【作者】 邵伟；

【导师】 厉有名；

【作者基本信息】 浙江大学 ， 内科学， 2003， 硕士

【摘要】 食管癌是常见的恶性肿瘤。食管癌疗效与病变早晚有关。及早揭示食管癌的本质是食管癌研究的重中之重。近年来的分子生物学进展表明，食管癌常见的基因改变是癌基因的激活和抑癌基因的失活，二者通过参与细胞周期的调控、信号传导、细胞分化及凋亡等事件，引起肿瘤发生和发展。近年来兴起的基因芯片技术在肿瘤研究中显示了巨大的应用潜力。基因芯片实质是一种高密度的核酸阵列，将大量特定序列的核酸片段有序地固定在玻片上，与荧光标记待测的核酸样品进行杂交分析。可以大量快速、准确地对核酸进行序列测定和定量分析。 目的： 本项研究拟采用寡核苷酸芯片技术筛选食管癌相关基因并探索它们之间的调控关系。 材料与方法： 1、病史资料： 浙江大学硕士研究生论文 l例组织标本由浙江大学医学院附属第一医院提供 患者，男性，58岁，住院号：361149。病理诊断：食管中分化鳞癌。TNM分类：T4NIMO2、实验方法： 分别提取食管癌、癌旁组织、正常食管组织总RNA，以总RNA进行逆转录，并分别用Cu3—duTP进行标记。根据文献报导的肿瘤相关基因共筛选288个，从Genebank中查出相应基因序歹，利用探针设计软件进行探针设计，探针长度为40hP。随后将寡核昔酸探针点样到经醛基化处理的载玻片上，制成 12 X 24矩阵。将上述制备的杂交荧光探针与芯片进行杂交，杂交温度为40℃，杂交时间为3小时。用ScanArray3000 激光扫描仪扫描芯片。所得图象再用haGene4．0分祈软件进行分析，比较荧光信号强度和比值。相应癌组织、癌旁组织、正常组织信号的荧光强度必须有一个大于1000，并且两者间相差两倍以上才能判断为差异表达的基因。荧光强度比大于2为表达增高，小于0．5为表达降低。结果： l、芯片杂交技术的可靠性：在芯片上共有288个CDNA，为保证芯片杂交结果的可靠性，在每块芯片上设定阴性对照：水稻基因u 个点）经芯片杂交，这些点的杂交信号均很低，证实了数据的可靠 性口 3 沙X「）学刨卜坝穴’h幻Z 2七．基回芯片筛3一吉5： ；义、分祈食管‘癌乡Z织／乏《应上常组织标本－与二 片杂交架Z果 竟达上调基沾g共6条二B厂乏叶一二、BTK、iN 比三弓八2、（二＊3、＊WI、且－ltjITI 三L13MHC《－间lLS三；INL 个一ACSh卜I乙l厂lZl（口、 A（l…eJ二口， 表H互二丁调基因J共8条二S们｝、；卜二人汕h订、SO厂S一Z、D什入 ＊即S一2’Z一、mS H卜Dinn、Am’RIA。 Q 山．士F一＞一芬灯滂ZZ叶门4I厂，I十卜二；丁七十州斗口个下、－太I＿二汀卜二九：八十上田 ’、，，刀w【民－曰？拴）了匀Z一飞八。人’川“工上币宁H三少H川。。’卜－’－＿）。U／1讨℃义》Z爿穴 二漠达二二调基L共8条；．1VJ一L’门MZ欠．inK：．V1欠 呈1飞K、、工m了1气K…I8；、比二mOSdpZe＊。一m卜入／ 上。）厂 工e川二＊ 厂＊（epIOr ｛己互e一1、elatedprC【－01厂【、＊＊ma厂Im卜N乌 几）r 卜。三ma比poe乙【C prOb〕Og。yCa门CO厂。n厂。，‘＋．Sn，淤p二 卜掀旧1、；D人1｝、’8。茶；引13、PR＊、一27、【］T刁、ml］卜讪P＿DP一一f9、川”6X龟 rAAD回入 h一！1llF－。nC（〕hreCts－L In。hall上C．ZI．5llel回pTC皿上elnllRN｝＼；； ＊ 5折食管癌组纤】（握卜别二织与芯寸白令茸台利畏 三Z达一 调基巨共亿。绦；C卜卜j、it一＊K…。。。s、RI乙、洲n。bC工一 了．、B了K、C—fOS、MCA山、卜（，Ic Sap1Gnsmm山、fo厂1（〕p上二r厂eCept（）rgC；1。；一 厂e＊t。，d厂）＊OtO。n；。O引上卜讣基因某5条：MM7、《WM乏、B邵lrA卜a二、于：））｛j S）］：：）1）1。；S f汕乞八 Z（）厂 ZyX：i．fi。bk ：／＼匕口 N）； 1、基L 芯＊ 技术旨 卜。呸高通哆 9 选与食管9 发生己能习 关的纂）j Ig 沏n八－学仙【研究生论义 ＊、表；达上 调的。篷因 X口：B山＊一1、附K、．JK主；＊ ＊T三JW。Hulnall（）CLass HS。A—．j＼k）1．155urtace Arl上igeii．”TI＄．MI、JNK3AI。Im了1、K—T·as、NSSS。［Ill以卜；工of，111lltopO（Zt1C口l、OtOOglyCZIC COI·1＊r（）t〔；dn、n1上In1一2、Ru、．＊知3、C一上＊S，、＊C八D、HOm〕S＊p让门。InRNA八）厂 leptl．n厂e；。ep七。工ge。l。，一。。fat。。（S pro上。。tri向育在食管癌的发一生和发展过程中＠负0进、厂个用C ＊、表达－干订的互。区1奶［：S了卜、K工＊OI＊、SOC｝一1、D12：5、P＊S～27、IRS．l。DRm＿、儿cTRZA、1＊UCS＊Ct ＊P一79、AP巳＊气SMAD3－。NU＊＊nCO10了、eCt＊I］＊Ut＊门t Q、。＊1y二。。Z厂＊TOteln m＊＊A、：＊C＊7、＊＊NBI、mZFI－刁厂八PZC、H；二）。；S。p。更多还原

