肝硬化患者发生的肝细胞癌差异表达基因文库的构建及初步筛选

Initial screening and library-making of differentially expressed genes in hepatocellular carcinoma based on cirrhosis

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。

Aim To study the differentially expressed genes in hepatocellular carcinoma(HCC) compared with cirrhosis and make the differentially expressed cDNAs subtracted library of malignant hepatocytes based on cirrhosis. Methods The differentially expressed fragments were obtained by using of suppression subtractive hybridization(SSH), then T/A cloning was accomplished. The positive clones were screened through blue/white visual assay and sequenced on automated sequencing system, finally homological analysis was performed on computers. Results Differentially expressed cDNAs were produced by Nested-PCR amplification, and after the electrophoresis on agarose gel it looked like a smear. All of 35 differentially expressed clones were isolated . The size of expressed sequence tags ranged from 112bp to 755bp. Sequence analysis revealed that 28 clones were from previously described genes, but the remaining 7 clones did not match any sequences in GenBank/EMBL/DDBJ database, indicating that they may be novel genes. Conclusions The experimental findings demonstrated that the HCC derived from cirrhosis involved the differentially expressed genes; whilst the subtracted gene library was constructed successfully, the technigue of SSH is proved to be applicable to seek differentially expressed genes.