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目的应用寡核苷酸芯片技术进行高通量分析原发性肝细胞癌与癌旁组织以及正常肝组织的差异表达基因,探索与肿瘤进展相关的靶基因。方法对原发性肝细胞癌患者的癌组织、癌旁组织以及正常肝组织,应用Trizol-步法进行总RNA抽提,10g/L琼脂糖凝胶电泳和芯片实验室进行RNA质量检测。总RNA纯化后进行逆转录cRNA合成、荧光标记和纯化,将癌组织和正常肝组织、癌组织和癌旁肝组织的cRNA探针分别与Agilent寡核苷酸芯片(21 074探针)进行杂交,洗涤后应用Agilent扫描仪获取图像,特征提取软件进行定量分析处理。挑选明显差异表达的基因进行SYBR Green I染料掺入的荧光实时逆转录聚合酶链反应验证。结果(1)配对组织总RNA质量高,反转录cRNA及荧光标记质量好；(2)2倍差异表达基因中,上调基因共420个,下调基因共552个,其中包括5倍上调基因DKK1；(3)以β-肌动蛋白为内对照的实时逆转录聚合酶链反应结果提示,DKK1在癌、癌旁和正常肝组织中的2~(-ΔCt)值分别为0．089 504、0．007 65和0．000 631。结论应用Agilent寡核苷酸芯片高通量、高效率地分析原发性肝细胞癌发生发展过程中的基因表达差异,可以筛选新的治疗靶点；原发性肝细胞癌的发生发展涉及多基因、多步骤,是一个复杂的过程；DKK1可能是原发性肝细胞癌发展过程中的新的分子靶点,其表达变化涉及肿瘤进展。
OBJECTIVE: To use oligonucleotide microarray to analyze differentially expressed genes in primary hepatocellular carcinoma and adjacent non-cancerous tissues as well as in normal liver tissues and to explore target genes related to tumor progression. Methods The total RNA was extracted by Trizol-step method in 10 cases of primary hepatocellular carcinoma, adjacent tissues and normal liver tissues. The quality of RNA was detected by 10 g / L agarose gel electrophoresis and chip lab. Total RNA was purified and reverse transcribed cRNA was synthesized, fluorescently labeled and purified. The cRNA probes of cancer tissues, normal liver tissues, cancer tissues and adjacent liver tissues were respectively hybridized with Agilent oligonucleotide chips (21 074 probe) After washing, the scanner was used to acquire the image and the feature extraction software was used for quantitative analysis. Fluorescent real-time reverse transcription-polymerase chain reaction (PCR) was performed to identify genes that were significantly differentially expressed using SYBR Green I dye. Results (1) The total RNA of the paired tissues was of high quality with good reverse transcriptase (cRNA) and fluorescent labeling. (2) There were 420 up-regulated genes and 552 down-regulated genes in 2-fold differentially expressed genes including 5-fold up-regulated gene DKK1 ; (3) Real-time RT-PCR with β-actin as internal control suggested that the 2 ~ (-ΔCt) values of DKK1 in cancer, paracancer and normal liver tissues were 0.089 504, 0.007 65 and 0.000 631. Conclusion The high-throughput and efficient analysis of gene expression in primary hepatocellular carcinoma using Agilent oligonucleotide microarray can screen new therapeutic targets. The occurrence and development of primary hepatocellular carcinoma involve more Gene, multi-step, is a complex process; DKK1 may be new molecular targets in the development of primary hepatocellular carcinoma, the expression changes related to tumor progression.