胃癌差异蛋白的无标记定量蛋白质组学研究

发布时间：2018-04-22 00:28

本文选题：胃癌 + 定量蛋白质组学　；参考：《第四军医大学》2016年博士论文

【摘要】：癌症是导致人类死亡的一大顽症。2012年全球肿瘤流行病统计数据解析,肺癌、乳腺癌和结直肠癌是世界范围内诊断最常见的癌症,而肺癌、肝癌和胃癌则是最主要的致死癌症。报告中指出,中国新增和死亡癌症患者居世界第一。2014年全国肿瘤登记中心最新研究显示,肺癌、胃癌和肝癌是我国范围内诊断常见的前三位癌症,而肺癌、肝癌和胃癌则是前三位致死癌症。由此可见,癌症乃至胃癌严重危害着我国人口健康,对其进行预防、诊断和治疗显得等至关重要。胃癌是遗传因素和环境因素相互作用引发的恶性肿瘤。临床上用于治疗胃癌的方法有手术、化学疗法和放射疗法,但由于其缺乏明显的临床特征,大多数患者一旦确诊已是晚期,故手术和化疗的5年生存率均不高,约为10%-30%。近年来,靶向治疗在临床上引发了革命性的突破,成为肿瘤治疗的希望。但是,靶向治疗的发展仍然存在着许多障碍,特别是肿瘤细胞的关键信号通路不清、所知理想靶位不足,有效的药物不多。因此,早期诊断标志物、药物靶分子发现和临床治疗靶向药物研发是目前亟待解决的重要问题。阐明胃癌发生发展的相关分子及其调控机制,不仅有可能为该病的早期诊断提供标志物,还可能为临床治疗提供候选靶分子和新策略。本研究通过收集胃癌患者手术切除配对胃癌-癌旁的中-低分化腺癌组织,利用无标记定量蛋白组学方法获得配对癌旁-胃癌组织差异表达蛋白,并进行生物信息学的功能注释和疾病关联性分析,一方面阐述差异表达蛋白在胃癌发生发展过程参与的可能分子调控机制,另一方面确定有望成为生物标志物的候选分子,对其进行实时定量pcr、蛋白印迹和免疫组化多层次验证。此外,通过免疫荧光、基因转染、流式细胞术、mtt、transwell小室、实时定量pcr、免疫共沉淀与蛋白印迹等技术在细胞水平上首次探讨了核不均一核糖核蛋白d(hnrnpd)在胃癌发生过程中的分子机制,为其作为有效分子标志物提供相应的理论基础。通过以上一系列实验研究,我们获得了以下的结果与结论。首先,通过无标记定量蛋白质组学技术分析配对胃癌-癌旁组织,分别鉴定到3100-3700种蛋白质,进一步筛选后获得146种差异表达蛋白,其中81种在胃癌组织中表达下调,65种在胃癌组织中表达上调。其次,通过生物信息学工具分析,146种差异表达蛋白与癌症息息相关,最多一类为核酸结合蛋白,具有核苷酸结合活性、催化活性、结构分子活性和转运体活性等分子功能,形成相互关联网络,参与代谢物和能量产生、氧化磷酸化、基因表达、核苷酸合成、rna剪切与加工、转录调控等生物学过程及相关信号通路,调控胃癌的发生发展。第三,针对其中4种差异表达分子,利用实时定量pcr、蛋白印迹和免疫组化验证了hnrnpa2b1、hnrnpd、hnrnpl和ybx-1在胃癌组织中高表达,癌旁组织中低表达,且hnrnpa2b1与hnrnpd的表达分布特征与ybx-1在胃癌组织中高度一致,均发生了质-核转移。其中,对hnrnpd和hnrnpl在胃癌组织中高表达的鉴定属于首次报道。最后,为了探讨hnrnpd与胃癌发生发展的关系及其可能的分子机制,首先利用免疫荧光实验再次证实了hnrnpd在胃癌细胞表达主要分布于细胞核;其次利用基因转染、流式细胞术、mtt、transwell小室、实时定量pcr、免疫共沉淀与蛋白印迹等技术探讨了hnrnpd能够促进胃癌细胞增殖与迁移,调控细胞周期,抑制细胞凋亡;并结合一系列细胞周期调控因子和原癌基因mrna,介导相关mrna表达水平发生改变以及极有可能通过ybx-1-pi3k/akt信号通路参与胃癌细胞的增殖,影响胃癌的发生发展。
[Abstract]:Cancer is a global epidemic of cancer epidemic statistics that causes human death in.2012. Lung, breast and colorectal cancer are the most common cancers diagnosed worldwide, while lung, liver and gastric cancer are the most fatal cancers. The report points out that the new and dead cancer patients in China live in the first.2014 of the world. The latest research in the cancer registration center shows that lung cancer, gastric cancer and liver cancer are the most common first three cancers in our country, while lung cancer, liver cancer and gastric cancer are the top three fatal cancers. Thus, cancer and even gastric cancer are seriously endangering the health of our population. It is very important to prevent, diagnose and treat it. The malignant tumor caused by the interaction of factors and environmental factors. The methods of clinical use for the treatment of gastric cancer are surgery, chemotherapy and radiotherapy, but because of their lack of obvious clinical features, the 5 year survival rate of the operation and chemotherapy is not high, due to the lack of obvious clinical features. It is about 10%-30%. in recent years, and the targeted therapy is in clinical practice. However, there are still many obstacles to the development of targeted therapy, especially the poor key signal pathways of the tumor cells, lack of ideal targets and few effective drugs. Therefore, early diagnostic markers, drug target molecular discovery and clinical therapy target drug development are It is an important problem to be solved urgently. It is not only possible to provide markers for the early diagnosis of the disease, but also provide candidate