【作者】 徐璟达；

【导师】 许祖德； 刘秀萍；

【作者基本信息】 复旦大学 ， 病理学， 2010， 博士

【摘要】 前言胃癌是全球最常见的恶性肿瘤之一,更是引起恶性肿瘤患者死亡的第二大原因。胃癌在全球地理上的分布有广泛的国家间差异。高危险地区(男性年龄标准化发生率>20每100000人)包括东亚地区(中国,日本)、东欧和中南美洲的部分地区。在北美洲,胃癌相对少见,但其在恶性肿瘤死亡率中也占有相当大的比例。人类BCL2L10 (Bcl-B、Diva、Boo)基因位于人染色体15q12.2, mRNA全长887个核苷酸,cDNA全长615个核苷酸,表达含有204个氨基酸残基的蛋白,其分子量约为23kDa。BCL2L10表达有组织差异性,成人大脑、心脏、肾脏、脾脏、肝脏、结肠、肺脏、小肠、肌肉、胃、睾丸和胎盘中有表达,骨髓和所有的腺体有非常高的表达,然而,卵巢、子宫和前列腺组织中几乎没有BCL2L10的表达。同样,在不同癌组织中,其表达也存在着差异。滤泡性淋巴瘤不表达BCL2L10蛋白,18%的多发性骨髓瘤中表达BCL2L10,弥漫性大B细胞淋巴瘤则有近半数表达BCL2L10。而乳腺癌、前列腺癌、结直肠癌和小细胞肺癌中BCL2L10高表达。研究表明,BCL2L10表达与一些肿瘤不良预后的指标有相关性,如与乳腺癌的肿瘤高级别,与结直肠癌的微卫星稳定性和结直肠癌的解剖位置,与前列腺癌患者更高的死亡率,与小细胞肺癌更短的生存率之间均存在相关性。成人正常胃组织中,BCL2L10 mRNA表达水平非常高。也有研究报道胃癌组织中BCL2L10蛋白高表达,而其高表达与癌组织高分化有关,这样的结论显然是相互矛盾的。我们没有检索到BCL2L10与胃癌患者预后有意义联系的报道。BCL2L10是Bcl-2家族最近被发现的蛋白成员。Bcl-2家族至少有20种可以在哺乳动物细胞发挥促凋亡或者抗凋亡作用的蛋白。有趣的是,除了调节凋亡的中心作用,Bcl-2家族的一些蛋白也影响细胞周期,与凋亡调节功能一起,构成了它们在肿瘤发生中的重要作用。关于BCL2L10调节胃癌细胞生物学行为,国内外也未见相关报道。本课题试图从人胃癌组织、人胃癌细胞株和裸鼠移植瘤模型三方面对BCL2L10与胃癌患者预后、与胃癌细胞生物学行为的相关关系及其分子机制进行研究。本课题的研究不仅对了解肿瘤的生物学特性具有明显的理论意义,而且也具有显著的临床价值和社会效益。第一部分BCL2L10蛋白的低表达提示胃癌患者的预后不良目的：探究BCL2L10在胃癌细胞中的表达水平,以及它对胃癌患者临床和预后的意义。方法和结果：我们应用免疫组织化学方法检测了213例胃癌组织BCL2L10的蛋白表达。免疫组织化学结果显示了胃癌细胞中BCL2L10普遍低表达。BCL2L10表达等级数据分成3组(组0到组2),以利于统计分析。完成数据统计分析后发现,BCL2L10低表达与较短的肿瘤相关生存率有显著的相关性(x2=81.55,P=1.956×10-18)。多因素回归分析指出BCL2L10低表达是胃癌的独立预后指标(P=4.883x10-8,HR=0.252)。受试者工作特征曲线(ROC curve)显示BCL2L10的面积为0.817(P=8.331×10-14),这说明BCL2L10低表达是胃癌的预后指标。为了加强和证实免疫组织化学结果的可靠性,我们通过实时定量PCR和免疫印迹检测10例新鲜胃正常组织、30例胃癌组织和6种胃癌细胞株中BCL2L10的表达水平,并且通过亚硫酸氢钠甲基化测序方法检测了胃正常组织和癌组织中BCL2L10基因启动子甲基化状态。实时定量PCR和免疫印迹的结果与免疫组织化学的结果一致,并经数据统计分析发现,基因启动子CpG岛高甲基化是胃癌组织中BCL2L10低表达的重要原因。小结：人胃癌组织中BCL2L10的表达广泛低于正常人胃粘膜上皮；人胃癌细胞中BCL2L10基因启动子的高甲基化是其低表达的重要原因；BCL2L10的低表达提示胃癌患者的预后不良。第二部分BCL2L10蛋白通过不同的通路调节胃癌细胞的凋亡和增殖目的：探究BCL2L10蛋白调节癌细胞凋亡和增殖的作用及其相关机制。方法和结果：我们通过流式细胞计数、荧光染色、裸鼠移植瘤模型和免疫印迹的方法研究了BCL2L10对胃癌细胞凋亡和增殖的调节作用。我们也应用相应的通路抑制剂来证实涉及凋亡或者增殖的主要通路。我们观察到胃癌细胞中上调的BCL2L10通过线粒体途径诱导凋亡,下调的BCL2L10通过PI3K/Akt信号通路促进增殖。小结：上调的BCL2L10蛋白通过线粒体途径诱导胃癌细胞凋亡；下调的BCL2L10激活PI3K/Akt信号通路,并通过后者促进胃癌细胞快速通过细胞周期G1/S期完成促增殖作用。上调BCL2L10后的促凋亡作用和下调BCL2L10后的促增殖作用很大程度上提示了BCL2L10是一种抑癌蛋白。更多还原

