摘要
目的 应用基因表达谱芯片技术了解XBP1S在肝细胞中可能上调或下调的基因,了解其可能的调节功能线索.方法 构建pcDNA3.1（-）-XBP1S真核表达载体,转染HepG2细胞,同时以空载体pcDNA3.1（-）处理相同细胞系作为对照.48 h后制备细胞裂解液,提取mRNA,应用基因表达谱芯片技术对差异表达mRNA进行检测和分析.结果 构建的表达载体经过限制性内切酶分析和DNA序列测定,证实准确无误,提取高质量的总mRNA并进行逆转录成为cDNA,进行基因表达谱芯片技术分析.经过差异基因表达谱的筛选,发现HepG2细胞转染XBP1S以后,有38个基因表达水平显著上调,30个基因表达水平显著下调.结论 成功构建XBP1S的真核表达载体pcDNA3.1（-）-XBP1S,运用基因表达谱芯片技术成功筛选了XBP1S转染细胞后的差异表达基因,这些差异表达基因包括细胞周期、蛋白质的翻译合成及运输、能量代谢、体内免疫调节、细胞凋亡及细胞内的信号转导等方面起重要作用及肿瘤发生相关的基因,为进一步阐明XBP1S可能存在的调控机制及XBP1S蛋白可能的生物学功能提供理论依据.
Objective To understand some regulation genes mediated by XBP1S in hepatocytes with gene expression profile and to explore XBP1S regulatory function. Methods The plasmid pcDNA3.1 （ - ） -XBP1S was constructed followed by restriction enzyme digestion and DNA sequen- cing analysis, and then transfect into HepG2 cells. Cells were lyse and extract mRNA was extracted after 48hr. Then gene expression was analyzed with gene expression profile CHIP. Results 38 upregulated genes and 30 downregulated genes were found by gene expression profile CHIP analysis. Conclusion pcDNA3.1 （-） -XBP1S was constructed successfully. The differentially expressed genes include those involved in cell cycle, protein translation and transportation, energy metabolism, in vivo immunoregulation, cell apoptosis, cell transduction and tumorigenesis related genes. These results provide theoretical basis for further study of XBP1S regulation mechanism and its biological function.
出处
《医学分子生物学杂志》
CAS
CSCD
2010年第3期225-232,共8页
Journal of Medical Molecular Biology
基金
教育部留学人员基金（No.教外司留2009-1590号）,重庆市科委自然科学基金（No.CSTC 2009BB5062）,重庆医科大学重点项目（No.XBZD200803）Q
