中文摘要：

目的探讨胰腺癌相关基因寡核苷酸芯片的构建，初步验证其在检测胰腺癌基因表达谱方面的应用。方法有目的地筛选胰腺癌相关基因，采用合成后点样的方法，制成寡核苷酸基因芯片。TRIzol法抽提组织总RNA，在cDNA第一链合成过程中，通过反转录酶将CyDye标记核苷酸直接掺入到eDNA中制备荧光探针，其中用Cy3-dCTP标记胰腺癌组织，cy5-dCTP标记正常胰腺组织。将荧光标记探针与芯片杂交16—18h。用Agilent扫描仪进行扫描，Imagene3．0软件进行图像分析，计算两种荧光Cy3与cy5信号强度的比值。挑选差异基因CDC25B和TUSC3进行荧光定量PCR（Sybrgreen方法）验证，PCR产物的定量方法采用比较Ct法。结果芯片杂交扫描图像信号清晰，具有较低的整体背景和较高的信噪比，各阳性质控点信号均匀一致，阴性质控点和空白点信号低。与正常组织相比，胰腺癌组织中差异表达基因24条，占全部基因的25．5％，其中上调基因17条（18．1％），下调基因7条（7．4％）。荧光定量PCR验证，CDC25B和TUSC3基因在胰腺癌中的表达趋势与芯片实验的结果一致。结论本研究制备的胰腺癌相关基因芯片可同时、并行检测多种胰腺癌相关基因的表达改变，具有一定的特异性和敏感性。胰腺癌组织与正常胰腺组织相比，基因表达谱具有明显差异。

英文摘要：

Objective To investigate the construction of oligonucleotide mieroarray specialized for pancreatic adenocarcinoma-associated genes and its application. Methods Pancreatic cancer related genes were purposely selected, and oligenucleotide microarray was prepared by spotting oligonucleotide probes onto glass slides coated with APS-PDC. Total RNA were extracted from frozen tissues with TRIzol method according to the manufacturers protocol, and purified with QIAGEN Rneasy Kit. Labeled cDNA targets for hybridizations were synthesized by reverse transcription from control- and cancer-total RNA samples in the presence of CyS-dCTP and Cy3-dCTP, respectively. The labeled probes were hybridized with oligenucleotide microarray for 16 h to 18 h. Hybridized microarray was scanned by Agilent laser scanner, and the aquired image was analyzed by Imagene3. 0 software. The intensity ratio of Cy3 and Cy5 were calculated. To confirm the expression profiles of these genes, quantitative reverse transcription-PCR （QRT-PCR） was carried out with CDC25B and TUSC3 genes. The productd of PCR were quantitated by comparative Ct method. Results The signal of microarray hybridization was clear, and the images had a lower background and higher signalnoise ratio. The signal of positive control spots were uniform, and spots of negative control and blank signal were fairly low. In comparison with normal pancreas, 24 differential expressed genes were identified, which included 17 up-regnlated and 7 down-regulated genes. The results of QRT-PCR demonstrated that the expression of CDC25B and TUSC3 in pancreatic cancer wexe increased and decreased respectively, which consistent with microarray hybridization. Conclusions The oligonucleotide microarray specialized for pancreatic cancer are desirable for its specialty, flexibility and sensitivity, which can simultaneously and parallelly detect multiple pancreatic cancer-associated genes. In contrast to normal pancreatic tissues, the genes expression profile are different significantly in pancreatic canc