【作者】 王学虎；

【导师】 傅仲学；

【作者基本信息】 重庆医科大学 ， 外科学， 2010， 博士

【摘要】 大肠癌是最常见的恶性肿瘤之一,在国内外都有较高的发病率。在美欧死亡率高居恶性肿瘤的第2位,我国已从第6位上升为第3位。肿瘤的发生、发展和多种因素相关,其中细胞凋亡基因的突变起着重要作用。凋亡抑制蛋白家族(Inhibitor of apoptosis proteins,IAPs)在抑制细胞凋亡中有重要作用。Survivin是近年发现的具有独特结构的IAPs家族的成员,在恶性肿瘤中具有较高的表达水平,并且和肿瘤的临床病理分期、淋巴节转移等具有密切的关系,是目前已知的最强的凋亡抑制因子。然而,越来越多的研究发现,Survivin不仅参与凋亡的抑制,还影响肿瘤细胞的增殖、分裂以及转移和侵袭等。但有关Survivin的研究观点仍存在较多的分歧,如:有学者认为Survivin在细胞质中表达与肿瘤的治疗效果和不良预后相关;而也有学者提出相反的观点,在细胞质中分布提示肿瘤预后良好。有学者认为Survivin在G1/S期发挥作用,但也有认为调控G2/M期。因此Survivin作用机制复杂,需要从整体水平深入探讨Survivin调控机制。蛋白质组学是在大规模水平上研究蛋白质变化的特征,通过观察干预前后组织细胞水平蛋白质表达变化及差异,了解特定因素对肿瘤细胞的蛋白质表达影响,明确干预因素的作用机理。另外,RNA干扰(RNA interference,RNAi)是可诱导目的基因同源mRNA降解阻断特定基因的表达,具有高度特异性和高效性。本研究应用RNAi技术,沉默Survivin基因的表达,分析结肠癌细胞生物学特性的变化;提取RNAi前后的细胞总蛋白,采用蛋白质组学技术研究Survivin调控下的差异蛋白变化,寻找与Survivin功能相关的蛋白质。第一部分Survivin在结肠癌中的表达及分布目的:探讨Survivin在结肠癌临床标本和细胞株中的表达及分布。方法:采用免疫组织化学和免疫细胞化学的方法研究Survivin在结肠癌细胞中的表达水平及分布,明确Survivin表达与肿瘤临床特点的关系。结果:在结肠癌临床组织标本中,Survivin阳性表达率为57.6%,结肠癌细胞株LoVo、HCT116、SW480中均有Survivin表达。Survivin以细胞质表达为主。Spearman相关分析显示Survivin表达与肿瘤患者的临床Ducks分期存在相关性。结论:在结肠癌临床组织和细胞株中Survivin表达均较高,并以细胞质表达为主。Survivin与肿瘤的Ducks分期具有相关性。第二部分携带针对人Survivin的shRNA的腺病毒的转染及对结肠癌细胞生物学特性的影响目的:研究Survivin基因对结肠癌细胞增殖、凋亡、周期和侵袭的影响。方法:扩增、纯化携带针对人Survivin的shRNA的增殖缺陷型腺病毒,利用流式细胞技术、RT-PCR、Western-bolt等检测对结肠癌细胞的感染及基因抑制效果。针对Survivin基因沉默后的结肠癌细胞,采用流式细胞仪、MTT、单克隆平板、细胞侵袭实验等,检测Survivin基因在体外对细胞增殖、凋亡、细胞周期和侵袭的影响。另外研究Survivin基因体内对肿瘤生长的抑制作用。结果:增殖缺陷型腺病毒在MOI为50时,转染率为92.67%,并具有明显基因沉默作用。Survivin基因沉默后,凋亡率明显增加,G1/S期细胞增多,G2/M期细胞减少,细胞侵袭能力减弱,Survivin在体内外均可抑制结肠癌细胞的生长。结论:构建的增殖缺陷型腺病毒具有较高的转染率和基因沉默高效性,Survivin对结肠癌细胞的凋亡、增殖、周期及侵袭有明显的影响。第三部分蛋白质样品制备、双向电泳及图谱分析目的:探讨RNAi后的蛋白质样品制备、双向电泳的条件及方法,为Survivin调控下差异蛋白的分析奠定基础。方法:免疫荧光分析Survivin在SW480中的分布;采用液氮冻融,RNase、DNase酶解及超声裂解等方法获取蛋白质样品,在不同蛋白浓度和聚焦强度下进行双向电泳,采用硝酸银染色并对图像进行分析。结果:在结肠癌细胞SW480中,Survivin主要分布在细胞质中。液氮冻融,RNase、DNase酶解及超声裂解等处理可明显降低核酸的影响,促进蛋白的溶解。250ug上样量及70000Vh聚焦后,图像分辨率和重复性均较好,在pI=4-8和Mr(15-120)×103分布的蛋白质点较多,蛋白质点数为1400个左右,超过80个蛋白质点存在明显差异。结论:成功建立了分辨率及重复性均较好的双向电泳图谱。第四部分差异蛋白质的质谱鉴定及Survivin调控机制分析目的;对Survivin基因沉默前后的差异蛋白进行质谱分析,探讨Survivin在结肠癌中的作用机制。方法:采用质谱分析及数据库信息检索,明确与Survivin相互作用蛋白质的变化;Western-bolt对兴趣蛋白质进行表达验证;免疫沉淀检测与Survivin直接作用的蛋白质。结果:经质谱分析有36种差异蛋白质被发现,差异蛋白质存在于多种细胞器,参与多种通路的调控,具有凋亡、周期、细胞代谢及侵袭等不同功能。对Caspase-10及CDK9进行表达验证。CDC2及HSPD1可能与Survivin存在相互作用。结论:细胞质中Survivin参与多种通路的调控。更多还原

