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目的 与肝硬化对比 ,研究肝细胞癌中差异表达的基因 ,构建在肝硬化基础上发生癌变的肝细胞之差异表达的cDNA消减文库。方法 采用抑制性消减杂交获得差异表达基因片段 ,T/A克隆 ,蓝白斑筛选 ,在自动测序仪上进行基因测序 ,做同源性分析。结果 经过巢式PCR扩增后 ,得到差异表达的cDNA ,琼脂糖凝胶电泳显示为一弥散条带 ;3 5个差异表达的克隆被分离 ,基因表达序列标签长度在 112～ 75 5bp之间。序列分析揭示 2 8个克隆是来自以前描述过的基因 ,而另外 7个在GenBank/EMBL/DDBJ数据库没有匹配序列 ,可能代表新基因。结论 实验结果表明从肝硬化发展至肝细胞癌涉及到多个基因的差异表达 ;本研究在成功地构建出消减基因文库的同时 ,也证明了抑制性消减杂交技术对寻找差异表达基因是十分适用的。
Objective To compare the expression of genes differentially expressed in hepatocellular carcinoma with cirrhosis and construct a cDNA subtractive library with differential expression of hepatocytes that have undergone canceration on the basis of cirrhosis. Methods Suppression subtractive hybridization was used to obtain differentially expressed gene fragments, T/A clones, blue and white spot screenings, and gene sequencing was performed on an automated sequencer to perform homology analysis. Results After nested PCR amplification, differentially expressed cDNAs were obtained and agarose gel electrophoresis showed a diffuse band; 35 differentially expressed clones were isolated and the length of the gene expression sequence was between 112 and 75 bp. Sequence analysis revealed that 28 clones were from the previously described genes, while the other 7 had no matching sequences in the GenBank/EMBL/DDBJ database and may represent new genes. Conclusion The experimental results show that the development of hepatocellular carcinoma from liver cirrhosis involves the differential expression of multiple genes; this study successfully constructed a subtracted gene library and also proved that suppression subtractive hybridization technology is very suitable for finding differentially expressed genes. of.