应用抑制性消减杂交技术筛选甘草酸苷下调大鼠肝星状细胞的靶基因

Screening and Cloning of Genes Down-regulated in Hepatic Stellate Cells Treated with Glycyrrhizin Using Suppression Subtractive Hybridization Technique

目的应用抑制性消减杂交（SSH）技术构建甘草酸苷处理的大鼠肝星状细胞（HSC）下调基因的cDNAs消减文库，从分子生物学角度阐明甘草酸苷治疗肝硬化的作用机制。方法以未用甘草酸苷处理的HSC作为对照组，同时以甘草酸苷作用的相同细胞系作为实验组；24h后制备细胞裂解液，提取mRNA并逆转录为cDNAs，经Rsa I酶切后。将时照组cDNAs分成两组，分别与两种不同的接头衔接，再与实验组cDNAs进行两次消减杂交及两次抑制性多聚酶链反应（PCR），将产物与T／A载体连接，构建cDNAs消减文库，并转染大肠埃希菌进行文库扩增，随机挑选克隆PCR扩增后进行测序及同源性分析。结果成功构建甘草酸苷作用的HSC下调基因的cDNAs消减文库。文库扩增后得到15个阳性克隆，进行菌落PCR分析，均得到200～1000bp插入片段。挑取含有插入片段的10个克隆进行测序，并通过生物信息学分析获得4种已知基因序列，包括损伤特异性的DNA结合蛋白（Ddbl）、Toll／白细胞介素-1样受体3（TIL3）、鲨烯环氧酶、结合肌动蛋白的双锌指蛋白（abLIM）。结论应用SSH技术成功构建了甘草酸苷下调HSC基因的cDNAs消减文库。该文库的建立为进一步阐明甘草酸苷治疗肝硬化的作用机制提供了理论依据。

Objective To construct a subtractive cDNAs library of genes down-regulated in hepatic stellate cells treated with glycyrrhizin using suppression subtractive hybridization （SSH） technique so as to illustrate the mechanism of glycyrrhizin in liver cirrhosis from the angle of molecular biology. Methods The mRNAs were isolated from hepatic stellate cells untreated and treated with glycyrrhizin and by reverse transcription the cDNAs were synthesized and designated as tester and driver, respectively. After restriction enzyme RsaI digestion, small sizes cDNAs were obtained. Tester cDNAs were subdivided into two portions and each was ligated with different cDNAs adaptor. After tester cDNAs were hybridized with driver cDNAs twice and underwent nested polymerase chain reaction （PCR） twice, the DNA fragments were subeloned into T/A plasmid vectors to set up the subtractive cDNAs library. Amplification of the library was carried out with DH5α. The cDNAs were sequenced and analyzed in GenBank with Blast search after colony PCR. Results The subtractive cDNAs library of genes down-regulated in hepatic stellate cells treated with glycyrrhizin was constructed successfully. The amplified library contained 15 positive clones. Colony PCR showed that these clones contained 200～1 000 bp inserts. Ten clones were analyzed by sequencing and bioinformaties. Four known genes genes were obtained, including damage-specific DNA binding protein 1 （Ddbl）, Toll/intedeukin-1 receptor-like protein 3 （TIL3）, squalene epoxidase and actin-binding double-zinc-finger protein （abLIM）. Conclusion A sublractivc cDNAs library of genes down-regulated in hepatic stellate cells treated with glycyrrhizin using SSH technique is constructed successfully, which provides theoretic base for illustrating the regulatory mechanism of glyeyrrhizin in liver cirrhosis.