANF1和Galectin-1基因在浆液性卵巢癌组织中表达下调,GGCT和GMFB在浆液性卵巢癌组织和正常卵巢上皮组织中表达没有显著差异。结论：1、6个蛋白质表达验证结果与双向电泳及质谱结果完全相符,前期差异蛋白质分离鉴定过程可信。2、HDGF,ARHGDIB,BANF1,Galectin-1基因表达变化趋势与蛋白质相同；GGC与GMFB基因水平没有明显变化,提示此两种蛋白表达可能受翻译或翻译后水平机制的调控。第三部分GMFB在浆液性卵巢肿瘤中的表达分析及预后相关分析目的：扩大样本量,检测GMFB在卵巢上皮组织,良性浆液性囊腺瘤,交界性浆液性腺瘤及浆液性卵巢癌中的表达；分析GMFB与浆液性卵巢癌患者临床病理参数及生存率的关系。方法：制作44例正常卵巢上皮组织,51例良性浆液性囊腺瘤,40例交界性浆液性腺瘤,110例浆液性卵巢癌石蜡切片,收集该组浆液性卵巢癌患者的临床病理资料及生存资料,采用免疫组织化学的方法检测GMFB的表达,采用多种统计学方法进行结果分析。结果：GMFB蛋白质在正常卵巢上皮组织及三种卵巢上皮性病变中表达有显著差异,且从正常卵巢上皮组织,到良性浆液性囊腺瘤,交界性浆液性腺瘤,再到浆液性卵巢癌,表达逐渐上调,并与浆液性卵巢癌患者的临床分期相关。高GMFB表达与浆液性卵巢癌患者的低总体生存率和低无瘤生存率相关,多元回归模型分析揭示GMFB是浆液性卵巢癌患者总体生存和无瘤生存(P=0.026)的独立预后因子。结论：1、GMFB可能参与浆液性卵巢癌的发生发展过程。2、GMFB是浆液性卵巢癌独立预后因子,还可能是潜在的的肿瘤分子标志物和靶向治疗靶点。第四部分GMFB表达对SKOV3细胞株增殖的影响目的：探讨GMFB对SKOV3细胞增殖力的影响,初步阐明GMFB在浆液性卵巢癌中的作用机制。方法：合成特异性的siRNA,转染入SKOV3细胞株中,沉默其中GMFB的表达,观察沉默前后肿瘤细胞增殖能力的变化。结果：GMFB基因沉默组SKOV3细胞增殖力明显下降。结论：GMFB可能通过促进肿瘤细胞增殖从而在浆液性卵巢癌发展过程中起作用。Vll更多还原

【Abstract】 Serous ovarian carcinoma is the most common subtype of epithelial ovarian cancer which remains the leading cause of death from gynecologic malignancy. The pathogenesis of serous ovarian carcinoma remains largely unknown. Recently, the viewpoint that malignancy is a kind of molecular disease is generally accepted. Authors believe that altered expression of certain clusters of genes/proteins probably contribute to the gain and maintain of specific features of malignant tumors. Quantitative changes in protein expression but not gene expression can eventually reflect the phenotypic biologic properties of ovarian cancer. Although a large number of studies have demonstrated various molecules including genes and proteins associated with the development or progress of cancers, few of them are recognized to be specific for epithelial ovarian cancer. In this study, we applied proteomic techniques to analyze the protein expression profiles of serous ovarian carcinoma and normal ovarian epithelium tissue aiming to characterize tumor-specific changes in the proteome of serous ovarian cancer, which may bring out new valuable diagnostic biomarkers and/or promising therapeutic targets for serous ovarian cancer, and further more, may provide new insight into carcinogenesis of serous ovarian caner. Using two-dimensional electrophoresis and silver staining, combined with PDQUEST analysis, approximately 1800 proteins spots were detected in normal ovarian epithelium and serous ovarian carcinoma tissue groups. Compared with the normal control group,63 protein spots exhibited significantly differential expression, including 38 up-regulated spots and 25 down-regulated spots.60 of them were successfully identified by MALDI-TOF/TOF MS, representing 54 unambiguous and unique proteins. To further confirm the protein alterations in SOC revealed by proteomic analysis, BANF1, galectin-1, GMFB, GGCT, HDGF, and RhoGDI 2 were selected for validation. The expression changes of these selected proteins were consistent with the 2-DE and silver-staining results, which validated that the differential expressions of proteins obtained from proteomic analysis were convincing. Corresponding gene expression analysis of these proteins was also performed using real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). The levels of ARHGDIB and HDGF mRNA expression were upregulated and those of BANF1 and LAGLS1 mRNA were downregulated in serous ovarian carcinoma compared with normal epithelium. But no changes of GMFB and GGCT mRNA expression were found in serous ovarian carcinoma, compared with its normal counterpart, which was not consistent with the alterations of protein expression.Additionally, we analyzed glia maturation factor beta (GMFB) protein expression by immunohistochemistry in 246 patients with various degrees of ovarian epithelial lesions including 45 normal ovarian epithelia,51 benign ovarian serous adenomas,40 borderline ovarian serous adenomas, and 110 serous ovarian carcinomas. GMFB expression was found to be gradually elevated from normal epithelium, to benign serous adenoma, to borderline serous adenoma, to serous ovarian carcinoma tissues, and was positively correlated with FIGO stage (P=0.012). High GMFB expression was associated with poor disease-free survival (P=0.010) and overall survival (P=0.003), while multivariate analysis revealed GMFB to be an independent prognostic factor for disease-free survival (P=0.026) and overall survival (P=0.006) in patients with SOC. Using RNAi technique, GMFB expression in SKOV3 cell line was silenced and the cells consequently exhibited significantly decreased proliferation (P＜0.05).We therefore propose that proteins identified here may be involved in the development or progression of serous ovarian carcinoma, and GMFB can be considered as a prognostic predictor for SOC patients, as well as a potential promising therapy target. PART I Identification of differentially expressed proteins