原发性肝细胞癌消减杂交文库构建及差异表达基因筛选

Construction and screening of suppression subtractive hybridization library of human primary hepatocellular carcinoma

目的构建人原发性肝细胞癌(HCC)消减杂交文库,筛选差异表达基因。方法以癌组织为检测者(tester)、癌旁组织为参照者(driver),应用抑制性消减杂交(SSH)方法构建消减杂交cDNA文库,随机挑选56个阳性克隆进行鉴定、测序及同源性分析。结果文库获得130个阳性克隆,鉴定96％有200～1 500 bp插入片断。获得的35个基因中包括已知基因26个。功能未知基因5个,新基因4个。已知基因中包含酶、细胞信号转导、基因调控因子、转录调节因子、癌基因、抗凋亡因子及细胞的黏附与生长调节因子等相关基因。结论应用SSH方法成功构建HCC差异表达基因cDNA文库,筛选出低丰度和新基因在内的HCC差异表达基因。

Objective To construct the suppression subtractive hybridization （SSH） library of human primary hepatocellular carcinoma （HCC） and screen the differential gene expression. Methods RNA from both HCC tissue （tester） and the corresponding non-HCC tissue （driver） Was isolated. By using SSH, the subtractive hybridization cDNA library was constructed. Fifty-six clones were picked randomly and amplified by colony PCR, then sequenced to identify the homology against the human UniGene database. Results Among 130 clones screened, 96% of them had the inserts with 200-1500 bp;Among 35 genes identified, 26 had known functions, 5 unknown functions and 4 with unknown genes which had no homology sequence on the human UniGene database. The known genes contained enzymes, cellular signal molecules, gene regulators, transcriptional factors, oncogenes, genes related with anti-apoptosis factors, adhesion and growth regulators. Conclusion The subtractive HCC library was established successfully by using SSH method and the differentiaUy expressed genes for HCC were screened.