【Abstract】 Colorectal cancer is the second leading cause of cancer related mortality in the Western world. It is important to find a new way to effectively inhibit cancer cell growth and metastasis. Studies suggest that survivin is one of the most tumor-specific genes. Survivin is a member of the inhibitory apoptosis protein (IAP) family. It deserves attention as a selective target gene for cancer therapy due to the fact that it is widely expressed in most malignancies.Some studies have shown that a sustained over-expression of survivin is a characteristic feature of colon cancer. Survivin has attracted abundant interest from several viewpoints of biomedical sciences for dual involvement in apoptosis regulation and mitotic progression. But several studies indicated that the relationship between survivin expression and tumor behavior is still not fully understood due to contradictory results. Some studies showed a positive relationship but others showed negative outcomes or even a lack of correlation. For example, cytoplasmic survivin expression is an unfavorable prognostic indicator. But in contrast, a favorable outcome associated has been reported. For this reason, this study was designed to globally identify the mechanism of survivin.Proteomics is an effective platform to globally detect and characterize proteins. A comparative proteomic approach is the main strategy of proteomics used to analyze and compare the differentially expressed proteins. On the other hand, RNA interference (RNAi), as a sequence-specific and posttranscriptional gene silencing method, is increasingly being used to determine the functions of specific genes. Thus, we silenced the expression of Survivin by the recombinant adenovirus. We detected the survivin gene expression, apoptosis, cell cycle, etc. after shRNA interference. Then, a comparative proteomic approach was performed to identify the differential proteins between SW480/Survivin(-) and SW480/Survivin(+) cells. Our proteomic findings may provide clues to further study the mechanism of the function of survivin in tumors.PARTⅠThe Expression and Localization of Survivin in Colon CanerObjective: To observe the expression and localization of Survivin in colon cancer.Methods: We screened the expression and localization of Survivin by immunohistochemistry and immunocytochemistry in colon cancer cells (including colon cancer tissues and cell lines).Results: Survivin protein expression was 57.6% in colon cancer tissue samples and cell lines (LoVo, HCT116, SW480). Survivin protein expressed mainly in the cytoplasm. Spearman correlation analysis showed that there are positive correlation between Survivin expression and Ducks stage.Conclusion: In the colon carcinoma tissue and cell lines, Survivin expressed at a high level and the localization was in cytoplasm. Survivin plays an important role in control tumor growth.PARTⅡConstruct of Recombinant Adenovirus Vector and its Effect in Colon Cancer CellsObjective: To construct the adenovirus vector which contains the shRNA of Survivin and investigate its effect in colon cancer cellsMethods: We constructed the recombinant adenovirus, which contains the shRNA of Survivin and transfected it into colon cancer cells. Then, we detected Survivin gene expression after shRNA interference. We detected the influence of Ad-Survivin/shRNA on the proliferation, apoptosis and invasion in vitro and vivo by MTT, colony-formation, flow cytometry, transewell and tumorigenic formation.Results: Ad-Survivin/shRNA displayed high transfection efficiency and suppressed Survivin expression efficiency. Survivin slience could induce apoptosis, effect the mitotic cycle and decrease invasion. Cell proliferation was significantly inhibited by Ad-Survivin/shRNA in vitro and vivo.Conclusion: RNAi of Survivin would be a potential therapeutic approach for colon cancer. Survivin plays an important role in control tumor growth by a variety of molecular regulatory mechanisms.PARTⅢProtein Sample Preparation, 2-DE and Image Analysis for ProteomeObjective: To study the protein sample preparation methods and to estabilish 2-DE profiles for SW480 cells under Survivin intervention.Methods: We used liquid nitrogen, RNase-DNase digestion and ultrasonication method to get the protein samples. Protein was focused at different voltagy intensities for two-dimensional electrophoresis.The PDquest sofeware was used to analysis the image.Results: Liquid nitrogen freeze thawing, RNase-DNase digestion and ultrasonication can significantly reduce the impact of nucleic acid and promote the dissolution of protein. The 2-D gels of SW480 displayed about 1,400 protein spots which distributed of pI=4-8 and Mr (15-120)×103. We found more than 80 protein spots were apparently different between before and after survivin gene silencing. Among these protein spots, 36 species were identified.Conclusion: The attained 2-D gels patterns of each cell line were highly reproducible and well-resolved. Optimized protein and 2-DE preparation was established.PARTⅣAnalysis on Differences Proteins by Mass Spectrometry and the Regulation Mechanisms of SurvivinObjective: To identify proteins and analysis their characteristics by MALDI-TOF-MS and HPLC-CHIP-MS/MS.Methods: The differential protein spots were analyzed by MALDI-TOF-MS and HPLC-CHIP-MS/MS and database searching. Gene ontology (GO) lists were downloaded for the functional analysis of proteins. Western-blot method was used to check the different proteins.Results: The differentially expressed proteins identified by 2-D proteome analysis were related to various cellular programs involving cell proliferation, cell cycle, apoptosis, expression of nucleic acid metabolic gene, and regulation of signal transduction.Colon cancer cells effected apoptosis by the regulation of Caspase-10 and cell cycle by the regulation by CDK9.Conclusion: Survivin plays an important role in control tumor growth by a variety of molecular regulatory mechanisms.更多还原