between normal ovarian epithelium and serous ovarian carcinoma tissuesObjective:To identify the differentially expressed proteins between normal ovarian epithelium and serous ovarian carcinoma tissues.Methods:Total proteins were extracted from 10 cases of serous ovarian carcinoma and 10 cases of normal ovarian epithelia. The proteins were separated using Two-dimensional electrophoresis and the gels were subjected to modified silver staining method compatible with MS. The stained gels were scanned using the high-resolution scanner GS-800 calibrated densitometer followed by analysis with PDQUEST analysis. Student’s t-test statistical analysis with 99% significance level has been applied to the replicate groups. Significantly differentially expressed protein spots match the threshold (>2 fold) and statistical analysis standard were selected.Results:Approximately 1800 proteins spots were detected in both groups (Figure 1 A and 1B). Compared with the normal control group,63 protein spots exhibited significantly differential expression, including 38 up-regulated spots and 25 down-regulated spots.60 spots were successfully identified by MALDI-TOF/TOFMS, representing 54 unambiguous and unique proteins.Conclusions:1、The differentially expressed proteins identified here may involve in the carcinogenesis of serous ovarian carcinoma.2、Proteome technique is fairly powerful for the detection of tumor-specific proteins.PART II Validation of differentially expressed proteins and quantization of corresponding gene expression level of selected proteins.Objective:To further confirm the protein alterations in serous ovarian carcinoma revealed by proteomic analysis and to compare protein expression with their corresponding mRNA expression.Methods:Six proteins of interest, BANF1, galectin-1, GMFB, GGCT, HDGF, and RhoGDI 2 were selected. Total proteins and RNA were extracted from 20 cases of serous ovarian carcinoma and 20 cases of normal ovarian epithelia. Protein expressions were examined using western blot and gene expression levels were quantified employing real time RT-PCR.Results:GMFB, GGCT, HDGF, and RhoGDI 2 protein expression were upregulated in serous ovarian carcinoma; BANF1, galectin-1 protein expression were decreased in serous ovarian carcinoma; HDGF, and ARHGDIB mRNA expression were elevated in serous ovarian carcinoma; BANF1, galectin-1 mRNA expression were downregualted in serous ovarian carcinoma; no changes of GMFB and GGCT mRNA expression were found in serous ovarian carcinoma. Conclusions:1、The expression changes of these selected proteins were consistent with the 2-DE and silver-staining results, which validated that the differential expressions of proteins obtained from proteomic analysis were convincing.2、GMFB and GGCT mRNA expression alterations were not consistant with that of proteins, suggesting that the expressions of the two proteins are likely to be influenced by translation and post-translational mechanisms in these tissues.PARTⅢGMFB expression in various serous ovarian epithelial lesions and survival analysisObjective:To analyze GMFB protein expression in various degrees of ovarian epithelial lesions and to determine the correlation between GMFB protein expression with clinicopathologic features and survival of patients with serous ovarian carcinoma.Methods:Employing immunohistochemistry to detect GMFB expression in 45 normal ovarian epithelia,51 benign ovarian serous adenomas,40 borderline ovarian serous adenomas, and 110 serous ovarian carcinomas; Collection the clinicopathologic features and survival data of these patients with serous ovarian carcinoma; using diverse statistical processes to analyze the results. Results:GMFB expression was found to be gradually elevated from normal epithelium, to benign serous adenoma, to borderline serous adenoma, to serous ovarian carcinoma tissues, and was positively correlated with FIGO stage. High GMFB expression was associated with poor disease-free survival and overall survival, while multivariate analysis revealed GMFB to be an independent prognostic factor for disease-free survival and overall survival in patients with SOC.Conculsions:1、GMFB may contribute to the initiation and development of serous ovarian carcinoma.2、GMFB can be considered as a prognostic predictor for SOC patients, as well as a potential promising therapy target.PART IV The effect of GMFB expression upon proliferation of SKOV3 cell lineObjective:To investigate the effect of GMFB expression upon proliferation of SKOV3 cell line.Methods:Using sequencing-targeting siRNA to silence GMFB expression in SKOV3 cell line and cellular proliferation was detected. Results:GMFB expression silenced cells showed decreased celluar proliferation.Conclusions:GMFB may contribute to the progression of serous ovarian carcinoma due to promotion of cellular proliferation.更多还原