【摘要】 目的筛选并克隆转化生长因子β1(TGF-β1)刺激肝星状细胞差异表达下调基因,阐明TGF-β1导致肝纤维化的分子生物学机制。方法以TGF-β1及磷酸盐缓冲液分别刺激大鼠肝星状细胞(即实验组和对照组),提取mRNA并逆转录为cDNA,经RsaⅠ酶切后,将对照组细胞cDNA分成两组,分别与两种不同的接头衔接,再与实验组细胞cDNA进行2次消减杂交及2次抑制性PCR扩增,将产物与pGEM-Teasy载体连接,构建cDNA消减文库,并转染大肠埃希菌进行文库扩增,随机挑选克隆经PCR扩增后进行测序及同源性分析。结果成功构建了TGF-β1刺激肝星状细胞差异表达基因的cDNA消减文库。文库扩增后得到98个阳性克隆,进行菌落PCR分析,均得到200～1000bp插入片段。选取含有插入片段的35个克隆进行测序,并通过生物信息学分析获得了19种已知基因序列和2个未知功能基因。结论应用抑制性消减杂交技术成功构建了TGF-β1刺激的肝星状细胞差异表达基因的cDNA消减文库,为进一步阐明TGF-β1参与肝纤维化的分子生物学机制提供了理论依据。更多还原

【Abstract】 Objective To screen and clone the down-regulated genes transactivated by transforming growth factor β1 (TGF-β1),further to elucidate the molecular biological mechanisms of hepatic fibrosis mediated by TGF-β1. Methods mRNA were isolated from hepatic stellate cells (HSCs) treated with TGF-β1 or PBS,respectively (experimental and control groups). Suppression subtractive hybridization (SSH) technique was employed to analyze the differentially expressed DNA sequences between the two groups. After restriction enzyme RsaⅠdigestion,small sizes cDNAs were obtained. Then control cDNA was divided into two groups and ligated to the specific Adaptor 1 and Adaptor 2,respectively. After experimental cDNA was hybridized with control cDNA and underwent subtractive PCR twice,respectively,and then subcloned into pGEM-Teasy plasmid vectors to construct the subtractive library. Amplification of the library was carried out with E.coli strain DH5α. The cDNA was sequenced and analyzed in GenBank by Blast search. Results The subtractive cDNA library of differentially expressed genes in HSCs treated with TGF-β1 was constructed successfully. The amplified library contained 98 positive clones,which were identified as 200-1000 bp inserts by PCR. Thirty-five clones were analyzed by sequencing and bioinformatic analysis,there were 19 known genes and 2 unknown genes. ConclusionsThe subtractive cDNA library of differentially expressed genes in HSCs treated with TGF-β1 by SSH technique was constructed successfully,which provide new insights into the study on mechanisms of liver fibrosis.更多还原