【Abstract】 Esophageal carcinoma is a common malignancy. Staging of the tumor has been proved to be associated with prognosis. So, it is most important for us to reveal the inbeing of esophageal carcinoma. Recently, with the development of molecular biology, it has been proved that the most common shifts of gene in the esophageal carcinoma are the activation of oncogene and inactivation of tumor suppressor gene, which lead to the occurrence and development of the cancer through taking part of the regulation of cell cycle, signal transfer, cell differentiation, apoptosis and so on. The gene chip technique, which is springing up recently, has shown great potential in the cancer research. The gene chip, in fact, is a high-density nucleic acid array, which fix up a large number of nucleic acid segments (probes) with special sequences on the slides, analyzing the sequences of samples of nucleic acid unkown by hybridization with probes. This technique can be used to sequences and quantitative analysis rapidly, largely and exactly.Purpose: To filtrate the genes associated with esophageal carcinoma and investigate the relationship between the genes and esophageal carcinoma by oligonucleotide chip technique. Materials and methods: 1.Material: one tissue sample supplied by the first affiliated hospital, medical college, Zhejianguniversity.Male ,58Y ,hospital number: 361149 , Pathologic diagnosis: medium-differentiatedsquamous cell carcinoma of esophagus.TNM type: T4N1M02.Methods:Pick up total RNA from esophggeal carcinoma, pericancerous tissues and normaltissue of esophagus, then retro-iranscribe cDNA and lable them with Cy3-duTP.According to literatures, 238 cancer-related genes were filtrated, whose sequenceswas found out from the Genebank, and were designed as probes by the software withthe length of 40 bp. These: oligonucleoti.de probes were arranged on the silylatedslides, composing a 12X24 matrix. The probes were hybridized with the gene chips(temperature: 40℃, Time: 3 hr). Fluorescence signals were scanned by laser scannerand farther analyzed by ImaGene4.0 software with parallel comparison among thesethree gene profiles.The difference of gene expression was judged when onefluorescence intensity, at least, among the three gene profiles should be larger than1000, and the ratio of fluorescence intensity was more than 2 between any two geneprofiles/when the ratio is more than 2,We think the expression is up ,otherwise theexpression is down.Results:1. The reliability of the chip technique: There were 288 cDNA on the chips in comparison with negative rice genome sequence (2 points). The low expression of rice genome by hybridization proved the reliability of data.2. Results of the filtration with gene chip:A. Comparison with esophagcal carcinoma tissue and normal esophagealepitheliumThere were 6 genes expressed up in esophageal carcinoma including: BCL-l, BTK, JNK3A2, CST3, JAW1, Human MHC Class f HLA-A Cell Surface Antigen.There were 8 genes expressed down in esophagea! carcinoma including: ST13, KIAA0161, SOCS-I, D123, PROS-27,1RS1, DRR1, ACTR1A.B. Comparison with pericancerous tissue and normal esophageal epithelium. There were 8 genes expressed up in peri cancerous tissue including: BCL-1, TIAM1, JNK3AI, BTK, IGF1, K-ras, Homo sapiens mRNA for leptin receptor gene-related protein, Human mRNA for hematopoetic proteoglycan core protein. There were 8 genes expressed down in pericancerous tissue including: ST13, PROS-27, IRS1, mucSac, DP-79, APEX, SMAD3, Human colorecta! mutant Cancer protein mRNA.C. Comparison with esophageal carcinoma tissue and pericancerous tissue.There were 10 genes expressed up in esophageal carcinoma including: CD44, int-2,K-ras, R1Z, MRP3, bcl-1, BTK, C-fos, MCAD, Homo Sapiens mRNA for leptinreceptor gene-related protein.There were 5 genes expressed down in esophageal carcinoma including: MCM7,CNNB1, BRF1, TFAP2C, Homo Sapiens mRNA for更多还原