【Abstract】 IntroductionThough it is no longer the second most common cancer worldwide, gastric cancer is still the second most common cause of death from cancer. The geographical distribution of gastric cancer is characterized by wide international variations. High-risk areas (age-standardized rate in men> 20 per 100,000) include East Asia (China, Japan), Eastern Europe, and parts of Central and South America. It is a relatively uncommon neoplasm in North America, but gastric cancer is the third most lethal neoplasm overall in North America, contributing substantially to the burden of cancer deaths.BCL2L10 gene is located on human chromesome 15q12.2 and expresses 204 amino acids protein, about 23kDa. BDL2L10 protein is strongly expressed in all normal plasma cells but found in only 18% of multiple myelomas. BCL2L10 immunostaining is also present in approximately half of diffuse large B-cell lymphoma specimens, whereas follicular lymphomas do not contain BCL2L10. In normal adult human stomach tissues, BCL2L10 mRNA is expressed at high levels (three-plus). However, no significant association with survival was observed in gastric cancer patients.BCL2L10 is the last protein member found in Bcl-2 family. There are at least 20 Bcl-2-related proteins in the life or death decision of mammalian cells. Intriguingly, in addition to its central role in regulating apoptosis, the Bcl-2 family influences the cell cycle or more specifically, the transit between quiescence and proliferation, which, together with apoptosis regulation, makes its important role in oncogenesis. However, no such research on BCL2L10 regulation on gastric cancer cells.Using three experimental models - retrospective clinical specimens of human gastric carcinoma, gastric cancer cell lines, and mouse xenograft model - our study investigated impact of BCL2L10 on gastric cancer cells.PartⅠLoss of BCL2L10 Protein Expression as Prognostic Predictor for Poor Clinical Outcome in Gastric CarcinomaAims: BCL2L10 protein is an apoptosis-related member of the Bcl-2 protein family. The clinical significance of its expression in gastric carcinoma is poorly understood. Our aim was to investigate BCL2L10 expression and its clinical and prognostic significance in gastric carcinoma patients.Methods and results: Immunohistochemistry revealed extensive loss of BCL2L10 expression in gastric cancer cells. The scaled BCL2L10 expression data was categorized into 3 groups (groups 0-2) to facilitate statistical analysis. We observed a significant correlation between the lower BCL2L10 expression group and shorter disease-free survival (P= 1.956×10-18). Multivariate regression analysis showed that loss of BCL2L10 protein expression (P= 4.883×10-8, HR= 0.252) is an independent prognostic predictor of gastric carcinoma. The receiver operator characteristic (ROC) curve showed that the area for BCL2L10 protein was 0.817 (P= 8.331×10-14), indicating that loss of BCL2L10 protein expression is an excellent prognostic predictor of gastric carcinoma. We re-investigated BCL2L10 expression by quantitative real time PCR, immunoblotting and examined methylation status of BCL2L10 gene promoter by bisulfite sequencing in fresh gastric normal and carcinoma tissues. We observed a significant correlation of lower BCL2L10 expression with hypermethylated CpG island of gene promoter in gastric carcinoma.Conclusions: To our knowledge, it is the first report that loss of BCL2L10 protein expression predicts poor clinical outcome in gastric carcinoma. Hypermethylated gene promoter is responsible for loss of BCL2L10 in gastric carcinoma. Part II BCL2L10 Protein Regulates Apoptosis/Proliferation through Differential Pathways in Gastric Cancer CellsAims: The reason and consequence of BCL2L10 downregulation in gastric carcinoma is poorly understood. Our aim was to investigate the function of BCL2L10 protein in gastric carcinoma.Methods and results: We studied its apoptosis and proliferation regulation in gastric cancer cell lines by flow cytometry, fluorescence staining, murine xenograft model and immunoblotting. Pathway inhibitors were also applied to confirm the major pathway involved in apoptosis or proliferation regulation. We observed apoptosis induced by upregualted BCL2L10 through the mitochondrial pathway and proliferation accelerated by BCL2L10 siRNA via PI3K/Akt signaling pathway in gastric cancer cell lines.Conclusions: The pro-apoptotic effect of BCL2L10 and growth promotion by BCL2L10 siRNA in gastric cancer cells might suggest a tumor suppressor of BCL2L10.更多还原