用基因芯片结合荧光差异显示-PCR筛选和识别胃腺癌转移相关的基因  

Screening and Identification of Gastric Adenocarcinoma Metastasis-related Genes by Using cDNA Microarray Coupled to FDD-PCR

作　　者：王建华[1] 陈诗书[1] 

机构地区：[1]上海第二医科大学分子生物学实验室,人类基因治疗研究中心,上海200025

出　　处：《生物化学与生物物理学报》2002年第4期475-481,共7页

摘　　要：使用来源于同一个胃腺癌病人的原发灶RF 1(ATCC编号 :CRL 186 4 )和转移灶RF 4 8细胞系 (ATCC编号 :CRL 186 3)作为研究肿瘤转移分子机制的模型。RF 1(实验组 )和RF 4 8(对照组 )的mRNA通过逆转录方法 ,将Cy3和Cy5两种荧光染料分别标记到两种细胞的cDNA上 ,制备成cDNA探针 ,并与表达谱芯片 (双点 4 0 96条基因 )进行杂交与扫描 ,重复 2次实验 ,利用计算机数据处理判断基因是否在上述两种细胞中有表达差异 ,共筛选出差异表达的基因共 138条 ,其中 81条在RF 4 8细胞中表达明显上调 ,5 7条在RF 4 8细胞中表达显著下调。同时也通过荧光差异显示 PCR(FDD PCR)技术 ,克隆了 4 5个涉及胃腺癌转移相关基因 ,包括未被发现的基因 3个。在两种筛选方法中都存在差异表达的基因共有 7条。对部分可能与肿瘤转移机制有关的差异表达基因的作用进行了分析和讨论。基因芯片技术可高通量、大规模地研究基因表达水平 ,FDD PCR技术可克隆出未发现的新基因 ,二者结合 ,初步筛选出与转移相关的基因 ,有助于揭示胃腺癌转移的分子机制。To clone gastric adenocarcinoma metastasis related genes, RF 1 cell line (primary tumor of a gastric adenocarcinoma patient ) and RF 48 cell line (its metastatic counterpart) were used as a model for studying the molecular mechanism of tumor metastasis. Two fluorescent cDNA probes, labeled with Cy3 and Cy5 dyes, were prepared from RF 1 and RF 48 mRNA samples by reverse transcription method. The two color probes were then mixed and hybridized to the cDNA chip constructed by double dots of 4 096 human genes, and scanned at two wavelengths. The experiment was repeated for 2 times. Differential expression genes from the above two cells were analyzed using the computer. 138 in all genes (3.4%) revealed differential expression in RF 48 cells compared with RF 1 cells: 81(2.1%) genes revealed apparent up regulation, and 56(1.3%) genes revealed down regulation. 45 genes involved in gastric adenocarcinoma metastasis were cloned using fluorescent differential display PCR (FDD PCR), including 3 novel genes. There were 7 differential expression genes that agreed with each other in two detection methods. The possible roles of some differential expressed genes, which maybe involved in the mechanism of tumor metastasis, were discussed. cDNA chip was used to analyze gene expression in a high throughput and large scale manner, in combination with FDD PCR for cloning unknown novel genes. In conclusion, some genes related to metastasis were preliminarily scanned, which would contribute to disclose the molecular mechanism of gastric adenocracinoma metastasis.

关 键 词：基因芯片 荧光差异显示-PCR 筛选 识别 胃腺癌 转移相关基因 

分 类 号：R735.2[医药卫生—肿瘤]