应用基因芯片技术筛选胃固有肌层间质瘤差异表达基因

发布时间：2018-05-18 00:19

本文选题：胃固有肌层间质瘤 + 基因芯片　；参考：《青岛大学》2017年硕士论文

【摘要】：目的:利用基因芯片技术对起源于胃固有肌层间质瘤及瘤周组织标本基因表达谱进行对比分析,筛选出胃间质瘤发生和发展过程中异常表达的基因,寻找显著差异基因,初步探讨胃固有肌层间质瘤及其瘤周组织基因表达的变化特征及其可能存在的生物信息学意义,以期指导胃间质瘤精准诊断及药物靶向治疗。方法:选取青岛大学附属烟台毓璜顶医院经内镜及腹腔镜治疗、病理检查及免疫组化检测证实为胃间质瘤患者各3例。术前均行小探头超声胃镜检查提示病灶起源于胃固有肌层,术中收集胃固有肌层间质瘤组织及与其配对的瘤周组织标本,每例标本于离体后即刻置于液氮中冰冻保存以备用。分别抽提胃固有肌层组织及瘤周正常组织总RNA,反转录合成cDNA,经体外转录(IVT)同时掺入biotin生物素标记合成cRNA探针,然后进行cRNA纯化及片段化,将片段化后的cRNA配制成适宜杂交的反应体系,并与Affymetrix mRNA表达谱芯片进行杂交。按照差异倍数法标准筛选出差异基因;而对这些差异基因进行基因功能注释、富集及通路分析,则采用PANTHER/KEGG数据库及Gene Ontology(GO)功能分析软件实现,筛选出与肿瘤发生发展相关的基因功能类和涉及的信号通路。结果:1.按照差异显著性标准,与瘤周正常组织比较,发现胃固有肌层间质瘤组织中共找到3293条差异表达基因,其中上调基因有2588个,下调基因有705个,尤以DPP10、ETV1、DKK4、CXCL14、MT1M等基因差异表达最为明显。2.这些差异表达基因功能大致分为细胞分化、细胞增殖调节、细胞粘附、细胞信号转导、细胞骨架结构等等多种生物学过程,并涉及多条与肿瘤相关信号传导通路,如Wnt信号通路、Ras信号通路、Rap1信号通路、P13K-AKT信号通路、细胞外基质受体相互作用等。结论:胃固有肌层间质瘤组织与瘤周组织之间存在诸如DPP10、ETV1、DKK4、CXCL14、MT1M等多条明显差异性表达基因,这些差异表达基因可能调控着胃间质瘤的发生和发展,为寻找胃固有肌层间质瘤新的分子标记物或治疗新靶点提供了重要的生物学依据及参考。若能进一步深入探究这些差异表达基因及其所涉及的相关的调控通路,将对有效的及早诊治胃间质瘤、提高胃间质瘤患者长期生存率和生存质量具有重要的临床指导意义。
[Abstract]:Objective: to compare and analyze the gene expression profiles of specimens derived from gastric propria muscular stromal tumors (GIST) and surrounding tissues by using gene chip technique, and to screen out the abnormal expression genes during the occurrence and development of gastric stromal tumors (GIST), and to search for significant difference genes. To explore the characteristics of gene expression and its possible bioinformatics significance in gastric propria muscular stromal tumors and its surrounding tissues in order to guide the accurate diagnosis and drug targeted therapy of gastric stromal tumors. Methods: three cases of gastric stromal tumors were confirmed by endoscopy and laparoscopy in Yantai Yuzhouding Hospital affiliated to Qingdao University. Before operation, small probe gastroscopy showed that the lesion originated from the lamina propria muscularis of the stomach. During the operation, specimens of stromal tissue of gastric propria myometrium were collected and matched with them. Each specimen was frozen and stored in liquid nitrogen immediately after the operation. Total RNAs were extracted from the muscularis propria of stomach and normal tissues around the tumor respectively, and cDNAs were synthesized by reverse transcription. CRNA probes were synthesized by incorporation of biotin labeled by in vitro transcription, and then cRNA was purified and segmented. The segmented cRNA was prepared into a suitable hybridization reaction system and hybridized with Affymetrix mRNA expression microarray. The differentially expressed genes were screened according to the standard of differential multiple method, and the function analysis software of PANTHER/KEGG database and Gene Ontology goo was used to analyze the function of these differentially expressed genes. Gene functional classes and signaling pathways involved in tumorigenesis and progression were screened. The result is 1: 1. According to the standard of significant difference, 3293 differentially expressed genes were found in gastric propria muscular stromal tumors, including 2588 up-regulated genes and 705 down-regulated genes. The differential expression of DPP10, ETV1, DKK4, CXCL14, MT1M and other genes was the most obvious. The function of these differentially expressed genes can be divided into many biological processes, such as cell differentiation, regulation of cell proliferation, cell adhesion, cell signal transduction, cytoskeleton structure, and many signal transduction pathways related to tumor. For example Wnt signaling pathway Ras signaling pathway Rap1 signal pathway P13K-AKT signal pathway extracellular matrix receptor interaction and so on. Conclusion: there are many distinct differentially expressed genes, such as DPP10, ETV1, DKK4, CXCL14, MT1M, between the tissues of gastric intrinsic muscular stromal tumors and surrounding tissues. These differentially expressed genes may regulate the occurrence and development of gastric stromal tumors. It provides important biological basis and reference for finding new molecular markers or new targets for treatment of gastric propria muscular stromal tumors. If we can further explore these differentially expressed genes and their related regulatory pathways, it will be of great clinical significance for the diagnosis and treatment of gastric stromal tumors as soon as possible, and for improving the long-term survival rate and quality of life of patients with gastric stromal tumors.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.2

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