targets and new strategies for the clinical treatment of gastric cancer. On the one hand, the possible molecular regulation mechanism of differential expression proteins involved in the development and development of gastric cancer is described. On the other hand, the possible molecular markers in the carcinogenesis and development of gastric cancer are discussed. In addition, the nuclear inhomogeneous ribonucleoprotein D (hnrnpd) in gastric cancer was first discussed at the cell level by immunofluorescence, gene transfection, flow cytometry, MTT, Transwell chamber, real-time quantitative PCR, immunoprecipitation and Western blotting, in addition to the real-time quantitative PCR, Western blot and immunohistochemistry. The molecular mechanism in the course provides a theoretical basis for its effective molecular markers. Through a series of experimental studies above, we have obtained the following results and conclusions. First, 3100-3700 kinds of proteins were identified by unmarked quantitative proteomic techniques, respectively. 146 differentially expressed proteins, of which 81 were down regulated in gastric cancer tissues, and 65 were up-regulated in gastric cancer tissues. Secondly, through bioinformatics analysis, 146 differentially expressed proteins were closely related to cancer, the most one was nucleic acid binding protein, with nucleotides binding activity, catalytic activity, structural molecular activity and transporter activity. Sex and other molecular functions, form interrelated networks, participate in metabolites and energy production, oxidative phosphorylation, gene expression, nucleotide synthesis, RNA shearing and processing, transcriptional regulation and other biological processes and related signaling pathways to regulate the occurrence and development of gastric cancer. Third, 4 differentially expressed molecules are used in real time quantitative PCR, Western blot and Immunohistochemistry showed that hnrnpa2b1, hnrnpd, hnrnpl and ybx-1 were highly expressed in gastric cancer tissues, low expression in para cancerous tissues, and the distribution characteristics of hnrnpa2b1 and hnrnpd were highly consistent with ybx-1 in gastric cancer tissues, and both of them had a mass nuclear transfer. Among them, the identification of high expression of hnrnpd and hnrnpl in gastric cancer was the first report. Finally, In order to explore the relationship between hnrnpd and the development of gastric cancer and its possible molecular mechanism, first of all, the expression of hnrnpd in gastric cancer cells was mainly distributed in the nucleus by immunofluorescence, and then h was explored by gene transfection, flow cytometry, MTT, Transwell chamber, real-time PCR, immunoprecipitation and Western blotting. Nrnpd can promote the proliferation and migration of gastric cancer cells, regulate cell cycle and inhibit cell apoptosis, and combine a series of cell cycle regulators and proto oncogene mRNA, mediate the change of related mRNA expression level and possibly participate in the proliferation of gastric cancer cells through the ybx-1-pi3k/akt signaling pathway, and affect the development of gastric cancer.

【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.2

【相似文献】