cDNA-RDA技术分离新的肝硬化相关基因片段

Novel differential gene expression in human cirrhosis detected by opt imal cDNA representational difference analysis

目的 :分离在肝硬化组织上调表达的新基因 ,以阐述肝硬化发生的分子机制。方法 :利用优化的代表性差异分析方法分析正常肝脏及肝硬化组织表达基因的差异。两轮杂交后的产物克隆到T载体中 ,斑点杂交筛选阳性克隆以获得差异表达基因片段 ,并进一步以半定量RT PCR鉴定。结果 :上调表达的克隆包括与一些序列和功能完全未知的基因同源的cDNA以及未曾报道过的在肝脏病变中发生表达变化的已知基因———钙结合酪氨酸磷酸化调节蛋白、雌激素应答B盒蛋白等。结论 :利用优化的代表性差异分析方法成功分离获得在肝硬化组织中上调表达的基因片段 ,其中一些新基因的克隆及其在肝硬化发生中的作用有待进一步研究。

Objective: To isolate new up-regulated ge ne s related to the pathogenesis of cirrhosis. Methods: Liver transcriptomes in normal liver tissue and cirrhosis were examined by opt imal cDNA representational difference analysis (cDNA-RDA). Resulting complement ary DNA (cDNA) clones were rescreened for differential expression by dot-blot h ybridization and then sequenced. Selected gene expression was identified with se mi-quantitative RT-PCR. Results: Three clones were homo logous only with expressed sequence tag (EST) sequences encoding genes having un known function or/and sequence. CABYR 〔calcium-binding tyrosine-(Y)-phosphor ylation regulated protein〕 and EBBP (estrogen-responsive B box protein) were first reported up-regulated in cirrhotic liver disease. Conclusion: Novel observations of differential gene expression in cirrhosis were made using cDNA-RDA. In particular,further studies of some novel genes and th eir product associated with cirrhosis are warranted.