摘要
目的:应用抑制性消减杂交技术(SSH)筛选γ-氨基丁酸(GABA)作用肝星状细胞系HSC-T6后的差异表达基因.方法:10 umol/L的GABA作用于HSC-T6细胞24 h,提取mRNA,用分光光度计进行定量分析.以GABA处理和未处理的T6细胞mRNA为模板逆转录合成双链cDNA(dscDNA),并分别标记为Tester和Drivet,酶切后与接头连接,经两次杂交,构建消减杂交文库,将消减文库的扩增产物进行转化、克隆分析后应用生物信息学技术将测得序列再进行同源性分析.结果:15种基因表达上调,其中包括与DNA合成、线粒体、肿瘤抑制以及凋亡相关的4类基因,结果显示GABA可能促进HSC-T6细胞增殖而抑制凋亡.结论:用SSH技术可以成功获得GABA刺激肝星状细胞基因上调的监测,证明GABA能够影响肝星状细胞的基因表达谱.
AIM： To screen for genes regulated by gamma aminobutyric acid （GABA） in hepatic stellate cells （HSC） using suppression subtractive hybridization （SSH）, and to investigate the biological function of GABA in the liver. METHODS： The hepatic stellate cell line HSC-T6 was co-cultured with 10μmol/L GABA for 24 hours, after which mRNA was extracted and reverse transcribed into cDNA. HSC-T6 cells co-cultured with phosphate buffer saline （PBS） were used as controls, mRNAs upregulated by GABA were identified by SSH. Thirty one randomly selected clones were sequenced and analyzed bioinformatically. RESULTS： Fifteen genes were found to be significantly up-regulated, including genes involved in DNA synthesis, apoptosis, mitochondrial function and tumor suppression. These results showed that GABA might promote HSC-T6 cell proliferation and inhibitapoptosis CONCLUSION： SSH technology successfully enabled the identification of genes that are differentially expressed in the presence and absence of GABA, demonstrating that GABA can affect the gene expression profiles of HSCs.
出处
《世界华人消化杂志》
CAS
北大核心
2007年第26期2831-2834,共4页
World Chinese Journal of Digestology
关键词
Γ-氨基丁酸
肝星状细胞
抑制性消减杂交
Gamma aminobutyric acid
Hepatic stellate cell
Suppression subtractive hybridization
