Analysis of liver diesease related gene expression using subtraction cloning

肝细胞癌的遗传学基础尚不完全清楚。虽然已经开发了各种方法来检测恶性疾病中的差异表达基因，但由于需要大量的样本，从临床标本中进行有效的分析通常是困难的。在本研究中，我们使用抑制消减杂交技术(SSH)分析了少量人肝细胞癌样本中差异表达的基因。总RNA取自丙型肝炎病毒相关的肝细胞癌组织和邻近的非肝细胞癌组织。用改进的RT-PCR方法合成cDNA，用两种不同的接头连接TestcDNA，并与过量的DRIVER基因进行杂交。通过抑制性聚合酶链式反应获得受试者特异性基因，并对扩增产物进行亚克隆和测序。已知的7个基因(粘着斑激酶，在结肠癌中缺失，鸟嘌呤结合抑制蛋白a，谷氨酰胺合成酶，鸟氨酸转氨酶，M130和胃蛋白酶原C)和2个未知基因在肝细胞癌中过表达，1个基因(核心蛋白)在肝细胞癌中抑制。利用定量RT-PCR对基因表达的定量分析表明，这些基因在原始肝癌样本和其他肝癌样本中的表达存在差异。这些发现表明，从少量的临床样本中识别以前未知的差异基因表达是可能的。有关基因表达变化的信息可能有助于阐明肝癌发病机制中的遗传事件，开发新的诊断标记，或确定新的治疗靶点。利用类似的技术，我们发现IP10在自身免疫性肝炎和慢性丙型肝炎中过表达。在PBC中，几个线粒体基因上调。

The genetic basis of hepatocellular carcinoma (HCC) has not yet been fully understood. Although various methods have been developed to detect differentially expressed genes in malignant diseases, efficient analysis from clinical specimens is generally difficult to perform due to the requirement of a large amount of samples. In the present study, we analyzed differentially expressed genes with a small amount of human HCC samples using suppression subtractive hybridization (SSH). Total RNA were obtained from the hepatitis C virus-associated HCC and adjacent to non-HCC liver tissues. cDNA was synthesized using modified RT-PCR, and then tester cDNA was ligated with two different kinds of adaptors and hybridized with an excess amount of driver cDNA. Tester specific cDNA was obtained by suppression PCR and the final PCR product was subcloned and sequenced. Seven known genes (focal adhesion kinase, deleted in colon cancer, guanine binding inhibitory protein a, glutamine synthetase, ornithine aminotransferase, M130, and pepsinogen C) and two previously unknown genes as being overexpressed in HCC, and 1 gene (decorin) as suppressed in HCC. Quantitative analysis of gene expression using quantitative RT-PCR demonstrated the differential expression of these genes in the original and other HCC samples. These findings demonstrated that it is possible to identify the previously unknown, differential gene expression from a small amount of clinical samples. Information about such alterations in gene expression could be useful for elucidating the genetic events in HCC pathogenesis, developing the new diagnosic markers, or determining novel therapeutic targets. Using similar technique, we found overexpression of IP10 in autoimmune hepatitis and chronic hepatitis C. In PBC, several mitochondrial genes are up-regulated.