基因芯片技术用于鼻咽癌及其放疗敏感性的研究

【作者】 杨树；

【导师】 李瑶； 裘敏燕；

【作者基本信息】 复旦大学 ， 生物工程（专业学位）， 2011， 硕士

【摘要】 鼻咽癌是一种上皮源性恶性肿瘤,绝大多数属于低分化鳞状细胞癌,恶性程度高,早期易发生淋巴结和全身远处转移,具有相当高的死亡率。在一些地区,特别是中国南部地区,鼻咽癌的发生非常普遍,其发生率是世界其他地区的10-30倍。已知的鼻咽癌的病因主要包括基因的易感性,环境中的致癌的化学物质,以及EB病毒的感染。放疗是鼻咽癌治疗的主要方法,而放疗后5年的生存率只有50-60%。在大多数病例中放疗抑制性成为阻碍治疗的一个重大障碍。所以找出放疗敏感型和抑制型之间的差异,提前预测放疗抑制型的病人,做出新的治疗方案能为鼻咽癌的治疗提供必要的帮助。我们利用能够检测14112个人类基因的cDNA芯片,对20例鼻咽癌病人组织样本与15例鼻咽慢性炎症病人组织样本进行表达谱分析比较。我们用ScanArray4000对芯片进行扫描,GenePix Pro3.0软件被用于芯片型号强度的分析和筛选。在完成芯片数据的归一化及筛选修补处理之后,利用SAM软件及Cluster软件进行差异基因的分析及聚类分析。表达谱芯片的分析结果显示,9个基因(q<0.01)至少在17例(85%)鼻咽癌组织中有2倍差异表达,其中8个下调基因(TAOK3, SLC16A2, PRB4, AMY2B, B3GALT4, MSMB, RPS27, CR2),1个上调基因(MXLIP)。为了进一步研究,我们选用CR2和MXLIP在另外50例低分化鼻咽癌组织中进行real-time RT-PCR验证。通过另外3例鼻咽慢性炎症组织的表达谱比较,real-time RT-PCR结果与芯片实验结果相同,CR2在41例(82%)鼻咽癌组织中下调,MXLIP在42例(84%)鼻咽癌组织中上调。随后,我们将这20例鼻咽癌病例对放疗敏感性进行分类,有8例鼻咽癌敏感型的病人组织样本与12例鼻咽癌放疗抑制型的病人组织样本。通过对这两组病人的表达谱数据进行比较,我们筛选出了111条在鼻咽癌放疗抑制型组织和敏感型组织中差异表达的基因,其中包括108条在抑制型中上调基因(ZNF608,PIZEO2, CSF1R等)和3条在抑制型中下调基因(ATP2C1, MUDENG, OLA1)。为了验证芯片结果的可靠性,我们应用定量RT-PCR的方法,在另外17例鼻咽癌病人样本中检测了差异表达基因ZNF608以及CSF1R的表达情况,RT-PCR结果与芯片结果吻合。我们将这些差异表达基因输入GenMAPP软件进行生物学通路分析。在通路分析中,我们发现了9类26条生物学通路与鼻咽癌的放疗抑制性有关。大部分靶基因富集与细胞离子平衡,细胞因子及免疫反应,体液免疫,细胞增殖,受体蛋白信号途径等通路。这里,我们认为鼻咽癌的放疗抑制性可能主要是由于细胞内钙离子的变化引起的。它能在放疗时抑制细胞凋亡,促进DNA修复达到拯救癌细胞的作用。另外,各种细胞内源性或者外源性因素引起的细胞增殖起到在治疗时维持肿瘤的大小的作用。更多还原

【Abstract】 Nasopharyngeal carcinoma (NPC), most of them are poorly differentiated, is one of epithelial origin malignant tumors. The cancer cells will be metastasis in the lymph dens covering the whole body, and the death rate is very high. In some regions, notably the southern parts of China, this cancer occurs in an endemic form with an incidence10-to30-fold higher than the other regions. The etiology of NPC involves multiple factors including genetic susceptibility, exposure to chemical carcinogens, and Epstein-Barr virus (EBV) infection. Radiotherapy is the major treatment modality for NPC, but in some cases, the disease is radiation resistant. The5-year survival rate after treatment is only50-60%. Hence, radiation resistance remains a serious obstacle to successful treatment in many cases. This study aims to screen the differently expressed genes based on cDNA microarrays NPC, then to estimate the probably mechanism of NPC radiation resistance.The total RNA of20NPC patient biopsy specimens, containing8radiation sensitive NPC and12radiation resistant NPC, and a pool of nasopharyngeal chronic inflammation (NPCI) tissues, as common reference for microarray experiments, was isolated by Trizol and then hybridized through cDNA Expression Profiling Panels. Then the microarrays were scanned using ScanArray4000Software.The method of Significance Analysis of Microarrays (SAM, version3.02) was performed involving one class comparison. Nine genes were found significantly aberrant expression in more than17(85%) NPC patients (q<0.01) with higher than2-fold change, included1over-expression gene and8under-expression genes. For further research, CR2and MXLIP expression were detected by real-time RT-PCR in additional50NPC specimens. Compared with3independent nasopharyngeal chronic phlogistic tissues, CR2was lower expression in41(82%) specimens and MXLIP was over expression in42(84%) specimens. This result was consistent with our microarray data.Then, the method of SAM was performed involving two class unpaired comparison to identified differentially expressed genes in radiation resistant NPC patients. The results of cDNA microarrays were verified by real time RT-PCR using17the other independent NPC tissues.111genes were detected as differentially expressed, in which108genes (ZNF608, PIZEO2, and CSF1R et al) were up-regulated, while3genes (ATP2C1, MUDENG and OLA1) were down-regulated in radiation resistant NPC tissue. Furthermore, we identified26enriched pathways (9categories) through GenMAPP analysis. Reflected by the targets, the results indicated that these differentially expressed genes were highly correlated with cell ion homeostasis, cytokines and inflammatory, humoral immune, cell proliferation et al. Here, we suggest the radiation resistant capacity of NPC was mostly due to the change of cell Ca2+homeostasis, which promoting anti-apoptosis, DNA repair and rescuing tumor cell under radiation therapy. Moreover, cell proliferation promotion which induced by extracellular and intracellular factors may maintain tumor size under radiotherapy, and lead to recurrence after treatment.更多还原