Analysis of liver diesease related gene expression using subtraction cloning

肝细胞癌（HCC）的遗传基础尚未完全了解。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，通常难以从临床标本中进行有效的分析。在本研究中，我们使用抑制减法杂交（SSH）分析了少量人类HCC样本的差异表达基因。从丙型肝炎病毒相关的HCC和邻近的非HCC肝组织中获得总RNA。利用改良的RT-PCR方法合成cDNA，然后将测试cDNA与两种不同的接头连接，并与过量的驱动cDNA杂交。通过抑制PCR获得测试者特异性cDNA，并对最终PCR产物进行亚克隆和测序。7个已知基因（局灶黏附激酶，结肠癌缺失，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个先前未知的基因在HCC中过表达，1个基因（decorin）在HCC中被抑制。使用定量RT-PCR对基因表达进行定量分析，证实了这些基因在原始和其他HCC样本中的差异表达。这些发现表明，从少量临床样本中识别以前未知的差异基因表达是可能的。有关基因表达改变的信息可能有助于阐明HCC发病机制中的遗传事件，开发新的诊断标志物，或确定新的治疗靶点。使用类似的技术，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，一些线粒体基因上调。

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.