基因表达谱芯片技术筛选肝再生增强因子反式调节基因

Screening and identification of genes transactivated by human augmenter of liver regeneration by microarray assay

目的:为了阐明人肝再生增强因子(augmenter of liver regeneration,ALR)的表达对于肝细胞基因表达谱的影响, 我们应用基因芯片技术,对于转染和未转染的HepG2细胞进行了分析. 方法:从HepG2细胞RNA中用反转录聚合酶链反应法(RT- PCR)扩增出ALR编码区DNA,常规分子生物学技术构建ALR的真核表达载体pcDNA3.1(-)-ALR,利用脂质体转染技术转染HepG2细胞,ALR的表达以Western blot杂交技术证实.从转染和非转染细胞HepG2种提取总mRNA,逆转录为cDNA,并进行基因芯片技术分析. 结果:经过限制性内切酶分析和序列测定,证实pcDNA3.1 (-)-ALR构建正确.ALR在HepG2细胞中的表达以Western blot杂交技术得到证实.对于ALR重组表达载体和空白载体转染的HepG2细胞的基因表达谱,利用基因芯片技术进行分析.结果表明,2种基因的表达水平上调,24种基因的表达水平下调.这些基因包括淀粉酶α、肿瘤坏死因子受体、金属蛋白酶1组织抑制因子、性激素相关蛋白等基因,相信这些类型的基因在ALR所发挥的生物学效应起到重要的作用. 结论:ALR对于肝细胞基因表达谱存在一定影响;基因芯片技术是分析蛋白反式调节基因表达谱的重要技术途径,有助于了解ALR对肝细胞和其他生物学功能的调节作用.

AIM: Augmenter of liver regeneration (ALR) is a protein that plays a role in liver regeneration. In order to clarify the effect of the expression of ALR on profile of the hepatocytic gene expression, we analyzed difference between the HepGZ cells transfected with ALR and controls by using gene chip technology. METHODS: Total RNA was extracted from HepG2 cells, and RT-PCR was performed to amplify the coding region of ALR. The expression of ALR in the transfected HepG2 cells was confirmed by Western blot. Total mRNA was isolated from the transfected HepG2 cells with pcDNA3.1(-) and pcDNA3-ALR, respectively. cDNA was prepared by reverse transcription. Microarray assay was conducted for screening of up-and down-regulated genes in both HepG2 cells. RESULTS: The expressive vector of pcDNA3(-)-ALR was constructed and confirmed by restriction enzyme digestion and DNA sequencing. The expression of ALR was confirmed by Western blot. After screening with cDNA microarray, we found 2 genes were up-regulated, and 24 genes including TNFRSFlA-associated via death domain, tissue inhibitor of metalloproteinase 1, epididymal androgen-related protein, down-regulated. CONCLUSION: ALR is a cell growth factor, which has some influences on gene expression profile of hepatocytes; Microarray technology is a method to analyze gene expression spectra of trans-regulation of a protein and conducive to understand the regulative effect of ALR on hepatocytes and other biological function.