基于基因表达谱芯片杂交分析Lamprey-PHB2转染前后Chang liver细胞的基因表达差异

Microarray analysis of gene expression profiling in Chang Liver cells associated with Lamprey-PHB2 transfection

选取了10个物种与本课题组前期克隆得到的东北七鳃鳗抗增殖蛋白2(Lm-PHB2)进行氨基酸序列相似性对比,检测PHB2基因进化水平,结果表明各物种的PHB2氨基酸序列在PHB结构域处高度保守,但在N-端和C-端氨基酸序列保守性较低.将重组质粒pEGFP-N1-Lm-PHB2瞬时转染入张氏肝(CHL)细胞后,利用基因表达谱芯片技术分析基因的表达差异.结果显示CHL细胞中共有270条显著差异表达基因,其中显著上调基因共141条,显著下调基因共129条,涉及细胞信号转导、细胞周期调节、细胞增殖、细胞代谢和细胞凋亡等多个方面.通过实时荧光定量聚合酶链式反应(PCR)对基因表达谱芯片分析结果进行验证,结果显示转染pEGFP-N1-Lm-PHB2质粒后,细胞周期基因CDC25C、氧化应激相关基因(CAT,SOD,GST)和抗细胞凋亡基因HAX1均有显著性差异.

In order to detect the evolutionary level of prohibitin 2(PHB2)gene,10 species were selected to compare the PHB2 amino acid sequence with lamprey PHB2(Lm-PHB2),which was obtained by cloning from Chinese northeast lamprey(Lampetra morii)in present work.The results showed that the PHB2 amino acid sequences of the different species were highly conserved at the PHB domain,but the sequence identity at the N-terminal and C-terminal amino acid sequences was low.Gene chip expression profiles were used to analysis the genes expression alter after the pEGFP-N1-PHB2 plasmids transfected in Chang Liver(CHL)cells.The results showed that there were 270 genes significantly altered expressed in CHL cells,among them,141 genes were significantly up-regulated and 129 genes were down-regulated,including cell signal transduction,cell cycle regulation,cell proliferation,cell metabolism and apoptosis aspect.The results of Affymetrix Gene Chip Array were verified by real-time quantitative(PCR).The results showed that Cell cycle gene CDC25C,oxidative stress-related genes(CAT,SOD,GST)and anti-apoptotic gene HAX1 expression were significantly altered after transfected pEGFP-N1-Lm-PHB2 plasmids in CHL cells.